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Adrenocortical nuclear progesterone-binding protein: Identification by photoaffinity labeling and evidence for deoxyribonucleic acid binding and stimulation by adrenocorticotropin
Nuclei of the guinea pig adrenal cortex contain a protein that specifically binds progesterone and that, biochemically, is clearly distinct from the classical progesterone receptor. The adrenocortical nuclear progesterone-binding protein has now been purified more than 2000-fold by steroid-affinity chromatography with a 75% yield. The purified protein preparation demonstrated three major bands on sodium dodecyl sulfate-polyacrylamide gel of 79K, 74K, and 50K. To determine which of the three might represent the progesterone-binding protein, steroid photoaffinity labeling was performed which resulted in the specific and exclusive labeling of a 50K band. Thus, the adrenocortical nuclear progesterone-binding protein appears to be distinct from the classical progesterone receptor not only biochemically, but also on the basis of molecular size. To test whether the adrenocortical nuclear progesterone-binding protein can be hormonally stimulated, guinea pigs were treated with ACTH. The chronic administration of ACTH caused a 4- to 6-fold increase in the specific progesterone binding capacity without a change in the binding affinity. There appeared to be no significant difference in nuclear progesterone binding between the zona fasciculata and zona reticularis. This finding suggests a mediating role for the progesterone-binding protein in ACTH action. In addition, the nuclear progesterone-binding protein bound to nonspecific DNA sequences, further suggesting a possible transcriptional regulatory role.
Demura, T.; Driscoll, W.J.; Lee, Y.C.; Strott, C.A. (National Institute of Child Health and Human Development, Bethesda, MD (USA))
Nuclear Pore Proteins and Cancer
Nucleocytoplasmic trafficking of macromolecules, a highly specific and tightly regulated process, occurs exclusively through the Nuclear Pore Complex. This immense structure is assembled from approximately 30 proteins, termed nucleoporins. Here we discuss the four nucleoporins that have been linked to cancers, either through elevated expression in tumors (Nup88) or through involvement in chromosomal translocations that encode chimeric fusion proteins (Tpr, Nup98, Nup214). In each case we consider the normal function of the nucleoporin and its translocation partners, as well as what is known about their mechanistic contributions to carcinogenesis, particularly in leukemias. Studies of nucleoporin-linked cancers have revealed novel mechanisms of oncogenesis and. in the future, should continue to expand our understanding of cancer biology.
Xu, Songli; Powers, Maureen A.
GAPDH Mediates Nitrosylation of Nuclear Proteins
S-nitrosylation by nitric oxide (NO) is a major mode of signaling to cellular proteins1, including prominent nuclear proteins such as HDAC22 and PARP13. The high reactivity of the NO group with protein thiols implies the existence of selective targeting mechanisms. Specificity of NO signaling is often achieved by the binding of NO synthase (NOS) to target proteins, either directly4 or through scaffolding proteins such as PSD-955 and CAPON6. As the three principal isoforms of NOS - neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS) - are primarily non-nuclear, the mechanisms by which nuclear proteins are selectively nitrosylated have been elusive. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is physiologically nitrosylated at its Cys150 residue, conferring upon it the ability to bind to Siah1, which possesses a nuclear localization signal and conveys nitrosylated GAPDH (SNO-GAPDH) to the nucleus7. We now show that SNO-GAPDH physiologically transnitrosylates nuclear proteins, including the deacetylating enzyme SIRT1, histone deacetylase-2 (HDAC2), and DNA-activated protein kinase (DNA-PK). Our findings reveal a novel mechanism for targeted nitrosylation of nuclear proteins and suggest that protein-protein transfer of NO groups may be a general mechanism in cellular signal transduction.
Kornberg, Michael D.; Sen, Nilkantha; Hara, Makoto R.; Juluri, Krishna R.; Van K. Nguyen, Judy; Snowman, Adele M.; Law, Lindsey; Hester, Lynda D.; Snyder, Solomon H.
Nuclear matrix proteins in human colon cancer.
The nuclear matrix is the nonchromatin scaffolding of the nucleus. This structure confers nuclear shape, organizes chromatin, and appears to contain important regulatory proteins. Tissue specific nuclear matrix proteins have been found in the rat, mouse, and human. In this study we compared high-resolution two-dimensional gel electropherograms of nuclear matrix protein patterns found in human colon tumors with those from normal colon epithelia. Tumors were obtained from 18 patients undergoing partial colectomy for adenocarcinoma of the colon and compared with tissue from 10 normal colons. We have identified at least six proteins which were present in 18 of 18 colon tumors and 0 of 10 normal tissues, as well as four proteins present in 0 of 18 tumors and in 10 of 10 normal tissues. These data, which corroborate similar findings of cancer-specific nuclear matrix proteins in prostate and breast, suggest that nuclear matrix proteins may serve as important markers for at least some types of cancer. Images
Keesee, S K; Meneghini, M D; Szaro, R P; Wu, Y J
Nuclear Pore Complex Protein Mediated Nuclear Localization of Dicer Protein in Human Cells
Human DICER1 protein cleaves double-stranded RNA into small sizes, a crucial step in production of single-stranded RNAs which are mediating factors of cytoplasmic RNA interference. Here, we clearly demonstrate that human DICER1 protein localizes not only to the cytoplasm but also to the nucleoplasm. We also find that human DICER1 protein associates with the NUP153 protein, one component of the nuclear pore complex. This association is detected predominantly in the cytoplasm but is also clearly distinguishable at the nuclear periphery. Additional characterization of the NUP153-DICER1 association suggests NUP153 plays a crucial role in the nuclear localization of the DICER1 protein.
Morinaga, Ayako; Burroughs, Alexander Maxwell; Kawaji, Hideya; Kubosaki, Atsutaka; Kimura, Ryuichiro; Tagata, Maiko; Ino, Yoko; Hirano, Hisashi; Chiba, Joe; Suzuki, Harukazu; Carninci, Piero; Hayashizaki, Yoshihide
Nuclear Export of Proteins and RNA
Several nuclear export receptors that facilitate the export of proteins and small RNAs from the nucleus to the cytoplasm have\\u000a been functionally characterized in Arabidopsis thaliana in the past few years. With the specific cargo molecules they transport, the export receptors supply the cytoplasm with information,\\u000a resulting in changes in cellular events. In this way, nuclear export receptors contribute to
Thomas Merkle
Sumoylation of heterogeneous nuclear ribonucleoproteins, zinc finger proteins, and nuclear pore complex proteins: A proteomic analysis
SUMO, a small ubiquitin-related modifier, is known to covalently attach to a number of nuclear regulatory proteins such as p53, IB, promyelocytic leukemia protein and c-Jun. The sumoylation reaction is catalyzed by the SUMO protease, which exposes the C-terminal active glycine residue of the nascent SUMO, the heterodimeric SUMO activating enzyme, the SUMO conjugating enzyme, Ubc9, and SUMO protein ligases,
Tianwei Li; Evgenij Evdokimov; Rong-Fong Shen; Chien-Chung Chao; Ephrem Tekle; Tao Wang; Earl R. Stadtman; David C. H. Yang; P. Boon Chock
Farnesylated Nuclear Proteins Kugelkern and Lamin Dm0 Affect Nuclear Morphology by Directly Interacting with the Nuclear Membrane
Nuclear shape changes are observed during a variety of developmental processes, pathological conditions, and ageing. The mechanisms underlying nuclear shape changes in the above-mentioned situations have mostly remained unclear. To address the molecular mechanism behind nuclear shape changes, we analyzed how the farnesylated nuclear envelope proteins Kugelkern and lamin Dm0 affect the structure of the nuclear membrane. We found that Kugelkern and lamin Dm0 affect nuclear shape without requiring filament formation or the presence of a classical nuclear lamina. We also could show that the two proteins do not depend on a group of selected inner nuclear membrane proteins for their localization to the nuclear envelope. Surprisingly, we found that farnesylated Kugelkern and lamin Dm0 protein constructs change the morphology of protein-free liposomes. Based on these findings, we propose that farnesylated proteins of the nuclear membrane induce nuclear shape changes by being asymmetrically inserted into the phospholipid bilayer via their farnesylated C-terminal part.
Polychronidou, Maria; Hellwig, Andrea
Nuclear Nonhistone Proteins in Murine Melanoma Cells
Nuclear nonhistone proteins (NHP's) have been implicated as regulatory agents involved in controlling genetic expression. Utilizing murine melanoma cells, we describe a method for isolating and fractionating NHP's which greatly increases the yield of these proteins as well as the level of resolution required for detecting small differences in particular NHP's. Mouse melanoma cells were grown in medium labeled with [3H]leucine. Following 48 hr of incubation, the cells were harvested and nuclei isolated. The NHP's were extracted from the nuclei in a series of steps which yielded four major fractions: NHP1, NHP2, NHP3, NHP4. This method solubilized 80-90% of the protein from the nuclear homogenate. The NHP fractions were then separated on DEAE-cellulose columns in a series of salt steps increasing in concentration from 0.05 to 0.50 M NaCl, followed by steps of 2 M NaCl and 4 and 7 M guanidine-hydrochloride. The 40 NHP fractions eluted from these columns were further separated on polyacrylamide-SDS gels and ranged in molecular weight from 9000 to 110,000 daltons. Differences were observed in the electrophoretic pattern of each of these 40 fractions. The high resolution of these fractionation procedures greatly enhances the possibility of observing small changes in proteins which may play a role in gene regulation. ImagesFIG. 2FIG. 5
Wikswo, Muriel A.; Mcguire, Joseph S.; Shansky, Janet E.; Boshes, Roger A.
Protein Dynamics: Implications for Nuclear Architecture and Gene Expression
NSDL National Science Digital Library
Studies of nuclear architecture reveal that the dynamic properties of proteins in the nucleus are critical for their function. The high mobility of proteins ensures their availability throughout the nucleus; their dynamic interplay generates an ever-changing, but overall stable, architectural framework, within which nuclear processes take place. As a consequence, overall nuclear morphology is determined by the functional interactions of nuclear components. The observed dynamic properties of nuclear proteins are consistent with a central role for stochastic mechanisms in gene expression and nuclear architecture.
Tom Mistelli (National Cancer Institute;)
The insulator binding protein CTCF associates with the nuclear matrix
Nuclear DNA is organized into chromatin loop domains. At the base of these loops, matrix-associated regions (MARs) of the DNA interact with nuclear matrix proteins. MARs act as structural boundaries within chromatin, and MAR binding proteins may recruit multiprotein complexes that remodel chromatin. The potential tumor suppressor protein CTCF binds to vertebrate insulators and is required for insulator activity. We
Katherine L Dunn; Helen Zhao; James R Davie
Nuclear Protein Isoforms: Implications for Cancer Diagnosis and Therapy
Post translational modifications (PTMs) of nuclear proteins play essential roles in the regulation of gene transcription and signal transduction pathways. Numerous studies have demonstrated a correlation between specific nuclear protein isoforms and cellular malignant process. This communication reviews the impact of major PTM events such as phosphorylation, acetylation, methylation, ubiquitination and sumoylation on several important nuclear proteins including p53, histones, proliferating cellular nuclear antigen (PCNA), and retinoblastoma protein (Rb) in the process. In addition, the implications of the PTMs as cancer biomarkers and therapeutic targets are considered.
Shen, Fei; Kirmani, Kashif Z.; Xiao, Zhimin; Thirlby, Benjamin H.; Hickey, Robert J.; Malkas, Linda H.
Nuclear protein isoforms: implications for cancer diagnosis and therapy.
Post-translational modifications (PTMs) of nuclear proteins play essential roles in the regulation of gene transcription and signal transduction pathways. Numerous studies have demonstrated a correlation between specific nuclear protein isoforms and cellular malignant process. This communication reviews the impact of major PTM events such as phosphorylation, acetylation, methylation, ubiquitination, and sumoylation on several important nuclear proteins including p53, histones, proliferating cellular nuclear antigen (PCNA), and retinoblastoma protein (Rb) in the process. In addition, the implications of the PTMs as cancer biomarkers and therapeutic targets are considered. PMID:21328449
Shen, Fei; Kirmani, Kashif Z; Xiao, Zhimin; Thirlby, Benjamin H; Hickey, Robert J; Malkas, Linda H
The nuclear envelope LEM-domain protein emerin
Emerin, a conserved LEM-domain protein, is among the few nuclear membrane proteins for which extensive basic knowledge�biochemistry, partners, functions, localizations, posttranslational regulation, roles in development and links to human disease�is available. This review summarizes emerin and its emerging roles in nuclear �lamina� structure, chromatin tethering, gene regulation, mitosis, nuclear assembly, development, signaling and mechano-transduction. We also highlight many open questions, exploration of which will be critical to understand how this intriguing nuclear membrane protein and its �family� influence the genome.
Berk, Jason M; Tifft, Kathryn E; Wilson, Katherine L
The nuclear envelope LEM-domain protein emerin.
Emerin, a conserved LEM-domain protein, is among the few nuclear membrane proteins for which extensive basic knowledge--biochemistry, partners, functions, localizations, posttranslational regulation, roles in development and links to human disease--is available. This review summarizes emerin and its emerging roles in nuclear "lamina" structure, chromatin tethering, gene regulation, mitosis, nuclear assembly, development, signaling and mechano-transduction. We also highlight many open questions, exploration of which will be critical to understand how this intriguing nuclear membrane protein and its "family" influence the genome. PMID:23873439
Berk, Jason M; Tifft, Kathryn E; Wilson, Katherine L
Nuclear protein import is reduced in cells expressing nuclear envelopathy-causing lamin A mutants
Lamins, which form the nuclear lamina, not only constitute an important determinant of nuclear architecture, but additionally play essential roles in many nuclear functions. Mutations in A-type lamins cause a wide range of human genetic disorders (laminopathies). The importance of lamin A (LaA) in the spatial arrangement of nuclear pore complexes (NPCs) prompted us to study the role of LaA mutants in nuclear protein transport. Two mutants, causing prenatal skin disease restrictive dermopathy (RD) and the premature aging disease Hutchinson Gilford progeria syndrome, were used for expression in HeLa cells to investigate their impact on the subcellular localization of NPC-associated proteins and nuclear protein import. Furthermore, dynamics of the LaA mutants within the nuclear lamina were studied. We observed affected localization of NPC-associated proteins, diminished lamina dynamics for both LaA mutants and reduced nuclear import of representative cargo molecules. Intriguingly, both LaA mutants displayed similar effects on nuclear morphology and functions, despite their differences in disease severity. Reduced nuclear protein import was also seen in RD fibroblasts and impaired lamina dynamics for the nucleoporin Nup153. Our data thus represent the first study of a direct link between LaA mutant expression and reduced nuclear protein import.
Busch, Albert; Kiel, Tilman; Heupel, Wolfgang-M. [University of Wuerzburg, Institute of Anatomy and Cell Biology, Koellikerstrasse 6, 97070 Wuerzburg (Germany)] [University of Wuerzburg, Institute of Anatomy and Cell Biology, Koellikerstrasse 6, 97070 Wuerzburg (Germany); Wehnert, Manfred [Institute of Human Genetics, University of Greifswald, Greifswald (Germany)] [Institute of Human Genetics, University of Greifswald, Greifswald (Germany); Huebner, Stefan, E-mail: firstname.lastname@example.com [University of Wuerzburg, Institute of Anatomy and Cell Biology, Koellikerstrasse 6, 97070 Wuerzburg (Germany)] [University of Wuerzburg, Institute of Anatomy and Cell Biology, Koellikerstrasse 6, 97070 Wuerzburg (Germany)
Proteome analysis of nuclear matrix proteins during apoptotic chromatin condensation.
The nuclear matrix (NM) is considered a proteinaceous scaffold spatially organizing the interphase nucleus, the integrity of which is affected during apoptosis. Caspase-mediated degradation of NM proteins, such as nuclear lamins, precedes apoptotic chromatin condensation (ACC). Nevertheless, other NM proteins remain unaffected, which most likely maintain a remaining nuclear structure devoid of chromatin. We, therefore, screened various types of apoptotic cells for changes of the nuclear matrix proteome during the process of apoptotic ACC. Expectedly, we observed fundamental alterations of known chromatin-associated proteins, comprising both degradation and translocation to the cytosol. Importantly, a consistent set of abundant NM proteins, some (e.g. hNMP 200) of which displaying structural features, remained unaffected during apoptosis and might therefore represent constituents of an elementary scaffold. In addition, proteins involved in DNA replication and DNA repair were found accumulated in the NM fraction before cells became irreversibly committed to ACC, a time point characterized in detail by inhibitor studies with orthovanadate. In general, protein alterations of a consistent set of NM proteins (67 of which were identified), were reproducibly detectable in Fas-induced Jurkat cells, in UV-light treated U937 cells and also in staurosporine-treated HeLa cells. Our data indicate that substantial alterations of proteins linking chromatin to an elementary nuclear protein scaffold might play an intriguing role for the process of ACC. PMID:12032676
Gerner, C; Gotzmann, J; Fr�hwein, U; Schamberger, C; Ellinger, A; Sauermann, G
Zyxin and paxillin proteins: focal adhesion plaque LIM domain proteins go nuclear
Zyxin and paxillin are the prototypes of two related subfamilies of LIM domain proteins that are localized primarily at focal adhesion plaques. However, recent work has shown that zyxin\\/paxillin family proteins also shuttle through the nucleus. These proteins may enter the nucleus by association with other proteins, but are exported from the nucleus by means of intrinsic leucine-rich nuclear export
Yuan Wang; Thomas D. Gilmore
Nesprin-3, a novel outer nuclear membrane protein, associates with the cytoskeletal linker protein plectin
espite their importance in cell biology, the mech- anisms that maintain the nucleus in its proper po- sition in the cell are not well understood. This is primarily the result of an incomplete knowledge of the proteins in the outer nuclear membrane (ONM) that are able to associate with the different cytoskeletal systems. Two related ONM proteins, nuclear envelope spectrin
Kevin Wilhelmsen; Sandy H. M. Litjens; Ingrid Kuikman; Ntambua Tshimbalanga; Hans Janssen; Iman van den Bout; Karine Raymond; Arnoud Sonnenberg
Nuclear organization of DNA replication initiation proteins in mammalian cells.
Origin recognition complex (ORC), CDC6, and MCM proteins assemble sequentially to form prereplication chromatin. However, their organization remains largely unclear in mammalian cells. Here we show that ORC1 proteins are associated with non-chromatin nuclear structures and assemble in nuclear foci in mammalian cells using an in vivo chemical cross-linking method. CDC6 proteins were also found to assemble in nuclear foci on non-chromatin nuclear structures, although their physical association with ORC1 has been undetectable. In contrast to the situation in yeast cells, CDC6 was found to remain associated with non-chromatin nuclear structures even after cells entered into S phase. Instead, ORC1 proteins were found to be degraded by a proteasome-dependent pathway during S phase. We also found that some ORC2 proteins are associated with non-chromatin nuclear structures like ORC1, although the remainder binds to nuclease-sensitive chromatin. Further analyses indicate that ORC2 physically interacts with ORC1 on non-chromatin nuclear structures. On the other hand, our results suggest that although a small proportion of MCM complexes are loaded onto chromatin regions near ORC foci, most of them are more widely distributed. Possible relations between such organization of prereplication chromatin and complicated origin specification in higher eukaryotic cells are discussed. PMID:11779870
Fujita, Masatoshi; Ishimi, Yukio; Nakamura, Hiromu; Kiyono, Tohru; Tsurumi, Tatsuya
Regulation of Neuronal Differentiation by Proteins Associated with Nuclear Bodies
Nuclear bodies are large sub-nuclear structures composed of RNA and protein molecules. The Survival of Motor Neuron (SMN) protein localizes to Cajal bodies (CBs) and nuclear gems. Diminished cellular concentration of SMN is associated with the neurodegenerative disease Spinal Muscular Atrophy (SMA). How nuclear body architecture and its structural components influence neuronal differentiation remains elusive. In this study, we analyzed the effects of SMN and two of its interaction partners in cellular models of neuronal differentiation. The nuclear 23 kDa isoform of Fibroblast Growth Factor � 2 (FGF-223) is one of these interacting proteins � and was previously observed to influence nuclear bodies by destabilizing nuclear gems and mobilizing SMN from Cajal bodies (CBs). Here we demonstrate that FGF-223 blocks SMN-promoted neurite outgrowth, and also show that SMN disrupts FGF-223-dependent transcription. Our results indicate that FGF-223 and SMN form an inactive complex that interferes with neuronal differentiation by mutually antagonizing nuclear functions. Coilin is another nuclear SMN binding partner and a marker protein for Cajal bodies (CBs). In addition, coilin is essential for CB function in maturation of small nuclear ribonucleoprotein particles (snRNPs). The role of coilin outside of Cajal bodies and its putative impacts in tissue differentiation are poorly defined. The present study shows that protein levels of nucleoplasmic coilin outside of CBs decrease during neuronal differentiation. Overexpression of coilin has an inhibitory effect on neurite outgrowth. Furthermore, we find that nucleoplasmic coilin inhibits neurite outgrowth independent of SMN binding revealing a new function for coilin in neuronal differentiation.
Forthmann, Benjamin; van Bergeijk, Jeroen; Lee, Yu-Wei; Lubben, Verena; Schill, Yvonne; Brinkmann, Hella; Ratzka, Andreas; Stachowiak, Michal K.; Hebert, Michael; Grothe, Claudia; Claus, Peter
Chikungunya virus capsid protein contains nuclear import and export signals
Background Chikungunya virus (CHIKV) is an alphavirus of the Togaviridae family. After autoproteolytic cleavage, the CHIKV capsid protein (CP) is involved in RNA binding and assembly of the viral particle. The monomeric CP is approximately 30�kDa in size and is small enough for passive transport through nuclear pores. Some alphaviruses are found to harbor nuclear localization signals (NLS) and transport of these proteins between cellular compartments was shown to be energy dependent. The active nuclear import of cytoplasmic proteins is mediated by karyopherins and their export by exportins. As nuclear and cytoplasmic trafficking may play a role in the life cycle of CHIKV, we have sought to identify nuclear localization and nuclear export signals in CHIKV CP in a virus-free system. Methods EGFP-fusion proteins of CHIKV CP and mutants thereof were created and used to monitor their intracellular localization. Binding of cellular proteins was confirmed in pull-down assays with purified CP using co-immuoprecipitation. Nuclear localization was demonstrated in a virus-free system using fluorescence microscopy. Results Here we show that CHIKV CP is a nuclear-cytoplasmic shuttling protein with an active NLS that binds to karyopherin ? (Kar?) for its nuclear translocation. We also found that the Kar?4 C-terminal NLS binding site is sufficient for this interaction. We further demonstrate that CHIKV CP interacts directly with the export receptor CRM1 to transport this viral protein out of the nucleus via a nuclear export signal (NES). The CHIKV CP NES was mapped between amino acids 143 and 155 of CP. Deduced from in silico analyses we found that the NES has a mode of binding similar to the snurportin-1 CRM1 complex. Conclusions We were able to show that in a virus-free system that the CHIKV capsid protein contains both, a NLS and a NES, and that it is actively transported between the cytoplasma and the nucleus. We conclude that CHIKV CP has the ability to shuttle via interaction with karyopherins for its nuclear import and, vice versa, by CRM1-dependent nuclear export.
PML, SUMO, and RNF4: Guardians of Nuclear Protein Quality.
In this issue of Molecular Cell, Guo et�al. (2014) report that misfolded or aggregated nuclear proteins, such as pathogenic polyQ proteins, are cleared by a SUMO-dependent quality control pathway, which involves the E3 SUMO ligase PML and the SUMO-targeted ubiquitin ligase RNF4. PMID:24996060
G�rtner, Anne; Muller, Stefan
Cellular maintenance of nuclear protein homeostasis.
The accumulation and aggregation of misfolded proteins is the primary hallmark for more than 45 human degenerative diseases. These devastating disorders include Alzheimer's, Parkinson's, Huntington's, and amyotrophic lateral sclerosis. Over 15 degenerative diseases are associated with the aggregation of misfolded proteins specifically in the nucleus of cells. However, how the cell safeguards the nucleus from misfolded proteins is not entirely clear. In this review, we discuss what is currently known about the cellular mechanisms that maintain protein homeostasis in the nucleus and protect the nucleus from misfolded protein accumulation and aggregation. In particular, we focus on the chaperones found to localize to the nucleus during stress, the ubiquitin-proteasome components enriched in the nucleus, the signaling systems that might be present in the nucleus to coordinate folding and degradation, and the sites of misfolded protein deposition associated with the nucleus. PMID:24305949
Gallagher, Pamela S; Oeser, Michelle L; Abraham, Ayelet-Chen; Kaganovich, Daniel; Gardner, Richard G
A transmembrane inner nuclear membrane protein in the mitotic spindle.
We have recently characterized a novel transmembrane protein of the inner nuclear membrane of mammalian cells. The protein has two very interesting features. First, despite being an integral membrane protein it is able to concentrate in the membranes colocalizing with the mitotic spindle in metaphase and anaphase. Hence, the protein was named Samp1, Spindle associated membrane protein 1. Secondly, it displays a functional connection to centrosomes. This article discusses various aspects of Samp1 in relation to possible cellular function(s). PMID:21327071
Figueroa, Ricardo; Gudise, Santhosh; Larsson, Veronica; Hallberg, Einar
Efficient NES-dependent protein nuclear export requires ongoing synthesis and export of mRNAs
The mechanisms regulating nuclear export of proteins are not fully understood. To investigate whether the efficiency of protein nuclear export may depend on ongoing RNA synthesis and\\/or mRNA nuclear export, we used a microinjection approach with a fluorescent reporter protein containing a nuclear export signal (NES) and scored protein export in human fibroblasts under conditions when the synthesis or export
Heather M. O'Hagan; Mats Ljungman
Nuclear localization of the PEP protein tyrosine phosphatase.
PEP is an intracellular protein tyrosine phosphatase expressed primarily by cells of hematopoietic origin that can be divided structurally into a catalytic domain and a large carboxy-terminal domain. The carboxy-terminal domain is enriched in proline, glutamic acid, serine, and threonine residues (PEST sequences) and contains a nonperfect tandem repeat sequence enriched in proline residues and a carboxy terminus enriched in basic amino acids. Here we show that PEP is diffusely expressed in lymphoid tissues, consistent with expression by many different cell types. Analysis of the PEP protein identifies a nuclear localization sequence within the extreme carboxy terminus. Transfer of 18 amino acids from the carboxy terminus of PEP to beta-galactosidase conferred nuclear localization, indicating that this sequence was sufficient for nuclear localization. Proteins enriched in PEST sequences are often rapidly degraded. However, pulse-chase analysis indicates that PEP has a half-life of greater than 5 h. Images
Flores, E; Roy, G; Patel, D; Shaw, A; Thomas, M L
HMG Nuclear Proteins: Linking Chromatin Structure to Cellular Phenotype
I. Summary Although the three families of mammalian HMG proteins (HMGA, HMGB and HMGN) participate in many of the same nuclear processes, each family plays its own unique role in modulating chromatin structure and regulating genomic function. This review focuses on the similarities and differences in the mechanisms by which the different HMG families impact chromatin structure and influence cellular phenotype. The biological implications of having three architectural transcription factor families with complementary, but partially overlapping, nuclear functions are discussed.
Reeves, Raymond
Identification and Characterization of Proteins Involved in Nuclear Organization Using Drosophila GFP Protein Trap Lines
Background Strains from a collection of Drosophila GFP protein trap lines express GFP in the normal tissues where the endogenous protein is present. This collection can be used to screen for proteins distributed in the nucleus in a non-uniform pattern. Methodology/Principal Findings We analyzed four lines that show peripheral or punctate nuclear staining. One of these lines affects an uncharacterized gene named CG11138. The CG11138 protein shows a punctate distribution in the nuclear periphery similar to that of Drosophila insulator proteins but does not co-localize with known insulators. Interestingly, mutations in Lamin proteins result in alterations in CG11138 localization, suggesting that this protein may be a novel component of the nuclear lamina. A second line affects the Decondensation factor 31 (Df31) gene, which encodes a protein with a unique nuclear distribution that appears to segment the nucleus into four different compartments. The X-chromosome of males is confined to one of these compartments. We also find that Drosophila Nucleoplasmin (dNlp) is present in regions of active transcription. Heat shock leads to loss of dNlp from previously transcribed regions of polytene chromosome without redistribution to the heat shock genes. Analysis of Stonewall (Stwl), a protein previously found to be necessary for the maintenance of germline stem cells, shows that Stwl is present in a punctate pattern in the nucleus that partially overlaps with that of known insulator proteins. Finally we show that Stwl, dNlp, and Df31 form part of a highly interactive network. The properties of other components of this network may help understand the role of these proteins in nuclear biology. Conclusions/Significance These results establish screening of GFP protein trap alleles as a strategy to identify factors with novel cellular functions. Information gained from the analysis of CG11138 Stwl, dNlp, and Df31 sets the stage for future studies of these proteins.
Rohrbaugh, Margaret; Clore, Alyssia; Davis, Julia; Johnson, Sharonta; Jones, Brian; Jones, Keith; Kim, Joanne; Kithuka, Bramwel; Lunsford, Krystal; Mitchell, Joy; Mott, Brian; Ramos, Edward; Tchedou, Maza R.; Acosta, Gilbert; Araujo, Mark; Cushing, Stuart; Duffy, Gabriel; Graves, Felicia; Griffin, Kyler; Gurudatta, B. V.; Jackson, Deaundra; Jaimes, Denis; Jamison, Kendall; Jones, Khali; Kelley, Dhaujee; Kilgore, Marquita; Laramore, Derica; Le, Thuy; Mazhar, Bakhtawar; Mazhar, Muhammad M.; McCrary, Britney; Miller, Teanndras; Moreland, Celethia; Mullins, Alex; Munye, Elyas; Okoorie, Sheila; Pittman, Elisha; Roberts, Nikkita; Rose, De'Warren; Rowland, Alex; Shagarabi, Anwar; Smith, Jamela; Stallworth, Tayler; Stroud, Nicole; Sung, Elizabeth; Sung, Kai; Takenaka, Naomi; Torre, Eduardo; Veira, Jarvis; Vu, Kim; Wagstaff, William; Wood, Ashley M.; Wu, Karen; Yang, Jingping; Corces, Victor G.
Functional Characterization of Nuclear Localization Signals in Yeast Sm Proteins
In mammals, nuclear localization of U-snRNP particles requires the snRNA hypermethylated cap structure and the Sm core complex. The nature of the signal located within the Sm core proteins is still unknown, both in humans and yeast. Close examination of the sequences of the yeast SmB, SmD1, and SmD3 carboxyl-terminal domains reveals the presence of basic regions that are reminiscent of nuclear localization signals (NLSs). Fluorescence microscopy studies using green fluorescent protein (GFP)-fusion proteins indicate that both yeast SmB and SmD1 basic amino acid stretches exhibit nuclear localization properties. Accordingly, deletions or mutations in the NLS-like motifs of SmB and SmD1 dramatically reduce nuclear fluorescence of the GFP-Sm mutant fusion alleles. Phenotypic analyses indicate that the NLS-like motifs of SmB and SmD1 are functionally redundant: each NLS-like motif can be deleted without affecting yeast viability whereas a simultaneous deletion of both NLS-like motifs is lethal. Taken together, these findings suggest that, in the doughnut-like structure formed by the Sm core complex, the carboxyl-terminal extensions of Sm proteins may form an evolutionarily conserved basic amino acid-rich protuberance that functions as a nuclear localization determinant.
Bordonne, Remy
Nuclear export dynamics of RNA-protein complexes
The central dogma of molecular biology � DNA makes RNA makes proteins � is a flow of information that in eukaryotes encounters a physical barrier: the nuclear envelope, which encapsulates, organizes and protects the genome. Nuclear-pore complexes, embedded in the nuclear envelope, regulate the passage of molecules to and from the nucleus, including the poorly understood process of the export of RNAs from the nucleus. Recent imaging approaches focusing on single molecules have provided unexpected insight into this crucial step in the information flow. This review addresses the latest studies of RNA export and presents some models for how this complex process may work.
Grunwald, David; Singer, Robert H.; Rout, Michael
GIP/MZT1 proteins orchestrate nuclear shaping
The functional organization of the nuclear envelope (NE) is only just emerging in plants with the recent characterization of NE protein complexes and their molecular links to the actin cytoskeleton. The NE also plays a role in microtubule nucleation by recruiting ?-Tubulin Complexes (?-TuCs) which contribute to the establishment of a robust mitotic spindle. ?-tubulin Complex Protein 3 (GCP3)-interacting proteins (GIPs) have been identified recently as integral components of ?-TuCs. GIPs have been conserved throughout evolution and are also named MZT1 (mitotic-spindle organizing protein 1). This review focuses on recent data investigating the role of GIP/MZT1 at the NE, including insights from the study of GIP partners. It also uncovers new functions for GIP/MZT1 during interphase and highlights a current view of NE-associated components which are critical for nuclear shaping during both cell division and differentiation.
Batzenschlager, Morgane; Herzog, Etienne; Houlne, Guy; Schmit, Anne-Catherine; Chaboute, Marie-Edith
Nuclear envelope proteomics: Novel integral membrane proteins of the inner nuclear membrane
The nuclear envelope (NE) is one of the least characterized structures of eukaryotic cells. The study of its functional roles is hampered by the small number of proteins known to be specifically located to it. Here, we present a comprehensive characterization of the NE proteome. We applied different fractionation procedures and isolated protein subsets derived from distinct NE compartments. We identified 148 different proteins by 16-benzyl dimethyl hexadecyl ammonium chloride (16-BAC) gel electrophoresis and matrix-assisted laser desorption ionization (MALDI) mass spectrometry; among them were 19 previously unknown or noncharacterized. The identification of known proteins in particular NE fractions enabled us to assign novel proteins to NE substructures. Thus, our subcellular proteomics approach retains the screening character of classical proteomic studies, but also allows a number of predictions about subcellular localization and interactions of previously noncharacterized proteins. We demonstrate this result by showing that two novel transmembrane proteins, a 100-kDa protein with similarity to Caenorhabditis elegans Unc-84A and an unrelated 45-kDa protein we named LUMA, reside in the inner nuclear membrane and likely interact with the nuclear lamina. The utility of our approach is not restricted to the investigation of the NE. Our approach should be applicable to the analysis of other complex membrane structures of the cell as well.
Dreger, Mathias; Bengtsson, Luiza; Schoneberg, Torsten; Otto, Henning; Hucho, Ferdinand
Molecular chaperone-mediated nuclear protein dynamics.
Homeostasis requires effective action of numerous biological pathways including those working along a genome. The variety of processes functioning in the nucleus is considerable, yet the number of employed factors eclipses this total. Ideally, individual components assemble into distinct complexes and serially operate along a pathway to perform work. Adding to the complexity is a multitude of fluctuating internal and external signals that must be monitored to initiate, continue or halt individual activities. While cooperative interactions between proteins of the same process provide a mechanism for rapid and precise assembly, the inherent stability of such organized structures interferes with the proper timing of biological events. Further prolonging the longevity of biological complexes are crowding effects resulting from the high concentration of intracellular macromolecules. Hence, accessory proteins are required to destabilize the various assemblies to efficiently transition between structures, avoid off-pathway competitive interactions, and to terminate pathway activity. We suggest that molecular chaperones have evolved, in part, to manage these challenges by fostering a general and continuous dynamic protein environment within the nucleus. PMID:24694369
Echtenkamp, Frank J; Freeman, Brian C
Caspase-dependent proteolysis of integral and peripheral proteins of nuclear membranes and nuclear pore complex proteins during apoptosis.
We have studied the fate of the nuclear envelope (NE) in different human cells committed to apoptosis by different chemical agents. Using a battery of antibodies against marker proteins of the three domains of the nuclear envelope, namely lamin B (LB) for the lamina, transmembrane proteins LBR and LAP2 for the inner nuclear membrane, and nucleoporins p62, Nup153 and gp210 for the nuclear pore complexes (NPCs), we observed a selective and conserved cleavage of LB, LAP2 and Nup153. In lymphoid cells, the rate of cleavage of these markers was independent of the apoptosis inducing agent, actinomycin D or etoposide, and more rapid than in attached epithelial cells. While lamin B is cleaved by caspase 6, the protease responsible for the cleavage of LAP2 and Nup153 was probably caspase 3, since (1) cleavage of both proteins was specifically prevented by in vivo addition of caspase 3 inhibitor Ac-DEVD-CHO and (2) consensus sites for these caspases are present in both proteins. As LB, LAP2 and Nup153 are exposed at the inner face of the nuclear envelope and all interact with chromatin, we suggest that their cleavage allows both the detachment of NE from chromatin and the clustering of NPCs in the plane of the membrane, two conserved morphological features of apoptosis observed in this study. PMID:10318766
Buendia, B; Santa-Maria, A; Courvalin, J C
In vitro nuclear interactome of the HIV-1 Tat protein
Background One facet of the complexity underlying the biology of HIV-1 resides not only in its limited number of viral proteins, but in the extensive repertoire of cellular proteins they interact with and their higher-order assembly. HIV-1 encodes the regulatory protein Tat (86�101aa), which is essential for HIV-1 replication and primarily orchestrates HIV-1 provirus transcriptional regulation. Previous studies have demonstrated that Tat function is highly dependent on specific interactions with a range of cellular proteins. However they can only partially account for the intricate molecular mechanisms underlying the dynamics of proviral gene expression. To obtain a comprehensive nuclear interaction map of Tat in T-cells, we have designed a proteomic strategy based on affinity chromatography coupled with mass spectrometry. Results Our approach resulted in the identification of a total of 183 candidates as Tat nuclear partners, 90% of which have not been previously characterised. Subsequently we applied in silico analysis, to validate and characterise our dataset which revealed that the Tat nuclear interactome exhibits unique signature(s). First, motif composition analysis highlighted that our dataset is enriched for domains mediating protein, RNA and DNA interactions, and helicase and ATPase activities. Secondly, functional classification and network reconstruction clearly depicted Tat as a polyvalent protein adaptor and positioned Tat at the nexus of a densely interconnected interaction network involved in a range of biological processes which included gene expression regulation, RNA biogenesis, chromatin structure, chromosome organisation, DNA replication and nuclear architecture. Conclusion We have completed the in vitro Tat nuclear interactome and have highlighted its modular network properties and particularly those involved in the coordination of gene expression by Tat. Ultimately, the highly specialised set of molecular interactions identified will provide a framework to further advance our understanding of the mechanisms of HIV-1 proviral gene silencing and activation.
Gautier, Virginie W; Gu, Lili; O'Donoghue, Niaobh; Pennington, Stephen; Sheehy, Noreen; Hall, William W
The protamine family of sperm nuclear proteins
The protamines are a diverse family of small arginine-rich proteins that are synthesized in the late-stage spermatids of many animals and plants and bind to DNA, condensing the spermatid genome into a genetically inactive state. Vertebrates have from one to 15 protamine genes per haploid genome, which are clustered together on the same chromosome. Comparison of protamine gene and amino-acid sequences suggests that the family evolved from specialized histones through protamine-like proteins to the true protamines. Structural elements present in all true protamines are a series of arginine-rich DNA-anchoring domains (often containing a mixture of arginine and lysine residues in non-mammalian protamines) and multiple phosphorylation sites. The two protamines found in mammals, P1 and P2, are the most widely studied. P1 packages sperm DNA in all mammals, whereas protamine P2 is present only in the sperm of primates, many rodents and a subset of other placental mammals. P2, but not P1, is synthesized as a precursor that undergoes proteolytic processing after binding to DNA and also binds a zinc atom, the function of which is not known. P1 and P2 are phosphorylated soon after their synthesis, but after binding to DNA most of the phosphate groups are removed and cysteine residues are oxidized, forming disulfide bridges that link the protamines together. Both P1 and P2 have been shown to be required for normal sperm function in primates and many rodents.
Balhorn, Rod
Nuclear trafficking of the POZ-ZF protein Znf131.
Znf131 is a member of the BTB/POZ family of transcription factors with roles in development and carcinogenesis. Like many members of this protein family, Znf131 displays robust nuclear localization in cultured cells, but the mechanism(s) of Znf131 nuclear trafficking is unknown. Here, we report the mechanism of Znf131 nuclear localization. Visual inspection of the Znf131 amino acid sequence revealed three basic regions (BR-1, -2 and -3) with the potential to serve as nuclear localization signals (NLS). Of the three basic regions, only BR-1 functioned independently to efficiently target heterologous beta-gal-GFP fusion proteins to HeLa cell nuclei. However, a Znf131 truncation mutant containing BR-2 and BR-3 efficiently targeted heterologous beta-gal-GFP fusion proteins to HeLa cell nuclei. Mutational analysis of full-length GFP-tagged Znf131 revealed that loss of any one BR alone did not prevent Znf131 nuclear localization. This apparent redundancy in NLS activity was due to the fact that intact BR-1 or BR-2 alone could target full-length Znf131 to nuclei. Consequently, simultaneous mutation of BR-1 and BR-2 abolished full-length Znf131 nuclear localization. Therefore, BR-1 and BR-2 are functional NLSs for Znf131 and as such are designated NLS-1 and NLS-2. Finally, wild type Znf131, and not a Znf131 NLS-defective mutant (NLS-1m/NLS-2m) interacted preferentially with the nuclear import receptor Importin-alpha3 in vitro. PMID:17306895
Donaldson, Nickett S; Daniel, Yasmin; Kelly, Kevin F; Graham, Monica; Daniel, Juliet M
Microtubule-associated nuclear envelope proteins in interphase and mitosis.
The LINC (linker of nucleoskeleton and cytoskeleton) complex forms a transcisternal bridge across the NE (nuclear envelope) that connects the cytoskeleton with the nuclear interior. This enables some proteins of the NE to communicate with the centrosome and the microtubule cytoskeleton. The position of the centrosome relative to the NE is of vital importance for many cell functions, such as cell migration and division, and centrosomal dislocation is a frequent phenotype in laminopathic disorders. Also in mitosis, a small group of transmembrane NE proteins associate with microtubules when they concentrate in a specific membrane domain associated with the mitotic spindle. The present review discusses structural and functional aspects of microtubule association with NE proteins and how this association may be maintained over the cell cycle. PMID:22103526
Figueroa, Ricardo A; Gudise, Santhosh; Hallberg, Einar
Maturation of cytosolic and nuclear iron-sulfur proteins.
Eukaryotic cells contain numerous cytosolic and nuclear iron-sulfur (Fe/S) proteins that perform key functions in metabolic catalysis, iron regulation, protein translation, DNA synthesis, and DNA repair. Synthesis of Fe/S clusters and their insertion into apoproteins are essential for viability and are conserved in eukaryotes. The process is catalyzed in two major steps by the CIA (cytosolic iron-sulfur protein assembly) machinery encompassing nine known proteins. First, a [4Fe-4S] cluster is assembled on a scaffold complex. This step requires a sulfur-containing compound from mitochondria and reducing equivalents from an electron transfer chain. Second, the Fe/S cluster is transferred from the scaffold to specific apoproteins by the CIA targeting complex. This review summarizes our molecular knowledge on CIA protein function during the assembly process. PMID:24314740
Netz, Daili J A; Mascarenhas, Judita; Stehling, Oliver; Pierik, Antonio J; Lill, Roland
The BRO proteins of Bombyx mori nucleopolyhedrovirus are nucleocytoplasmic shuttling proteins that utilize the CRM1-mediated nuclear export pathway
The BRO proteins of Bombyx mori nucleopolyhedrovirus (BmNPV) display a biphasic pattern of intracellular localization during infection. At early times, they reside in the nucleus but then show both cytoplasmic and nuclear localization as the infection proceeds. Therefore, we examined the possibility of nuclear export. Using inhibitors, we reveal that BmNPV BRO proteins shuttle between the nucleus and cytoplasm. Mutations on the leucine-rich region of BRO proteins resulted in nuclear accumulation of transiently expressed proteins, suggesting that this region functions as a CRM1-dependent nuclear export signal (NES). On the contrary, mutant BRO-D with an altered NES did not show nuclear accumulation in infected cells, although protein production seemed to be reduced. RT-PCR analysis showed that the lower level of protein production was due to a reduction in RNA synthesis. Taken together, our results suggest that BRO proteins are nucleocytoplasmic shuttling proteins that utilize the CRM1-mediated nuclear export pathway.
Kang, Won Kyung [Molecular Entomology Laboratory, RIKEN, 2-1 Hirosawa, Wako 351-0198 (Japan)]. E-mail: email@example.com; Kurihara, Masaaki [Molecular Entomology Laboratory, RIKEN, 2-1 Hirosawa, Wako 351-0198 (Japan)]. E-mail: firstname.lastname@example.com; Matsumoto, Shogo [Molecular Entomology Laboratory, RIKEN, 2-1 Hirosawa, Wako 351-0198 (Japan)]. E-mail: email@example.com
Nuclear localization of endogenous RGK proteins and modulation of cell shape remodeling by regulated nuclear transport.
The members of the RGK small GTP-binding protein family, Kir/Gem, Rad, Rem and Rem2, are multifunctional proteins that regulate voltage-gated calcium channel activity and cell shape remodeling. Calmodulin (CaM) or CaM 14-3-3 are regulators of RGK functions and their association defines the subcellular localization of RGK proteins. Abolition of CaM association results in the accumulation of RGK proteins in the nucleus, whereas 14-3-3 binding maintains them in the cytoplasm. Kir/Gem possesses nuclear localization signals (NLS) that mediate nuclear accumulation through an importin alpha5-dependent pathway (see Mahalakshmi RN, Nagashima K, Ng MY, Inagaki N, Hunziker W, B�guin P. Nuclear transport of Kir/Gem requires specific signals and importin alpha5 and is regulated by Calmodulin and predicted service phosphorylations. Traffic 2007; doi: 10.1111/j.1600-0854.2007.00598.x). Because the extent of nuclear localization depends on the RGK protein and the cell type, the mechanism and regulation of nuclear transport may differ. Here, we extend our analysis to the other RGK members and show that Rem also binds importin alpha5, whereas Rad associates with importins alpha3, alpha5 and beta through three conserved NLS. Predicted phosphorylation of a serine residue within the bipartite NLS affects, as observed for Kir/Gem, nuclear accumulation of Rem, but not that of Rad or Rem2. We also identify an additional regulatory phosphorylation for all RGK proteins that prevents binding of 14-3-3 and thereby interferes with their cytosolic relocalization by 14-3-3. Functionally, nuclear localization of RGK proteins contributes to the suppression of RGK-mediated cell shape remodeling. Importantly, we show that endogenous RGK proteins are localized predominantly in the nucleus of individual cells of the brain cortex 'in situ' as well as in primary hippocampal cells, indicating that transport between the nucleus and their site of action in the cytoplasm (i.e., cytoskeleton, endoplasmic reticulum or plasma membrane) is of physiological relevance for the regulation of RGK protein function. PMID:17605760
Mahalakshmi, Ramasubbu N; Ng, Mei Yong; Guo, Ke; Qi, Zeng; Hunziker, Walter; B�guin, Pascal
ER membrane protein complex required for nuclear fusion
Diploid cells of the yeast Saccharomyces cerevisiae form after the mating of two haploid cells of the opposite mating type. After fusion of the two plasma membranes of the mating cells, a dinucleated cell forms initially in which the two haploid nuclei then rapidly fuse to form a single diploid nucleus. This latter event, called karyogamy, can be divided into two distinct steps: the microtubule-based movement that causes the two nuclei to become closely juxtaposed and the fusion of the nuclear membranes. For the membrane fusion step, one required component, the ER luminal protein Kar2p (BiP), has been identified. For topological reasons, however, it has been unclear how Kar2p could function in this role. Kar2p is localized to the luminal (i.e., noncytoplasmic) face of the ER membrane, yet nuclear fusion must initiate from the cytosolic side of the outer nuclear membrane or the ER membrane with which it is contiguous. There is both genetic and biochemical evidence that Kar2p interacts with Sec63p, an ER membrane protein containing both luminal and cytosolic domains that is involved in protein translocation across the membrane. We have isolated novel sec63 mutant alleles that display severe karyogamy defects. Disruption of the genes encoding other Sec63p-associated proteins (Sec71p and Sec72p) also results in karyogamy defects. A suppressor mutant (sos1-1) partially corrects the translocation defect but does not alleviate the karyogamy defect. sec61 and sec62 mutant alleles that cause similar or more severe protein translocation defects show no karyogamy defects. Taken together, these results suggest a direct role for Sec63p, Sec71p, and Sec72p in nuclear membrane fusion and argue against the alternative interpretation that the karyogamy defects result as an indirect consequence of the impaired membrane translocation of another component(s) required for the process. We propose that an ER/nuclear membrane protein complex composed of Sec63p, Sec71p, and Sec72p plays a central role in mediating nuclear membrane fusion and requires ER luminally associated Kar2p for its function.
Identification and Characterization of the Ubiquitously Occurring Nuclear Matrix Protein NMP 238
By systematic comparison of two-dimensional electrophoretic patterns of nuclear matrix proteins an ubiquitously occurring (common) nuclear matrix protein, termed NMP 238, was detected. Localization of the protein in isolated nuclear matrices and in nuclear and cytoplasmic regions of cells was determined by confocal immunofluorescence microscopy. N-terminal protein sequencing, mass spectrometry, and sequencing of a human EST cDNA clone showed identity
Klaus Holzmann; Christopher Gerner; Thomas Korosec; Angelika P�ltl; Rudolf Grimm; Georg Sauermann
Nuclear protein spreading: implication for pathophysiology of neuromuscular diseases.
While transfer of a protein encoded by a single nucleus to nearby nuclei in multinucleated cells has been known for almost 25 years, the biological consequences for gain-of-function diseases have not been considered. Here, we have investigated nuclear protein spreading and its potential consequences in two of the three most prevalent neuromuscular diseases. By performing co-cultures between diseased or control human myoblasts and murine C2C12 myoblasts, we demonstrate that in facioscapulohumeral dystrophy, although the transcription of the toxic protein DUX4 occurs in only a limited number of nuclei, the resulting protein diffuses into nearby nuclei within the myotubes, thus spreading aberrant gene expression. In myotonic dystrophy type 1, we observed that in human-mouse heterokaryons, the expression of a mutated DMPK from human nuclei titrates splicing factors produced by neighboring nuclei, inducing the mis-splicing of several pre-mRNAs in murine nuclei. In both cases, the spreading of the pathological phenotypes from one nucleus to another is observed, highlighting an additional mechanism that contributes to the dissemination and worsening of the muscle pathogenesis. These results indicate that nuclear protein spreading may be an important component of pathophysiology of gain of function muscular diseases which should be taken into consideration in the design of new therapeutic approaches. PMID:24659496
Ferreboeuf, Maxime; Mariot, Virginie; Furling, Denis; Butler-Browne, Gillian; Mouly, Vincent; Dumonceaux, Julie
Nuclear protein IK undergoes dynamic subcellular translocation and forms unique nuclear bodies during the cell cycle.
IK is a nuclear protein containing a unique domain named RED due to the presence of a repetitive arginine (R), aspartic (E), and glutamic acid (D) sequence. To date, the function of this protein remains largely unknown despite of a couple of previous studies in the literature. Here we report that depletion of IK via RNA interference results in mitotic arrest. We also demonstrate that IK undergoes dynamic translocation during interphase and mitosis. In particular, IK is primarily present in some interphase cells as nuclear foci/bodies which do not co-localize with nucleoli, PMA bodies and Cajal bodies. Pull-down analysis coupled with mass spectrometry reveals that IK is associated with DHX15, a putative ATP-dependent RNA helicase. Our results strongly suggest that IK may participate in pre-mRNA splicing and that it may be a useful biomarker for a new nuclear structure in the cell. PMID:24252166
Hu, Liyan; Yang, Feikun; Liu, Xianan; Xu, Dazhong; Dai, Wei
Nuclear magnetic resonance analysis of protein-DNA interactions.
Recent methodological and instrumental advances in solution-state nuclear magnetic resonance have opened up the way to investigating challenging problems in structural biology such as large macromolecular complexes. This review focuses on the experimental strategies currently employed to solve structures of protein-DNA complexes and to analyse their dynamics. It highlights how these approaches can help in understanding detailed molecular mechanisms of target recognition. PMID:21389020
Campagne, S; Gervais, V; Milon, A
Identification of a Nuclear Stat1 Protein Tyrosine Phosphatase
Upon interferon (IFN) stimulation, Stat1 becomes tyrosine phosphorylated and translocates into the nu- cleus, where it binds to DNA to activate transcription. The activity of Stat1 is dependent on tyrosine phos- phorylation, and its inactivation in the nucleus is accomplished by a previously unknown protein tyrosine phosphatase (PTP). We have now purified a Stat1 PTP activity from HeLa cell nuclear
J. ten Hoeve; M. de Jesus Ibarra-Sanchez; Y. Fu; W. Zhu; M. Tremblay; M. David; K. Shuai
Whole-genome screening identifies proteins localized to distinct nuclear bodies
The nucleus is a unique organelle that contains essential genetic materials in chromosome territories. The interchromatin space is composed of nuclear subcompartments, which are defined by several distinctive nuclear bodies believed to be factories of DNA or RNA processing and sites of transcriptional and/or posttranscriptional regulation. In this paper, we performed a genome-wide microscopy-based screening for proteins that form nuclear foci and characterized their localizations using markers of known nuclear bodies. In total, we identified 325 proteins localized to distinct nuclear bodies, including nucleoli (148), promyelocytic leukemia nuclear bodies (38), nuclear speckles (27), paraspeckles (24), Cajal bodies (17), Sam68 nuclear bodies (5), Polycomb bodies (2), and uncharacterized nuclear bodies (64). Functional validation revealed several proteins potentially involved in the assembly of Cajal bodies and paraspeckles. Together, these data establish the first atlas of human proteins in different nuclear bodies and provide key information for research on nuclear bodies.
Fong, Ka-wing; Li, Yujing; Wang, Wenqi; Ma, Wenbin; Li, Kunpeng; Qi, Robert Z.; Liu, Dan; Songyang, Zhou
p95vav associates with the nuclear protein Ku-70.
The proto-oncogene vav is expressed solely in hematopoietic cells and plays an important role in cell signaling, although little is known about the proteins involved in these pathways. To gain further information, the Src homology 2 (SH2) and 3 (SH3) domains of Vav were used to screen a lymphoid cell cDNA library by the yeast two-hybrid system. Among the positive clones, we detected a nuclear protein, Ku-70, which is the DNA-binding element of the DNA-dependent protein kinase. In Jurkat and UT7 cells, Vav is partially localized in the nuclei, as judged from immunofluorescence and confocal microscopy studies. By using glutathione S-transferase fusion proteins derived from Ku-70 and coimmunoprecipitation experiments with lysates prepared from human thymocytes and Jurkat and UT7 cells, we show that Vav associates with Ku-70. The interaction of Vav with Ku-70 requires only the 150-residue carboxy-terminal portion of Ku-70, which binds to the 25 carboxy-terminal residues of the carboxy SH3 domain of Vav. A proline-to-leucine mutation in the carboxy SH3 of Vav that blocks interaction with proline-rich sequences does not modify the binding of Ku-70, which lacks this motif. Therefore, the interaction of Vav with Ku-70 may be a novel form of protein-protein interaction. The potential role of Vav/Ku-70 complexes is discussed. PMID:8524317
Romero, F; Dargemont, C; Pozo, F; Reeves, W H; Camonis, J; Gisselbrecht, S; Fischer, S
p95vav associates with the nuclear protein Ku-70.
The proto-oncogene vav is expressed solely in hematopoietic cells and plays an important role in cell signaling, although little is known about the proteins involved in these pathways. To gain further information, the Src homology 2 (SH2) and 3 (SH3) domains of Vav were used to screen a lymphoid cell cDNA library by the yeast two-hybrid system. Among the positive clones, we detected a nuclear protein, Ku-70, which is the DNA-binding element of the DNA-dependent protein kinase. In Jurkat and UT7 cells, Vav is partially localized in the nuclei, as judged from immunofluorescence and confocal microscopy studies. By using glutathione S-transferase fusion proteins derived from Ku-70 and coimmunoprecipitation experiments with lysates prepared from human thymocytes and Jurkat and UT7 cells, we show that Vav associates with Ku-70. The interaction of Vav with Ku-70 requires only the 150-residue carboxy-terminal portion of Ku-70, which binds to the 25 carboxy-terminal residues of the carboxy SH3 domain of Vav. A proline-to-leucine mutation in the carboxy SH3 of Vav that blocks interaction with proline-rich sequences does not modify the binding of Ku-70, which lacks this motif. Therefore, the interaction of Vav with Ku-70 may be a novel form of protein-protein interaction. The potential role of Vav/Ku-70 complexes is discussed.
Romero, F; Dargemont, C; Pozo, F; Reeves, W H; Camonis, J; Gisselbrecht, S; Fischer, S
Protein Sub-Nuclear Localization Prediction Using SVM and Pfam Domain Information
The nucleus is the largest and the highly organized organelle of eukaryotic cells. Within nucleus exist a number of pseudo-compartments, which are not separated by any membrane, yet each of them contains only a specific set of proteins. Understanding protein sub-nuclear localization can hence be an important step towards understanding biological functions of the nucleus. Here we have described a method, SubNucPred developed by us for predicting the sub-nuclear localization of proteins. This method predicts protein localization for 10 different sub-nuclear locations sequentially by combining presence or absence of unique Pfam domain and amino acid composition based SVM model. The prediction accuracy during leave-one-out cross-validation for centromeric proteins was 85.05%, for chromosomal proteins 76.85%, for nuclear speckle proteins 81.27%, for nucleolar proteins 81.79%, for nuclear envelope proteins 79.37%, for nuclear matrix proteins 77.78%, for nucleoplasm proteins 76.98%, for nuclear pore complex proteins 88.89%, for PML body proteins 75.40% and for telomeric proteins it was 83.33%. Comparison with other reported methods showed that SubNucPred performs better than existing methods. A web-server for predicting protein sub-nuclear localization named SubNucPred has been established at http://188.8.131.52/mkumar/subnucpred/. Standalone version of SubNucPred can also be downloaded from the web-server.
Kumar, Ravindra; Jain, Sohni; Kumari, Bandana; Kumar, Manish
The Nuclear Envelope Protein, LAP1B, Is a Novel Protein Phosphatase 1 Substrate
Protein phosphatase 1 (PP1) binding proteins are quintessential regulators, determining substrate specificity and defining subcellular localization and activity of the latter. Here, we describe a novel PP1 binding protein, the nuclear membrane protein lamina associated polypeptide 1B (LAP1B), which interacts with the DYT1 dystonia protein torsinA. The PP1 binding domain in LAP1B was here identified as the REVRF motif at amino acids 55-59. The LAP1B:PP1 complex can be immunoprecipitated from cells in culture and rat cortex and the complex was further validated by yeast co-transformations and blot overlay assays. PP1, which is enriched in the nucleus, binds to the N-terminal nuclear domain of LAP1B, as shown by immunocolocalization and domain specific binding studies. PP1 dephosphorylates LAP1B, confirming the physiological relevance of this interaction. These findings place PP1 at a key position to participate in the pathogenesis of DYT1 dystonia and related nuclear envelope-based diseases.
Santos, Mariana; Rebelo, Sandra; Van Kleeff, Paula J. M.; Kim, Connie E.; Dauer, William T.; Fardilha, Margarida; da Cruz e Silva, Odete A.; da Cruz e Silva, Edgar F.
Nuclear Actin-Related Proteins in Epigenetic Control
The nuclear actin-related proteins (ARPs) share overall structure and low-level sequence homology with conventional actin. They are indispensable subunits of macromolecular machines that control chromatin remodeling and modification leading to dynamic changes in DNA structure, transcription, and DNA repair. Cellular, genetic, and biochemical studies suggest that the nuclear ARPs are essential to the epigenetic control of the cell cycle and cell proliferation in all eukaryotes, while in plants and animals they also exert epigenetic controls over most stages of multicellular development including organ initiation, the switch to reproductive development, and senescence and programmed cell death. A theme emerging from plants and animals is that in addition to their role in controlling the general compaction of DNA and gene silencing, isoforms of nuclear ARP-containing chromatin complexes have evolved to exert dynamic epigenetic control over gene expression and different phases of multicellular development. Herein, we explore this theme by examining nuclear ARP phylogeny, activities of ARP-containing chromatin remodeling complexes that lead to epigenetic control, expanding developmental roles assigned to several animal and plant ARP-containing complexes, the evidence that thousands of ARP complex isoforms may have evolved in concert with multicellular development, and ARPs in human disease.
Meagher, Richard B.; Kandasamy, Muthugapatti K.; McKinney, Elizabeth C.; Roy, Eileen
Binding of triiodothyronine to rat liver nuclear matrix. influence of thyroid hormones on the phosphorylation of nuclear matrix proteins
The interaction of thyroid hormones with rat liver nuclear matrix proteins was investigated. It was shown that the nuclear matrix contains sites that bind triiodothyronine with high affinity (K = 1.07.10/sup 9/ M/sup -1/) and limited capacity (the maximum binding capacity is equal to 28 /SUP a/ .5 fmoles of triiodothyronine per 100 ug protein). Electrophoretic identification of the matrix proteins that bind triiodothyronine was performed. The molecular weight of the main triiodothyronine-binding fraction is 50,000-52,000. It was shown that the administration of triiodothyronine to thyroidectomized rats stimulates the phosphorylation of all the protein fractions of the nuclear matrix.
Adylova, A.T.; Atakhanova, B.A.
Large-scale identification of mammalian proteins localized to nuclear sub-compartments
Many nuclear components participating in related pathways appear concentrated in specific areas of the mammalian nucleus. The importance of this organization is attested to by the dysfunction that correlates with mis-localization of nuclear proteins in human disease and cancer. Determining the sub- nuclear localization of proteins is therefore important for understanding genome regulation and function, and it also provides clues
Heidi G. E. Sutherland; Gail K. Mumford; Kathryn Newton; Lisa V. Ford; Rachel Farrall; Graham Dellaire; Javier F. C�ceres; Wendy A. Bickmore
The Ty1 integrase protein can exploit the classical nuclear protein import machinery for entry into the nucleus
Like its retroviral relatives, the long terminal repeat retrotransposon Ty1 in the yeast Saccharomyces cerevisiae must traverse a permanently intact nuclear membrane for successful transposition and replication. For retrotransposition to occur, at least a subset of Ty1 proteins, including the Ty1 integrase, must enter the nucleus. Nuclear localization of integrase is dependent upon a C-terminal nuclear targeting sequence. However, the nuclear import machinery that recognizes this nuclear targeting signal has not been defined. We investigated the mechanism by which Ty1 integrase gains access to nuclear DNA as a model for how other retroelements, including retroviruses like HIV, may utilize cellular nuclear transport machinery to import their essential nuclear proteins. We show that Ty1 retrotransposition is significantly impaired in yeast mutants that alter the classical nuclear protein import pathway, including the Ran-GTPase, and the dimeric import receptor, importin-?/?. Although Ty1 proteins are made and processed in these mutant cells, our studies reveal that an integrase reporter is not properly targeted to the nucleus in cells carrying mutations in the classical nuclear import machinery. Furthermore, we demonstrate that integrase coimmunoprecipitates with the importin-? transport receptor and directly binds to importin-?. Taken together, these data suggest Ty1 integrase can employ the classical nuclear protein transport machinery to enter the nucleus.
McLane, Laura M.; Pulliam, Kanika F.; Devine, Scott E.; Corbett, Anita H.
The human protein PRR14 tethers heterochromatin to the nuclear lamina during interphase and mitotic exit.
The nuclear lamina is a protein meshwork that lies under the inner nuclear membrane of metazoan cells. One function of the nuclear lamina is to organize heterochromatin at the inner nuclear periphery. However, very little is known about how heterochromatin attaches to the nuclear lamina and how such attachments are restored at mitotic exit. Here, we show that a previously unstudied human protein, PRR14, functions to tether heterochromatin to the nuclear periphery during interphase, through associations with heterochromatin protein 1 (HP1) and the nuclear lamina. During early mitosis, PRR14 is released from the nuclear lamina and chromatin and remains soluble. Strikingly, at the onset of anaphase, PRR14 is incorporated rapidly into chromatin through HP1 binding. Finally, in telophase, PRR14 relocalizes to the reforming nuclear lamina. This stepwise reassembly of PRR14 suggests a function in the selection of HP1-bound heterochromatin for reattachment to the nuclear lamina as cells exit mitosis. PMID:24209742
Poleshko, Andrey; Mansfield, Katelyn M; Burlingame, Caroline C; Andrake, Mark D; Shah, Neil R; Katz, Richard A
The PWWP domain: a potential protein�protein interaction domain in nuclear proteins influencing differentiation?
Upon characterization of WHSC1, a gene mapping to the Wolf�Hirschhorn syndrome critical region and at its C-terminus similar to the Drosophila ASH1\\/trithorax group proteins, we identified a novel protein domain designated PWWP domain. To gain insight into its structure, evolutionary conservation and its potential functional role, we performed database searches to identify other PWWP domain-containing proteins. We retrieved 39 proteins,
Ingrid Stec; Sylvia B. Nagl; Gert-Jan B. van Ommen; Johan T. den Dunnen
Nuclear transit of human zipcode-binding protein IMP1.
The human IMPs (insulin-like growth factor II mRNA-binding proteins) belong to a vertebrate zipcode-binding protein family consisting of two RNA recognition motifs and four K homology domains and have been implicated in cytoplasmic mRNA localization, turnover and translational control. In the present study, we show that IMP1 is capable of translocating into nuclei of NIH 3T3 fibroblasts and its immunoreactivity is present in the nuclei of human spermatogenic cells. IMP1 does not contain a simple import signal, but nuclear entry was facilitated by disruption of RNA binding and cytoplasmic granule formation. IMP1 contains two NESs (nuclear export signals) within the RNA-binding K homology domains 2 and 4. The former is a leucine-rich leptomycin B-sensitive NES, whereas the latter is a leptomycin B-insensitive NES. Taken together, these results indicate that IMP1 may attach to its target mRNAs in the nucleus and thereby define the cytoplasmic fate of the transcripts.
Nielsen, Jacob; Adolph, Sidsel K; Rajpert-De Meyts, Ewa; Lykke-Andersen, Jens; Koch, Grete; Christiansen, Jan; Nielsen, Finn C
Cellular distribution of ACT domain repeat protein 9, a nuclear localizing protein, in rice (Oryza sativa).
Regulatory ACT domains serve as amino acid-binding sites in certain amino acid metabolic enzymes and transcriptional regulators in bacteria. The ACT domain repeat protein (ACR) family in plants is primarily composed of four copies of the domain homologous to those of the bacteria Gln sensor GLND. In the current study, to evaluate the possible involvement of the protein OsACR9 in the Gln-sensing system related to nitrogen (N) metabolism in rice (Oryza sativa L.), subcellular localization of OsACR9 and its accumulation and cellular distribution in various rice organs were examined by transient expression analysis and immunological methods using a monospecific antibody, respectively. Transient expression analysis of OsACR9 fused with a synthetic green fluorescent protein in cultured rice cells suggested nuclear localization of OsACR9. In rice roots, OsACR9 protein was distributed in epidermis, exodermis, sclerenchyma and vascular parenchyma cells, and its accumulation markedly increased after supply of NH(+)(4). In rice leaf samples, OsACR9 protein was abundant in the vascular parenchyma and mestome-sheath cells of young leaf blades at the early stage of development and in the vascular parenchyma and phloem-companion cells of mature leaf sheaths. OsACR9 protein also showed a high level of accumulation in vascular parenchyma cells of dorsal vascular bundles and aleurone cells in young rice grains at the early stage of ripening. The possibility of the nuclear protein OsACR9 acting as a Gln sensor in rice is subsequently discussed through comparison of its spatiotemporal expression with that of Gln-responsive N-assimilatory genes. PMID:18282189
Kudo, Toru; Kawai, Akiko; Yamaya, Tomoyuki; Hayakawa, Toshihiko
Transcription-Dependent and Transcription-Independent Nuclear Transport of hnRNP Proteins
Heterogeneous nuclear RNAs and specific nuclear proteins form heterogeneous nuclear ribonucleoprotein complexes (hnRNPs), one of the most abundant components of the nucleus. In mitosis, as the nuclear envelope breaks down, hnRNPs disperse throughout the cell. At the end of mitosis, hnRNPs dissociate and their proteins are transported into the daughter cell nuclei separately. Some are transported immediately (early group), while
Serafin Pinol-Roma; Gideon Dreyfuss
A novel nuclear zinc finger protein EZI enhances nuclear retention and transactivation of STAT3
A novel cDNA EZI isolated as an oncostatin M- inducible gene encoded a protein containing 12 C2H2-type zinc fingers. EZI was found to transactivate the promoters that are also responsive to STAT3 and activated the acute phase response element (APRE) synergistically with STAT3. Co-immunoprecipitation demonstrated the association of EZI with STAT3, which was mediated by the N-terminal region (1�183) of EZI. The EZI mutant lacking this region showed reduced transcriptional activity, indicating that EZI and STAT3 function cooperatively through physical interaction. While EZI predominantly localized in the nucleus and enhanced the nuclear localization of STAT3, the EZI mutant lacking 11 zinc finger motifs failed to translocate into the nucleus and also inhibited nuclear localization of STAT3 as well as STAT3-mediated transactivation. These results indicate that EZI is a novel nuclear zinc finger protein that augments STAT3 activity by keeping it in the nucleus.
Nakayama, Koh; Kim, Kyung-Woon; Miyajima, Atsushi
Protein targeting to the nuclear pore. What can we learn from plants. [Nuclear pore complex
Characteristic of eukaryotic cells are the numerous types of membrane-bound organelles or compartments found in the cytoplasm, with each type carrying out an essential function for the cell. The spatial separation of proteins and biochemical pathways typical of the various types of organelles requires selective targeting apparatuses. Because each type of organelle contains its own targeting apparatus, proteins destined for a particular organelle must contain the proper targeting signal(s) for entry. These signal-dependent targeting pathways ensure that proteins are targeted to the proper organelle. Understanding how proteins are targeted to the different types of organelles is an important goal in the field of cell biology. In plants recent studies have highlighted a number of unusual features, and as the understanding of import in plants increases, the authors have gained new insights, such as a model for the targeting of proteins from the cytoplasm to the NPC. These advances will contribute to further expansion of the knowledge of nuclear import in eukaryotes.
Smith, H.M.S.; Raikhel, N.V. (Michigan State Univ., East Lansing, MI (United States). DOE Plant Research Lab.)
Specific nuclear envelope transmembrane proteins can promote the location of chromosomes to and from the nuclear periphery
Background Different cell types have distinctive patterns of chromosome positioning in the nucleus. Although ectopic affinity-tethering of specific loci can be used to relocate chromosomes to the nuclear periphery, endogenous nuclear envelope proteins that control such a mechanism in mammalian cells have yet to be widely identified. Results To search for such proteins, 23 nuclear envelope transmembrane proteins were screened for their ability to promote peripheral localization of human chromosomes in HT1080 fibroblasts. Five of these proteins had strong effects on chromosome 5, but individual proteins affected different subsets of chromosomes. The repositioning effects were reversible and the proteins with effects all exhibited highly tissue-restricted patterns of expression. Depletion of two nuclear envelope transmembrane proteins that were preferentially expressed in liver each reduced the normal peripheral positioning of chromosome 5 in liver cells. Conclusions The discovery of nuclear envelope transmembrane proteins that can modulate chromosome position and have restricted patterns of expression may enable dissection of the functional relevance of tissue-specific patterns of radial chromosome positioning.
The K nuclear shuttling domain: a novel signal for nuclear import and nuclear export in the hnRNP K protein.
Protein import into the nucleus and export from the nucleus are signal-mediated processes that require energy. The nuclear transport process about which the most information is currently available is classical nuclear localization signal (NLS)-mediated nuclear import. However, details concerning the signal-mediated export of proteins and RNAs as well as alternative nuclear import pathways are beginning to emerge. An example of this is the heterogeneous nuclear ribonucleoprotein (hnRNP) A1 protein which, by virtue of its M9 domain, is actively exported from the nucleus and imported into the nucleus via a novel pathway mediated by the recently characterized transportin protein. Here we report that the shuttling hnRNP K protein contains a novel shuttling domain (termed KNS) which has many of the characteristics of M9, in that it confers bi-directional transport across the nuclear envelope. KNS-mediated nuclear import is dependent on RNA polymerase II transcription, and we show that a classical NLS can override this effect. Furthermore, KNS accesses a separate import pathway from either classical NLSs or M9. This demonstrates the existence of a third protein import pathway into the nucleus and thereby defines a new type of nuclear import/export signal.
Michael, W M; Eder, P S; Dreyfuss, G
A nuclear localization domain in the hnRNP A1 protein
The heterogeneous nuclear RNP (hnRNP) A1 protein is one of the major pre-mRNA/mRNA binding proteins in eukaryotic cells and one of the most abundant proteins in the nucleus. It is localized to the nucleoplasm and it also shuttles between the nucleus and the cytoplasm. The amino acid sequence of A1 contains two RNP motif RNA-binding domains (RBDs) at the amino terminus and a glycine-rich domain at the carboxyl terminus. This configuration, designated 2x RBD-Gly, is representative of perhaps the largest family of hnRNP proteins. Unlike most nuclear proteins characterized so far, A1 (and most 2x RBD-Gly proteins) does not contain a recognizable nuclear localization signal (NLS). We have found that a segment of ca. 40 amino acids near the carboxyl end of the protein (designated M9) is necessary and sufficient for nuclear localization; attaching this segment to the bacterial protein beta- galactosidase or to pyruvate kinase completely localized these otherwise cytoplasmic proteins to the nucleus. The RBDs and another RNA binding motif found in the glycine-rich domain, the RGG box, are not required for A1 nuclear localization. M9 is a novel type of nuclear localization domain as it does not contain sequences similar to classical basic-type NLS. Interestingly, sequences similar to M9 are found in other nuclear RNA-binding proteins including hnRNP A2.
Radioprotective Thiolamines WR-1065 and WR-33278 Selectively Denature Nonhistone Nuclear Proteins
NASA Technical Reports Server (NTRS)
Differential scanning calorimetry was used to study the interactions of nuclei isolated from Chinese hamster V79 cells with the radioprotector WR-1065, other thiol compounds, and polyamines. Differential scanning calorimetry monitors denaturation of macromolecules and resolves the major nuclear components (e.g. constrained and relaxed DNA, nucleosome core, and nuclear matrix) of intact nuclei on the basis of thermal stability. WR-1065 treatment (0.5-10 mM) of isolated nuclei led to the irreversible denaturation of nuclear proteins, a fraction of which are nuclear matrix proteins. Denaturation of 50% of the total nonhistone nuclear protein content of isolated nuclei occurred after exposure to 4.7 mM WR-1065 for 20 min at 23 C. In addition, a 22% increase in the insoluble protein content of nuclei isolated from V79 cells that had been treated with 4 mM WR-1065 for 30 min at 37 C was observed, indicating that WR-1065-induced protein denaturation occurs not only in isolated nuclei but also in the nuclei of intact cells. From the extent of the increase in insoluble protein in the nucleus, protein denaturation by WR-1065 is expected to contribute to drug toxicity at concentrations greater than approximately 4 mM. WR-33278, the disulfide form of WR1065, was approximately twice as effective as the free thiol at denaturing nuclear proteins. The proposed mechanism for nucleoprotein denaturation is through direct interactions with protein cysteine groups with the formation of destabilizing protein-WR-1065 disulfides. In comparison to its effect on nuclear proteins in isolated nuclei, WR-1065 had only a very small effect on non-nuclear proteins of whole cells, isolated nuclear matrix, or the thiol-rich Ca (2+) ATPase of sarcoplasmic reticulum, indicating that WR-1065 can effectively denature protein only inside an intact nucleus, probably due to the increased concentration of the positively charged drug in the vicinity of DNA.
Booth, Valerie K.; Roberts, Jeanette C.; Warters, Raymond L.; Wilmore, Britta H.; Lepock, James R.
The P1 family: a new class of nuclear mammalian proteins related to the yeast Mcm replication proteins.
Monospecific antibodies against an oligopeptide, conserved among the Mcm class of yeast replication proteins, were used to screen a human cDNA library. Eight of the isolated cDNA clones have the potential to code for sections of proteins with high sequence similarities to the yeast proteins Mcm3 and Cdc46 from Saccharomyces cerevisiae and Cdc21 from S. pombe. Our results establish a novel and highly conserved family of nuclear proteins in mammalian cells. Images
Hu, B; Burkhart, R; Schulte, D; Musahl, C; Knippers, R
Interaction between a plasma membrane-localized ankyrin-repeat protein ITN1 and a nuclear protein RTV1
Highlights: Black-Right-Pointing-Pointer ITN1, a plasma membrane ankyrin protein, interacts with a nuclear DNA-binding protein RTV1. Black-Right-Pointing-Pointer The nuclear transport of RTV1 is partially inhibited by interaction with ITN1. Black-Right-Pointing-Pointer RTV1 can promote the nuclear localization of ITN1. Black-Right-Pointing-Pointer Both overexpression of RTV1 and the lack of ITN1 increase salicylic acids sensitivity in plants. -- Abstract: The increased tolerance to NaCl 1 (ITN1) protein is a plasma membrane (PM)-localized protein involved in responses to NaCl stress in Arabidopsis. The predicted structure of ITN1 is composed of multiple transmembrane regions and an ankyrin-repeat domain that is known to mediate protein-protein interactions. To elucidate the molecular functions of ITN1, we searched for interacting partners using a yeast two-hybrid assay, and a nuclear-localized DNA-binding protein, RTV1, was identified as a candidate. Bimolecular fluorescence complementation analysis revealed that RTV1 interacted with ITN1 at the PM and nuclei in vivo. RTV1 tagged with red fluorescent protein localized to nuclei and ITN1 tagged with green fluorescent protein localized to PM; however, both proteins localized to both nuclei and the PM when co-expressed. These findings suggest that RTV1 and ITN1 regulate the subcellular localization of each other.
Sakamoto, Hikaru [Department of Bioproduction, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri-shi, Hokkaido 093-2422 (Japan)] [Department of Bioproduction, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri-shi, Hokkaido 093-2422 (Japan); Sakata, Keiko; Kusumi, Kensuke [Department of Biology, Faculty of Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581 (Japan)] [Department of Biology, Faculty of Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581 (Japan); Kojima, Mikiko; Sakakibara, Hitoshi [RIKEN Plant Science Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045 (Japan)] [RIKEN Plant Science Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045 (Japan); Iba, Koh, E-mail: firstname.lastname@example.com [Department of Biology, Faculty of Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581 (Japan)] [Department of Biology, Faculty of Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581 (Japan)
Signals and structural features involved in integral membrane protein targeting to the inner nuclear membrane
We have examined transfected cells by im- munofluorescence microscopy to determine the signals and structural features required for the targeting of in- tegral membrane proteins to the inner nuclear mem- brane. Lamin B receptor (LBR) is a resident protein of the nuclear envelope inner membrane that has a nu- cleoplasmic, amino-terminal domain and a carboxyl- terminal domain with eight putative
Bruno Soullam; Howard J. Worman
Expression of nervous tissue nuclear protein and glial fibrillary acidic protein during morphogenesis of the neocortex.
We studied the distribution of glial fibrillary acidic protein (GFAP) and neuron-specific nuclear histone protein NeuN in sulci of the brain cortex during the pre- and postnatal ontogeny. The expression of GFAP during morphogenetic development of the sulci and gyri is cyclic. After functional reorientation of GFAP in human fetuses at weeks 28-30 it ceases to be a marker of morphogenetically active glia and becomes a marker of glial cells exclusively. Redistribution of NeuN expression in different layers of sulci during their formation was found: enhanced expression of NeuN in the cortical layer 6 of sulci and its reduced expression in the upper layers were noted, whereas outside the cortical sulci NeuN expression was similar in all layers. At weeks 24-25 of gestation, NeuN serves as a marker of ingrowth of secondary visual fibers from the dorsal thalamus. PMID:21165411
Godovalova, O S
Nuclear and nucleolar localization of Saccharomyces cerevisiae ribosomal proteins S22 and S25
Nuclear import usually relies on the presence of nuclear localization sequences (NLSs). NLSs are recognized by NLS receptors (importins), which target their substrates to the nuclear pore. We identified the NLSs of the yeast ribosomal proteins S22 and S25 and studied the former by mutational analysis. Furthermore, in S25 the nucleolar targeting information was found to overlap with its NLS.
Antonius C. J. Timmers; Rogier Stuger; Peter J. Schaap; Jan van �t Riet; Hendrik A Rau�
Cloning and sequence of the human nuclear protein cyclin: homology with DNA-binding proteins
A full-length cDNA clone for the human nuclear protein cyclin has been isolated by using polyclonal antibodies and sequenced. The sequence predicts a protein of 261 amino acids (M/sub r/ 29,261) with a high content of acidic (41, aspartic and glutamic acids) versus basic (24, lysine and arginine) amino acids. The identity of the cDNA clone was confirmed by in vitro hybrid-arrested translation of cyclin mRNA. Blot-hybridization analysis of mouse 3T3 and human MOLT-4 cell RNA revealed a mRNA species of approximately the same size as of the cDNA insert. Expression of cyclin mRNA was undetectable or very low in quiescent cells, increasing after 8-10 hr of serum stimulation. Inhibition of DNA synthesis by hydroxyurea in serum-stimulated cells did not affect the increase in cyclin mRNA but inhibited 90% the expression of H3 mRNA. These results suggest that expression of cyclin and histone mRNAs are controlled by different mechanisms. A region of the cyclin sequence shows a significant homology with the putative DNA binding site of several proteins, specially with the transcriptional-regulator cAMP-binding protein of Escherichia coli, suggesting that cyclin could play a similar role in eukaryotic cells.
Almendral, J.M.; Huebsch, D.; Blundell, P.A.; Macdonald-Bravo, H.; Bravo, R.
Mitotic Phosphorylation of Chromosomal Protein HMGN1 Inhibits Nuclear Import and Promotes Interaction with 14.3.3 Proteins
Progression through mitosis is associated with reversible phosphorylation of many nuclear proteins including that of the high-mobility group N (HMGN) nucleosomal binding protein family. Here we use immunofluorescence and in vitro nuclear import studies to demonstrate that mitotic phosphorylation of the nucleosomal binding domain (NBD) of the HMGN1 protein prevents its reentry into the newly formed nucleus in late telophase. By microinjecting wild-type and mutant proteins into the cytoplasm of HeLa cells and expressing these proteins in HmgN1?/? cells, we demonstrate that the inability to enter the nucleus is a consequence of phosphorylation and is not due to the presence of negative charges. Using affinity chromatography with recombinant proteins and nuclear extracts prepared from logarithmically growing or mitotically arrested cells, we demonstrate that phosphorylation of the NBD of HMGN1 promotes interaction with specific 14.3.3 isotypes. We conclude that mitotic phosphorylation of HMGN1 protein promotes interaction with 14.3.3 proteins and suggest that this interaction impedes the reentry of the proteins into the nucleus during telophase. Taken together with the results of previous studies, our results suggest a dual role for mitotic phosphorylation of HMGN1: abolishment of chromatin binding and inhibition of nuclear import.
Prymakowska-Bosak, Marta; Hock, Robert; Catez, Frederic; Lim, Jae-Hwan; Birger, Yehudit; Shirakawa, Hitoshi; Lee, Kyung; Bustin, Michael
Nucleus-Specific Importin Alpha Proteins and Nucleoporins Regulate Protein Import and Nuclear Division in the Binucleate Tetrahymena thermophila? �
The ciliate Tetrahymena thermophila, having both germ line micronuclei and somatic macronuclei, must possess a specialized nucleocytoplasmic transport system to import proteins into the correct nucleus. To understand how Tetrahymena can target proteins to distinct nuclei, we first characterized FG repeat-containing nucleoporins and found that micro- and macronuclei utilize unique subsets of these proteins. This finding implicates these proteins in the differential permeability of the two nuclei and implies that nuclear pores with discrete specificities are assembled within a single cell. To identify the import machineries that interact with these different pores, we characterized the large families of karyopherin homologs encoded within the genome. Localization studies of 13 putative importin (imp) ?- and 11 imp ?-like proteins revealed that imp ?-like proteins are nucleus specific�nine localized to the germ line micronucleus�but that most imp ?-like proteins localized to both types of nuclei. These data suggest that micronucleus-specific proteins are transported by specific imp ? adapters. The different imp ? proteins exhibit substantial sequence divergence and do not appear to be simply redundant in function. Disruption of the IMA10 gene encoding an imp ?-like protein that accumulates in dividing micronuclei results in nuclear division defects and lethality. Thus, nucleus-specific protein import and nuclear function in Tetrahymena are regulated by diverse, specialized karyopherins.
Malone, Colin D.; Falkowska, Katarzyna A.; Li, Alanna Y.; Galanti, Sarah E.; Kanuru, Reshi C.; LaMont, Elizabeth G.; Mazzarella, Kate C.; Micev, Alan J.; Osman, Morwan M.; Piotrowski, Nicholas K.; Suszko, Jason W.; Timm, Adam C.; Xu, Ming-Ming; Liu, Lucy; Chalker, Douglas L.
Large-scale identification of mammalian proteins localized to nuclear sub-compartments.
Many nuclear components participating in related pathways appear concentrated in specific areas of the mammalian nucleus. The importance of this organization is attested to by the dysfunction that correlates with mis-localization of nuclear proteins in human disease and cancer. Determining the sub-nuclear localization of proteins is therefore important for understanding genome regulation and function, and it also provides clues to function for novel proteins. However, the complexity of proteins in the mammalian nucleus is too large to tackle this on a protein by protein basis. Large-scale approaches to determining protein function and sub-cellular localization are required. We have used a visual gene trap screen to identify more than 100 proteins, many of which are normal, located within compartments of the mouse nucleus. The most common discrete localizations detected are at the nucleolus and the splicing speckles and on chromosomes. Proteins at the nuclear periphery, or in other nuclear foci, have also been identified. Several of the proteins have been implicated in human disease or cancer, e.g. ATRX, HMGI-C, NBS1 and EWS, and the gene-trapped proteins provide a route into further understanding their function. We find that sequence motifs are often shared amongst proteins co-localized within the same sub-nuclear compartment. Conversely, some generally abundant motifs are lacking from the proteins concentrated in specific areas of the nucleus. This suggests that we may be able to predict sub-nuclear localization for proteins in databases based on their sequence. PMID:11555636
Sutherland, H G; Mumford, G K; Newton, K; Ford, L V; Farrall, R; Dellaire, G; C�ceres, J F; Bickmore, W A
Proteomic analysis of high NaCl-induced changes in abundance of nuclear proteins
Mammalian cells are normally stressed by high interstitial NaCl in the renal medulla and by lesser elevation of NaCl in several other tissues. High NaCl damages proteins and DNA and can kill cells. Known protective responses include nuclear translocation of the transcription factor NFAT5 and other proteins. In order better to understand the extent and significance of changes in nuclear protein abundance, we extracted nuclear and cytoplasmic proteins separately from HEK293 cells and measured by LC-MS/MS (iTRAQ) changes of abundance of proteins in the extracts in response to high NaCl at three time points: 1 h, 8 h, and adapted for two passages. We confidently identified a total of 3,190 proteins; 163 proteins changed significantly at least at one time point in the nucleus. We discerned the biological significance of the changes by Gene Ontology and protein network analysis. Proteins that change in the nucleus include ones involved in protein folding and localization, microtubule-based process, regulation of cell death, cytoskeleton organization, DNA metabolic process, RNA processing, and cell cycle. Among striking changes in the nucleus, we found a decrease of all six 14-3-3 isoforms; dynamic changes of �cytoskeletal� proteins, suggestive of nucleoskeletal reorganization; rapid decrease of tubulins; and dynamic changes of heat shock proteins. Identification of these changes of nuclear protein abundance enhances our understanding of high NaCl-induced cellular stress, and provides leads to previously unknown damages and protective responses.
Li, Jinxi; Ferraris, Joan D.; Yu, Danni; Singh, Taruna; Izumi, Yuichiro; Wang, Guanghui; Gucek, Marjan
Importin-?-16 is a translocon-associated protein involved in sorting membrane proteins to the nuclear envelope
A viral inner nuclear membrane�sorting motif sequence (INM-SM) was used to identify proteins that recognize integral membrane proteins destined for the INM. Herein we describe importin-?-16, a membrane-associated isoform of Spodoptera frugiperda importin-? that contains the C-terminal amino acid residues comprising armadillo helical-repeat domains 7�10. In the endoplasmic reticulum (ER) membrane, importin-?-16 is adjacent to the translocon protein Sec61?. Importin-?-16
Suraj Saksena; Max D Summers; Jared K Burks; Arthur E Johnson; Sharon C Braunagel
Identification of a nuclear localization signal in the retinitis pigmentosa-mutated RP26 protein, ceramide kinase-like protein
Retinitis pigmentosa (RP) is a genetically heterogeneous disease characterized by degeneration of the retina. A mutation in a new ceramide kinase (CERK) homologous gene, named CERK-like protein (CERKL), was found to cause autosomal recessive retinitis pigmentosa (RP26). Here, we show a point mutation of one of two putative nuclear localization signal (NLS) sequences inhibited the nuclear localization of the protein. Furthermore, the tetra-GFP-tagged NLS, which cannot passively enter the nucleus, was observed not only in the nucleus but also in the nucleolus. Our results provide First evidence of the active nuclear import of CERKL and suggest that the identified NLS might be responsible for nucleolar retention of the protein. As recent studies have shown other RP-related proteins are localized in the nucleus or the nucleolus, our identification of NLS in CERKL suggests that CERKL likely plays important roles for retinal functions in the nucleus and the nucleolus.
Inagaki, Yuichi [Department of Biomembrane and Biofunctional Chemistry, Graduate School of Pharmaceutical Science, Hokkaido University, Kita 12, Nishi 6, Kita-ku, Sapporo 060-0812 (Japan); Mitsutake, Susumu [Department of Biomembrane and Biofunctional Chemistry, Graduate School of Pharmaceutical Science, Hokkaido University, Kita 12, Nishi 6, Kita-ku, Sapporo 060-0812 (Japan); Igarashi, Yasuyuki [Department of Biomembrane and Biofunctional Chemistry, Graduate School of Pharmaceutical Science, Hokkaido University, Kita 12, Nishi 6, Kita-ku, Sapporo 060-0812 (Japan)]. E-mail: firstname.lastname@example.com
Fate of the Inner Nuclear Membrane Protein Lamin B Receptor and Nuclear Lamins in Herpes Simplex Virus Type 1 Infection
During herpesvirus egress, capsids bud through the inner nuclear membrane. Underlying this membrane is the nuclear lamina, a meshwork of intermediate filaments with which it is tightly associated. Details of alterations to the lamina and the inner nuclear membrane during infection and the mechanisms involved in capsid transport across these structures remain unclear. Here we describe the fate of key protein components of the nuclear envelope and lamina during herpes simplex virus type 1 (HSV-1) infection. We followed the distribution of the inner nuclear membrane protein lamin B receptor (LBR) and lamins A and B2 tagged with green fluorescent protein (GFP) in live infected cells. Together with additional results from indirect immunofluorescence, our studies reveal major morphologic distortion of nuclear-rim LBR and lamins A/C, B1, and B2. By 8 h p.i., we also observed a significant redistribution of LBR-GFP to the endoplasmic reticulum, where it colocalized with a subpopulation of cytoplasmic glycoprotein B by immunofluorescence. In addition, analysis by fluorescence recovery after photobleaching reveals that LBR-GFP exhibited increased diffusional mobility within the nuclear membrane of infected cells. This is consistent with the disruption of interactions between LBR and the underlying lamina. In addition to studying stably expressed GFP-lamins by fluorescence microscopy, we studied endogenous A- and B-type lamins in infected cells by Western blotting. Both approaches reveal a loss of lamins associated with virus infection. These data indicate major disruption of the nuclear envelope and lamina of HSV-1-infected cells and are consistent with a virus-induced dismantling of the nuclear lamina, possibly in order to gain access to the inner nuclear membrane.
Scott, Emily S.; O'Hare, Peter
Multiple and surprising new functions for emerin, a nuclear membrane protein.
Emerin is an integral protein of the nuclear inner membrane. Emerin is not essential, but its loss of function causes Emery-Dreifuss muscular dystrophy. We summarize significant recent progress in understanding emerin, which was previously known to interact with barrier-to-autointegration factor and lamins. New partners include transcription repressors, an mRNA splicing regulator, a nuclear membrane protein named nesprin, nuclear myosin I and F-actin. These interactors imply multiple roles for emerin in the nucleus, some of which overlap with related LEM-domain proteins. PMID:15037308
Bengtsson, Luiza; Wilson, Katherine L
Nuclear Receptor Interaction Protein (NRIP) expression assay using human tissue microarray and immunohistochemistry technology confirming nuclear localization
Background��A novel human nuclear receptor interaction protein (NRIP) has recently been discovered by Chen SL et al, which may play a\\u000a role in enhancing the transcriptional activity of steroid nuclear receptors in prostate (LNCaP) and cervical (C33A) cancer\\u000a cell lines. However, knowledge about the biological functions and clinical implications of NRIP, is still incomplete. Our\\u000a aim was to determine the
Chih-Ping Han; Ming-Yung Lee; Shu-Ling Tzeng; Chung-Chin Yao; Po-Hui Wang; Ya-Wen Cheng; Show-Li Chen; Teresa S Wu; Yeu-Sheng Tyan; Lai-Fong Kok
Identification of Nuclear Phosphatidylinositol 4,5-Bisphosphate-Interacting Proteins by Neomycin Extraction*
Considerable insight into phosphoinositide-regulated cytoplasmic functions has been gained by identifying phosphoinositide-effector proteins. Phosphoinositide-regulated nuclear functions however are fewer and less clear. To address this, we established a proteomic method based on neomycin extraction of intact nuclei to enrich for nuclear phosphoinositide-effector proteins. We identified 168 proteins harboring phosphoinositide-binding domains. Although the vast majority of these contained lysine/arginine-rich patches with the following motif, K/R-(Xn = 3�7)-K-X-K/R-K/R, we also identified a smaller subset of known phosphoinositide-binding proteins containing pleckstrin homology or plant homeodomain modules. Proteins with no prior history of phosphoinositide interaction were identified, some of which have functional roles in RNA splicing and processing and chromatin assembly. The remaining proteins represent potentially other novel nuclear phosphoinositide-effector proteins and as such strengthen our appreciation of phosphoinositide-regulated nuclear functions. DNA topology was exemplar among these: Biochemical assays validated our proteomic data supporting a direct interaction between phosphatidylinositol 4,5-bisphosphate and DNA Topoisomerase II?. In addition, a subset of neomycin extracted proteins were further validated as phosphatidyl 4,5-bisphosphate-interacting proteins by quantitative lipid pull downs. In summary, data sets such as this serve as a resource for a global view of phosphoinositide-regulated nuclear functions.
Lewis, Aurelia E.; Sommer, Lilly; Arntzen, Magnus ?.; Strahm, Yvan; Morrice, Nicholas A.; Divecha, Nullin; D'Santos, Clive S.
Nuclear Transport of the Major Capsid Protein Is Essential for Adeno-Associated Virus Capsid Formation
Adeno-associated virus capsids are composed of three proteins, VP1, VP2, and VP3. Although VP1 is necessary for viral infection, it is not essential for capsid formation. The other capsid proteins, VP2 and VP3, are sufficient for capsid formation, but the functional roles of each protein are still not well understood. By analyzing a series of deletion mutants of VP2, we identified a region necessary for nuclear transfer of VP2 and found that the efficiency of nuclear localization of the capsid proteins and the efficiency of virus-like particle (VLP) formation correlated well. To confirm the importance of the nuclear localization of the capsid proteins, we fused the nuclear localization signal of simian virus 40 large T antigen to VP3 protein. We show that this fusion protein could form VLP, indicating that the VP2-specific region located on the N-terminal side of the protein is not structurally required. This finding suggests that VP3 has sufficient information for VLP formation and that VP2 is necessary only for nuclear transfer of the capsid proteins.
Hoque, Mainul; Ishizu, Ken-ichiro; Matsumoto, Akiko; Han, Song-Iee; Arisaka, Fumio; Takayama, Makoto; Suzuki, Kenji; Kato, Kenzo; Kanda, Tadahito; Watanabe, Hajime; Handa, Hiroshi
eEF1A Is a Novel Component of the Mammalian Nuclear Protein Export Machinery
The cytoplasmic translation factor eEF1A has been implicated in the nuclear export of tRNA species in lower eukaryotes. Here we demonstrate that eEF1A plays a central role in nuclear export of proteins in mammalian cells. TD-NEM (transcription-dependent nuclear export motif), a newly characterized nuclear export signal, mediates efficient nuclear export of several proteins including the von Hippel-Lindau (VHL) tumor suppressor and the poly(A)-binding protein (PABP1) in a manner that is dependent on ongoing RNA polymerase II (RNA PolII)-dependent transcription. eEF1A interacts specifically with TD-NEM of VHL and PABP1 and disrupting this interaction, by point mutations of key TD-NEM residues or treatment with actinomycin D, an inhibitor of RNA PolII-dependent transcription, prevents assembly and nuclear export. siRNA-induced knockdown or antibody-mediated depletion of eEF1A prevents in vivo and in vitro nuclear export of TD-NEM�containing proteins. Nuclear retention experiments and inhibition of the Exportin-5 pathway suggest that eEF1A stimulates nuclear export of proteins from the cytoplasmic side of the nuclear envelope, without entering the nucleus. Together, these data identify a role for eEF1A, a cytoplasmic mediator of tRNA export in yeast, in the nuclear export of proteins in mammalian cells. These results also provide a link between the translational apparatus and subcellular trafficking machinery demonstrating that these two central pathways in basic metabolism can act cooperatively.
Khacho, Mireille; Mekhail, Karim; Pilon-Larose, Karine; Pause, Arnim; Cote, Jocelyn
ANP32B Is a Nuclear Target of Henipavirus M Proteins
Membrane envelopment and budding of negative strand RNA viruses (NSVs) is mainly driven by viral matrix proteins (M). In addition, several M proteins are also known to be involved in host cell manipulation. Knowledge about the cellular targets and detailed molecular mechanisms, however, is poor for many M proteins. For instance, Nipah Virus (NiV) M protein trafficking through the nucleus is essential for virus release, but nuclear targets of NiV M remain unknown. To identify cellular interactors of henipavirus M proteins, tagged Hendra Virus (HeV) M proteins were expressed and M-containing protein complexes were isolated and analysed. Presence of acidic leucine-rich nuclear phosphoprotein 32 family member B (ANP32B) in the complex suggested that this protein represents a direct or indirect interactor of the viral matrix protein. Over-expression of ANP32B led to specific nuclear accumulation of HeV M, providing a functional link between ANP32B and M protein. ANP32B-dependent nuclear accumulation was observed after plasmid-driven expression of HeV and NiV matrix proteins and also in NiV infected cells. The latter indicated that an interaction of henipavirus M protein with ANP32B also occurs in the context of virus replication. From these data we conclude that ANP32B is a nuclear target of henipavirus M that may contribute to virus replication. Potential effects of ANP32B on HeV nuclear shuttling and host cell manipulation by HeV M affecting ANP32B functions in host cell survival and gene expression regulation are discussed.
Bauer, Anja; Neumann, Sebastian; Karger, Axel; Henning, Ann-Kristin; Maisner, Andrea; Lamp, Boris; Dietzel, Erik; Kwasnitschka, Linda; Balkema-Buschmann, Anne; Keil, Gunther M.; Finke, Stefan
Organellar proteomics reveals hundreds of novel nuclear proteins in the malaria parasite Plasmodium falciparum.
BACKGROUND: The post-genomic era of malaria research provided unprecedented insights into the biology of Plasmodium parasites. Due to the large evolutionary distance to model eukaryotes, however, we lack a profound understanding of many processes in Plasmodium biology. One example is the cell nucleus, which controls the parasite genome in a development- and cell cycle-specific manner through mostly unknown mechanisms. To study this important organelle in detail, we conducted an integrative analysis of the P. falciparum nuclear proteome. RESULTS: We combined high accuracy mass spectrometry and bioinformatic approaches to present for the first time an experimentally determined core nuclear proteome for P. falciparum. Besides a large number of factors implicated in known nuclear processes, one-third of all detected proteins carry no functional annotation, including many phylum- or genus-specific factors. Importantly, extensive experimental validation using 30 transgenic cell lines confirmed the high specificity of this inventory, and revealed distinct nuclear localization patterns of hitherto uncharacterized proteins. Further, our detailed analysis identified novel protein domains potentially implicated in gene transcription pathways, and sheds important new light on nuclear compartments and processes including regulatory complexes, the nucleolus, nuclear pores, and nuclear import pathways. CONCLUSION: Our study provides comprehensive new insight into the biology of the Plasmodium nucleus and will serve as an important platform for dissecting general and parasite-specific nuclear processes in malaria parasites. Moreover, as the first nuclear proteome characterized in any protist organism, it will provide an important resource for studying evolutionary aspects of nuclear biology. PMID:23181666
Oehring, Sophie C; Woodcroft, Ben J; Moes, Suzette; Wetzel, Johanna; Dietz, Olivier; Pulfer, Andreas; Dekiwadia, Chaitali; Maeser, Pascal; Flueck, Christian; Witmer, Kathrin; Brancucci, Nicolas Mb; Niederwieser, Igor; Jenoe, Paul; Ralph, Stuart A; Voss, Till S
Organellar proteomics reveals hundreds of novel nuclear proteins in the malaria parasite Plasmodium falciparum
Background The post-genomic era of malaria research provided unprecedented insights into the biology of Plasmodium parasites. Due to the large evolutionary distance to model eukaryotes, however, we lack a profound understanding of many processes in Plasmodium biology. One example is the cell nucleus, which controls the parasite genome in a development- and cell cycle-specific manner through mostly unknown mechanisms. To study this important organelle in detail, we conducted an integrative analysis of the P. falciparum nuclear proteome. Results We combined high accuracy mass spectrometry and bioinformatic approaches to present for the first time an experimentally determined core nuclear proteome for P. falciparum. Besides a large number of factors implicated in known nuclear processes, one-third of all detected proteins carry no functional annotation, including many phylum- or genus-specific factors. Importantly, extensive experimental validation using 30 transgenic cell lines confirmed the high specificity of this inventory, and revealed distinct nuclear localization patterns of hitherto uncharacterized proteins. Further, our detailed analysis identified novel protein domains potentially implicated in gene transcription pathways, and sheds important new light on nuclear compartments and processes including regulatory complexes, the nucleolus, nuclear pores, and nuclear import pathways. Conclusion Our study provides comprehensive new insight into the biology of the Plasmodium nucleus and will serve as an important platform for dissecting general and parasite-specific nuclear processes in malaria parasites. Moreover, as the first nuclear proteome characterized in any protist organism, it will provide an important resource for studying evolutionary aspects of nuclear biology.
Nuclear Trafficking of Retroviral RNAs and Gag Proteins during Late Steps of Replication
Retroviruses exploit nuclear trafficking machinery at several distinct stages in their replication cycles. In this review, we will focus primarily on nucleocytoplasmic trafficking events that occur after the completion of reverse transcription and proviral integration. First, we will discuss nuclear export of unspliced viral RNA transcripts, which serves two essential roles: as the mRNA template for the translation of viral structural proteins and as the genome for encapsidation into virions. These full-length viral RNAs must overcome the cell�s quality control measures to leave the nucleus by co-opting host factors or encoding viral proteins to mediate nuclear export of unspliced viral RNAs. Next, we will summarize the most recent findings on the mechanisms of Gag nuclear trafficking and discuss potential roles for nuclear localization of Gag proteins in retrovirus replication.
Stake, Matthew S.; Bann, Darrin V.; Kaddis, Rebecca J.; Parent, Leslie J.
Identification and characterization of the small nuclear ribonucleoprotein particle D' core protein.
The addition of urea to sodium dodecyl sulfate (SDS)-polyacrylamide gels has allowed the identification and characterization of the small nuclear ribonucleoprotein particle (snRNP) D' protein and has also improved resolution of the E, F, and G snRNP core proteins. In standard SDS-polyacrylamide gels, the D' and D snRNP core proteins comigrate at approximately 16 kilodaltons. The addition of urea to the separating gel caused the D' protein to shift to a slower electrophoretic mobility that is distinct from that of the D protein. The shift to a slower electrophoretic mobility in the presence of urea suggests that the D' protein has extensive secondary structure that is not totally disrupted by SDS alone. Both N-terminal sequencing and partial peptide maps indicate that the D and D' proteins are distinct gene products, and the sequence data have identified the faster moving of the two proteins as the previously cloned D protein (L. A. Rokeach, J. A. Haselby, and S. O. Hoch, Proc. Natl. Acad. Sci. USA 85:4832-4836, 1988). In the cytoplasm, the D protein is found primarily in the small-nuclear-RNA-free 6S protein complexes, while the D' protein is found primarily in the 20S protein complexes. Like the D protein, the D' protein is an autoantigen in patients with systemic lupus erythematosus and is recognized by some of the Sm class of autoimmune antisera. PMID:2143805
Andersen, J; Feeney, R J; Zieve, G W
A novel heterogeneous nuclear RNP protein with a unique distribution on nascent transcripts
Immediately after the initiation of transcrip- tion in eukaryotes, nascent RNA polymerase II tran- scripts are bound by nuclear proteins resulting in the formation of heterogeneous nuclear ribonucleoprotein (hnRNP) complexes, hnRNP complexes from HeLa cell nuclei contain >20 major proteins in the molecu- lar mass range of 34,000-120,000 D. Among these are the previously described A, B, and C groups
S. Pinol-Roma; Maurice S. Swanson; Joseph G. Gall; Gideon Dreyfuss
Characterization of a baculovirus nuclear localization signal domain in the late expression factor 3 protein
The baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) single-stranded DNA binding protein LEF-3 is a multi-functional protein that is required to transport the helicase protein P143 into the nucleus of infected cells where they function to replicate viral DNA. The N-terminal 56 amino acid region of LEF-3 is required for nuclear transport. In this report, we analyzed the effect of site-specific mutagenesis of LEF-3 on its intracellular distribution. Fluorescence microscopy of expression plasmid-transfected cells demonstrated that the residues 28 to 32 formed the core nuclear localization signal, but other adjacent positively-charged residues augmented these sequences. Comparison with other group I Alphabaculoviruses suggested that this core region functionally duplicated residues including 18 and 19. This was demonstrated by the loss of nuclear localization when the equivalent residues (18 to 20) in Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) LEF-3 were mutated. The AcMNPV LEF-3 nuclear localization domain was also shown to drive nuclear transport in mammalian cells indicating that the protein nuclear import systems in insect and mammalian cells are conserved. We also demonstrated by mutagenesis that two conserved cysteine residues located at 82 and 106 were not essential for nuclear localization or for interaction with P143. However, by using a modified construct of P143 that localized on its own to the nucleus, we demonstrated that a functional nuclear localization domain on LEF-3 was required for interaction between LEF-3 and P143.
Au, Victoria; Yu Mei [Department of Microbiology and Immunology, Queen's University, Kingston, ON, K7L 3N6 (Canada); Carstens, Eric B. [Department of Microbiology and Immunology, Queen's University, Kingston, ON, K7L 3N6 (Canada)], E-mail: email@example.com
The farnesylated nuclear proteins KUGELKERN and LAMIN B promote aging-like phenotypes in Drosophila flies.
The nuclear lamina consists of a meshwork of lamins and lamina-associated proteins, which provide mechanical support, control size and shape of the nucleus, and mediate the attachment of chromatin to the nuclear envelope. Abnormal nuclear shapes are observed in aging cells of humans and nematode worms. The expression of laminDelta50, a constitutively active lamin A splicing variant in Hutchinson-Gilford progeria syndrome patients, leads to the lobulation of the nuclear envelope accompanied by DNA damage, and loss of heterochromatin. So far, it has been unclear whether these age-related changes are laminDelta50 specific or whether proteins that affect nuclear shape such as KUGELKERN or LAMIN B in general play a causative role in senescence. Here we show that in adult Drosophila flies, the size of the nuclei increases with age and the nuclei assume an aberrant shape. Moreover, induced expression of the farnesylated lamina proteins Lamin B and Kugelkern cause aberrant nuclear shapes and reduce the lifespan of adult flies. The shorter lifespan correlates with an early decline in age-dependent locomotor behaviour. Expression of kugelkern or lamin B in mammalian cells induces a nuclear lobulation phenotype in conjunction with DNA damage, and changes in histone modification similar to that found in cells expressing laminDelta50 or in cells from aged individuals. We conclude that lobulation of the nuclear membrane induced by the insertion of farnesylated lamina-proteins can lead to aging-like phenotypes. PMID:18494863
Brandt, Annely; Krohne, Georg; Grosshans, J�rg
Studies in protein dynamics using heteronuclear nuclear magnetic resonance spectroscopy
Dynamic processes in proteins are important for their biological function. Several issues in protein dynamics are addressed by applying existing NMR methodologies to investigate dynamics of several small proteins. Amide H\\/D exchange rates have been measured for the N-terminal domain of the ribosomal protein L9, residues 1--56. The results suggest that the structure of the domain is preserved in isolation
Liliya Vugmeyster
Characterization of a Drosophila phosphorylation-dependent nuclear-localization-signal-binding protein.
A 94 kDa nuclear-localization-signal (NLS)-binding protein was purified from Drosophila embryos. The NLS of the simian-virus-40 T-antigen is specifically bound by the dephosphorylated form of the protein. After phosphorylation, the affinity of the protein for the NLS is sharply decreased. In the dephosphorylated form, p94 (protein of 94 kDa) is the major NLS-binding protein in Drosophila embryos. Immunoprecipitation confirmed the ATP-dependent phosphorylation of p94, and co-precipitation of two additional phosphorylated proteins, indicated that the NLS-binding protein is part of a larger complex in Drosophila embryos. In agreement with the immunoprecipitation results, cross-linking experiments demonstrated the interaction of p94 with three additional proteins. These protein-protein interactions were also phosphorylation-dependent.
Cserpan, I; Mathe, E; Patthy, A; Udvardy, A
Identification of a nuclear localization signal in mouse polycomb protein, M33.
The mouse Polycomb group (PcG) protein M33 forms nuclear complexes with the products of other PcG members and maintains repressed states of developmentally important genes, including homeotic genes. In this context, nuclear localization is a prerequisite for M33 to exert its function. However, we previously found that M33 in mouse liver shuttles dynamically between the nucleus and the cytoplasm, depending on the proliferative states of cells, coupled with phosphorylation and dephosphorylation of M33 protein. To understand the mechanism and significance of this phenomenon, we identified the functional nuclear localization signal (NLS) of M33 protein. Deletion mutants that lack a particular one of three putative NLS motifs failed to localize in the nucleus. Green fluorescent protein (GFP) fused to this motif specifically localized in the nucleus. We conclude that this amino-acid stretch in M33 acts as the functional NLS for this protein. PMID:17043400
Hirose, Sayako; Komoike, Yuta; Higashinakagawa, Toru
Dual localized mitochondrial and nuclear proteins as gene expression regulators in plants?
Mitochondria heavily depend on the coordinated expression of both mitochondrial and nuclear genomes because some of their most significant activities are held by multi-subunit complexes composed of both mitochondrial and nuclear encoded proteins. Thus, precise communication and signaling pathways are believed to exist between the two compartments. Proteins dual localized to both mitochondria and the nucleus make excellent candidates for a potential involvement in the envisaged communication. Here, we review the identified instances of dual localized nucleo-mitochondrial proteins with an emphasis on plant proteins and discuss their functions, which are seemingly mostly related to gene expression regulation. We discuss whether dual localization could be achieved by dual targeting and/or by re-localization and try to apprehend the signals required for the respective processes. Finally, we propose that in some instances, dual localized mitochondrial and nuclear proteins might act as retrograde signaling molecules for mitochondrial biogenesis.
Duchene, Anne-Marie; Giege, Philippe
Dual localized mitochondrial and nuclear proteins as gene expression regulators in plants?
Mitochondria heavily depend on the coordinated expression of both mitochondrial and nuclear genomes because some of their most significant activities are held by multi-subunit complexes composed of both mitochondrial and nuclear encoded proteins. Thus, precise communication and signaling pathways are believed to exist between the two compartments. Proteins dual localized to both mitochondria and the nucleus make excellent candidates for a potential involvement in the envisaged communication. Here, we review the identified instances of dual localized nucleo-mitochondrial proteins with an emphasis on plant proteins and discuss their functions, which are seemingly mostly related to gene expression regulation. We discuss whether dual localization could be achieved by dual targeting and/or by re-localization and try to apprehend the signals required for the respective processes. Finally, we propose that in some instances, dual localized mitochondrial and nuclear proteins might act as retrograde signaling molecules for mitochondrial biogenesis. PMID:23056004
Duch�ne, Anne-Marie; Gieg�, Philippe
Heat-Shock Protein 90 Promotes Nuclear Transport of Herpes Simplex Virus 1 Capsid Protein by Interacting with Acetylated Tubulin
Although it is known that inhibitors of heat shock protein 90 (Hsp90) can inhibit herpes simplex virus type 1 (HSV-1) infection, the role of Hsp90 in HSV-1 entry and the antiviral mechanisms of Hsp90 inhibitors remain unclear. In this study, we found that Hsp90 inhibitors have potent antiviral activity against standard or drug-resistant HSV-1 strains and viral gene and protein synthesis are inhibited in an early phase. More detailed studies demonstrated that Hsp90 is upregulated by virus entry and it interacts with virus. Hsp90 knockdown by siRNA or treatment with Hsp90 inhibitors significantly inhibited the nuclear transport of viral capsid protein (ICP5) at the early stage of HSV-1 infection. In contrast, overexpression of Hsp90 restored the nuclear transport that was prevented by the Hsp90 inhibitors, suggesting that Hsp90 is required for nuclear transport of viral capsid protein. Furthermore, HSV-1 infection enhanced acetylation of ?-tubulin and Hsp90 interacted with the acetylated ?-tubulin, which is suppressed by Hsp90 inhibition. These results demonstrate that Hsp90, by interacting with acetylated ?-tubulin, plays a crucial role in viral capsid protein nuclear transport and may provide novel insight into the role of Hsp90 in HSV-1 infection and offer a promising strategy to overcome drug-resistance.
Chen, Maoyun; Xiang, Yangfei; Jin, Fujun; Ma, Kaiqi; Qiu, Xianxiu; Wang, Qiaoli; Peng, Tao; Kitazato, Kaio; Wang, Yifei
Characterization of carbonic anhydrase IX interactome reveals proteins assisting its nuclear localization in hypoxic cells.
Carbonic anhydrase IX (CA IX) is a transmembrane protein affecting pH regulation, cell migration/invasion, and survival in hypoxic tumors. Although the pathways related to CA IX have begun to emerge, molecular partners mediating its functions remain largely unknown. Here we characterize the CA IX interactome in hypoxic HEK-293 cells. Most of the identified CA IX-binding partners contain the HEAT/ARM repeat domain and belong to the nuclear transport machinery. We show that the interaction with two of these proteins, namely XPO1 exportin and TNPO1 importin, occurs via the C-terminal region of CA IX and increases with protein phosphorylation. We also demonstrate that nuclear CA IX is enriched in hypoxic cells and is present in renal cell carcinoma tissues. These data place CA IX among the cell-surface signal transducers undergoing nuclear translocation. Accordingly, CA IX interactome involves also CAND1, which participates in both gene transcription and assembly of SCF ubiquitin ligase complexes. It is noteworthy that down-regulation of CAND1 leads to decreased CA IX protein levels apparently via affecting its stability. Our findings provide the first evidence that CA IX interacts with proteins involved in nuclear/cytoplasmic transport, gene transcription, and protein stability, and suggest the existence of nuclear CA IX protein subpopulations with a potential intracellular function, distinct from the crucial CA IX role at the cell surface. PMID:23181366
Buanne, Pasquale; Renzone, Giovanni; Monteleone, Francesca; Vitale, Monica; Monti, Simona Maria; Sandomenico, AnnaMaria; Garbi, Corrado; Montanaro, Donatella; Accardo, Marina; Troncone, Giancarlo; Zatovicova, Miriam; Csaderova, Lucia; Supuran, Claudiu T; Pastorekova, Silvia; Scaloni, Andrea; De Simone, Giuseppina; Zambrano, Nicola
Structure Determination of Membrane Proteins by Nuclear Magnetic Resonance Spectroscopy
Many biological membranes consist of 50% or more (by weight) membrane proteins, which constitute approximately one-third of all proteins expressed in biological organisms. Helical membrane proteins function as receptors, enzymes, and transporters, among other unique cellular roles. Additionally, most drugs have membrane proteins as their receptors, notably the superfamily of G protein�coupled receptors with seven transmembrane helices. Determining the structures of membrane proteins is a daunting task because of the effects of the membrane environment; specifically, it has been difficult to combine biologically compatible environments with the requirements for the established methods of structure determination. There is strong motivation to determine the structures in their native phospholipid bilayer environment so that perturbations from nonnatural lipids and phases do not have to be taken into account. At present, the only method that can work with proteins in liquid crystalline phospholipid bilayers is solid-state NMR spectroscopy.
Opella, Stanley J.
Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors.
We have developed an in vitro system involving digitonin-permeabilized vertebrate cells to study biochemical events in the transport of macromolecules across the nuclear envelope. While treatment of cultured cells with digitonin permeabilizes the plasma membranes to macromolecules, the nuclear envelopes remain structurally intact and nuclei retain the ability to transport and accumulate proteins containing the SV40 large T antigen nuclear location sequence. Transport requires addition of exogenous cytosol to permeabilized cells, indicating the soluble cytoplasmic factor(s) required for nuclear import are released during digitonin treatment. In this reconstituted import system, a protein containing a nuclear location signal is rapidly accumulated in nuclei, where it reaches a 30-fold concentration compared to the surrounding medium within 30 min. Nuclear import is specific for a functional nuclear location sequence, requires ATP and cytosol, and is temperature dependent. Furthermore, accumulation of the transport substrate within nuclei is completely inhibited by wheat germ agglutinin, which binds to nuclear pore complexes and inhibits transport in vivo. Together, these results indicate that the permeabilized cell system reproduces authentic nuclear protein import. In a preliminary biochemical dissection of the system, we observe that the sulfhydryl alkylating reagent N-ethylmaleimide inactivates both cytosolic factor(s) and also component(s) in the insoluble permeabilized cell fraction required for nuclear protein import. Because this permeabilized cell model is simple, efficient, and works effectively with cells and cytosol fractions prepared from a variety of different vertebrate sources, it will prove powerful for investigating the biochemical pathway of nuclear transport. PMID:2391365
Adam, S A; Marr, R S; Gerace, L
The Nuclear Shuttle Protein of Tomato Leaf Curl New Delhi Virus Is a Pathogenicity Determinant
The role of the movement protein (MP) and nuclear shuttle protein (NSP) in the pathogenicity of Tomato leaf curl New Delhi virus (ToLCNDV), a bipartite begomovirus, was studied. Both genes were expressed in Nicotiana benthamiana, Nicotiana tabacum, and Lycopersicon esculentum plants with the Potato virus X (PVX) expression vector or by stable transformation of gene constructs under the control of
Mazhar Hussain; Shahid Mansoor; Shazia Iram; Ayesha Naureen Fatima; Yusuf Zafar
A novel role for the nuclear membrane protein emerin in association of the centrosome to the outer nuclear membrane.
The type II inner nuclear membrane protein emerin is a component of the LINC complex that connects the nuclear lamina to the actin cytoskeleton. In emerin-null or -deficient human dermal fibroblasts we find that the centrosome is detached from the nucleus. Moreover, following siRNA knockdown of emerin in wild-type fibroblasts, the centrosome also becomes detached from the nucleus. We show that emerin interacts with tubulin, and that nocadozole-treated wild-type cells phenocopy the detached centrosome characteristic of emerin-null/deficient cells. We also find that a significant fraction of emerin is located at the outer nuclear membrane and peripheral ER, where it interacts directly with the centrosome. Our data provide the first evidence in mammalian cells as to the nature of the linkage of the centrosome, and therefore the tubulin cytoskeleton, with the outer nuclear membrane. PMID:17785515
Salpingidou, Georgia; Smertenko, Andrei; Hausmanowa-Petrucewicz, Irena; Hussey, Patrick J; Hutchison, Chris J
Nuclear domain 10-associated proteins recognize and segregate intranuclear DNA/protein complexes to negate gene expression
Background DNA viruses, such as herpes simplex virus type 1 (HSV-1), Simian virus 40 (SV40), and Cytomegaloviruses (CMV), start their replicative processes and transcription at specific nuclear domains known as ND10 (nuclear domain 10, also called PML bodies). It has been previously determined that for HSV-1 and SV40, a short DNA sequence and its binding protein are required and sufficient for cell localization of viral DNA replication and gene transcription. Results Our recent observations provide evidence that a foreign (not endogenous) DNA/protein complex in the nucleus recruits ND10 proteins. First, the complexes formed from the bacterial lac operator DNA and its binding protein (lac repressor), or from HPV11 (human papillomavirus 11) origin DNA and its binding protein (E2), co-localized with different ND10 proteins. Second, the HSV-1 amplicon without inserted lac operator DNA repeats distributed in the nucleus randomly, whereas the amplicon with lac operator DNA repeats associated with ND10, suggesting that DNA-binding proteins are required to localize at ND10. The cellular intrinsic DNA/protein complex (as detected for U2 DNA) showed no association with ND10. Furthermore, our examination of PML?/?, Daxx?/?, and Sp100-negative cells led to our discovering that DNA/protein complexes recruit ND10 protein independently. Using the GFP-LacI/Operator system, we were able to direct the transfected DNA to ND10 and found that gene expression was significantly repressed when the transfected DNA was directed to ND10. Conclusion Taken together, the results suggest that cells recognize DNA/protein complexes through a mechanism that involves interaction with the ND10-associated proteins.
A functional nuclear localization sequence in the VP1 capsid protein of coxsackievirus B3
The capsid proteins of some RNA viruses can translocate to the nucleus and interfere with cellular phenotypes. In this study we found that the VP1 capsid protein of coxsackievirus B3 (CVB3) was dominantly localized in the nucleus of the cells transfected with VP1-expressing plasmid. The VP1 nuclear localization also occurred in the cells infected with CVB3. Truncation analysis indicated that the VP1 nuclear localization sequence located near the C-terminal. The substitution of His220 with threonine completely abolished its translocation. The VP1 proteins of other CVB types might have the nuclear localization potential because this region was highly conserved. Moreover, the VP1 nuclear localization induced cell cycle deregulation, including a prolonged S phase and shortened G2-M phase. Besides these findings, we also found a domain between Ala72 and Phe106 that caused the VP1 truncates dotted distributed in the cytoplasm. Our results suggest a new pathogenic mechanism of CVB. - Highlights: Black-Right-Pointing-Pointer The VP1 protein of coxsackievirus B3 can specifically localize in the nucleus. Black-Right-Pointing-Pointer The nuclear localization signal of coxsackievirus B3 VP1 protein locates near its C-terminal. Black-Right-Pointing-Pointer The VP1 nuclear localization of coxsackievirus B3 can deregulate cell cycle. Black-Right-Pointing-Pointer There is a domain in the VP1 that determines it dotted distributed in the cytoplasm.
Wang, Tianying; Yu, Bohai; Lin, Lexun; Zhai, Xia; Han, Yelu; Qin, Ying; Guo, Zhiwei; Wu, Shuo; Zhong, Xiaoyan; Wang, Yan; Tong, Lei; Zhang, Fengmin; Si, Xiaoning [Department of Microbiology, Harbin Medical University, Harbin 150081 (China)] [Department of Microbiology, Harbin Medical University, Harbin 150081 (China); Zhao, Wenran, E-mail: email@example.com [Department of Cell Biology, Harbin Medical University, Harbin 150081 (China)] [Department of Cell Biology, Harbin Medical University, Harbin 150081 (China); Zhong, Zhaohua, E-mail: email@example.com [Department of Microbiology, Harbin Medical University, Harbin 150081 (China)] [Department of Microbiology, Harbin Medical University, Harbin 150081 (China)
Nesprins, but not Sun proteins, switch isoforms at the nuclear envelope during muscle development
Nesprins are a family of nuclear transmembrane proteins anchored via Sun proteins to the nuclear membrane. Analysis of nesprins during human muscle development revealed an increase in nesprin-1-giant during early myogenesis in vitro. During the transition from immature to mature muscle fibres in vivo, nesprin-2 partly replaced nesprin-1 at the nuclear envelope and short nesprin isoforms became dominant. Sun1 and Sun2 proteins remained unchanged during this fibre maturation. In emerin-negative skin fibroblasts, nesprin-2-giant was relocated from the nuclear envelope to the cytoplasm, not to the endoplasmic reticulum, while nesprin-1 remained at the nuclear envelope. In emerin-negative keratinocytes lacking nesprin-1, nesprin-2 remained at the nuclear envelope. HeLa cell nuclear envelopes lacked nesprin-1, which was the dominant form in myoblasts, while a novel 130kD nesprin-2 isoform dominated Ntera-2 cells. The results suggest the possibility of isoform-specific and tissue-specific roles for nesprins in nuclear positioning.
Randles, K. Natalie; Lam, Le Thanh; Sewry, Caroline A.; Puckelwartz, Megan; Furling, Denis; Wehnert, Manfred; McNally, Elizabeth M.; Morris, Glenn E.
Direct membrane protein-DNA interactions required early in nuclear envelope assembly
Among the earliest events in postmitotic nuclear envelope (NE) assembly are the interactions between chromatin and the membranes that will fuse to form the NE. It has been proposed that interactions between integral NE proteins and chromatin proteins mediate initial membrane recruitment to chromatin. We show that several transmembrane NE proteins bind to DNA directly and that NE membrane proteins as a class are enriched in long, basic domains that potentially bind DNA. Membrane fractions that are essential for NE formation are shown to bind directly to protein-free DNA, and our data suggest that these interactions are critical for early steps in NE assembly.
Ulbert, Sebastian; Platani, Melpomeni; Boue, Stephanie; Mattaj, Iain W.
Mitochondrial malate dehydrogenases in Brassica napus: altered protein patterns in different nuclear mitochondrial combinations
Two-dimensional analyses of mitochondrial proteins of Brassica napus revealed a set of differences in patterns of mitochondrial matrix proteins isolated from different nuclear backgrounds. One of these varying proteins was identified as mitochondrial malate dehydrogenase (mMDH; EC 18.104.22.168) by homology analyses of the partial amino acid sequence. Immunological detection identified additional mMDH subunits and detected different patterns of mMDH subunits
Uwe Witt; Renate L�hrs; Friedrich Buck; Kerstin Lembke; Marlies Gr�neberg-Seiler; Wolfgang Abel
Identification of a Functional, CRM-1-Dependent Nuclear Export Signal in Hepatitis C Virus Core Protein
Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. HCV core protein is involved in nucleocapsid formation, but it also interacts with multiple cytoplasmic and nuclear molecules and plays a crucial role in the development of liver disease and hepatocarcinogenesis. The core protein is found mostly in the cytoplasm during HCV infection, but also in the nucleus in patients with hepatocarcinoma and in core-transgenic mice. HCV core contains nuclear localization signals (NLS), but no nuclear export signal (NES) has yet been identified. We show here that the aa(109�133) region directs the translocation of core from the nucleus to the cytoplasm by the CRM-1-mediated nuclear export pathway. Mutagenesis of the three hydrophobic residues (L119, I123 and L126) in the identified NES or in the sequence encoding the mature core aa(1�173) significantly enhanced the nuclear localisation of the corresponding proteins in transfected Huh7 cells. Both the NES and the adjacent hydrophobic sequence in domain II of core were required to maintain the core protein or its fragments in the cytoplasmic compartment. Electron microscopy studies of the JFH1 replication model demonstrated that core was translocated into the nucleus a few minutes after the virus entered the cell. The blockade of nucleocytoplasmic export by leptomycin B treatment early in infection led to the detection of core protein in the nucleus by confocal microscopy and coincided with a decrease in virus replication. Our data suggest that the functional NLS and NES direct HCV core protein shuttling between the cytoplasmic and nuclear compartments, with at least some core protein transported to the nucleus. These new properties of HCV core may be essential for virus multiplication and interaction with nuclear molecules, influence cell signaling and the pathogenesis of HCV infection.
Cerutti, Andrea; Maillard, Patrick; Minisini, Rosalba; Vidalain, Pierre-Olivier; Roohvand, Farzin; Pecheur, Eve-Isabelle; Pirisi, Mario; Budkowska, Agata
Location of nuclear proteins on the chromosomes of newt oocytes
IN eukaryote cells chromosomal proteins are responsible for the organisational state of the DNA and the control of genetic expression, therefore knowledge of their location is an essential prerequisite for understanding chromosome function. Cytological localisation of chromosomal proteins is feasible using cells with giant chromosomes such as the oocytes of the newt Triturus cristatus carnifex where there are clearly defined
Sarah E. M. Scott; John Sommerville
Coordinate Nuclear Targeting of the FANCD2 and FANCI Proteins via a FANCD2 Nuclear Localization Signal
Fanconi anemia (FA) is a rare recessive disease, characterized by congenital defects, bone marrow failure, and increased cancer susceptibility. FA is caused by biallelic mutation of any one of sixteen genes. The protein products of these genes function cooperatively in the FA-BRCA pathway to repair DNA interstrand crosslinks (ICLs). A central step in the activation of this pathway is the monoubiquitination of the FANCD2 and FANCI proteins. Monoubiquitinated FANCD2 and FANCI localize to discrete chromatin regions where they function in ICL repair. Despite their critical role in ICL repair, very little is known about the structure, function, and regulation of the FANCD2 and FANCI proteins, or how they are targeted to the nucleus and chromatin. In this study, we describe the functional characterization of an amino-terminal FANCD2 nuclear localization signal (NLS). We demonstrate that the amino terminal 58 amino acids of FANCD2 can promote the nuclear expression of GFP and is necessary for the nuclear localization of FANCD2. Importantly, mutation of this FANCD2 NLS reveals that intact FANCD2 is required for the nuclear localization of a subset of FANCI. In addition, the NLS is necessary for the efficient monoubiquitination of FANCD2 and FANCI and, consequently, for their localization to chromatin. As a result, FANCD2 NLS mutants fail to rescue the ICL sensitivity of FA-D2 patient cells. Our studies yield important insight into the domain structure of the poorly characterized FANCD2 protein, and reveal a previously unknown mechanism for the coordinate nuclear import of a subset of FANCD2 and FANCI, a key early step in the cellular ICL response.
Boisvert, Rebecca A.; Rego, Meghan A.; Azzinaro, Paul A.; Mauro, Maurizio; Howlett, Niall G.
Identification and characterization of a nuclear scaffold protein that binds the matrix attachment region DNA.
The association of nuclear DNA with the nuclear matrix (scaffold) is mediated by defined segments of DNA called matrix association region (MAR). By using a plasmid harboring a portion of the Ig kappa gene within which MAR had been located, we searched for proteins recognizing MAR in the nuclear scaffold components electrophoretically separated and blotted onto a membrane. In the presence of nonspecific competitor DNA, the labeled plasmid selectively bound to a protein with apparent molecular weight of 120,000 (designated SP120). The protein was purified directly from SDS-polyacrylamide gels and renatured by a guanidine hydrochloride procedure. The DNA region in the plasmid responsible for the binding to the solubilized SP120 coincided with the 365-base pair HindIII-HinfI fragment that had been identified as MAR. In solution, SP120 exhibited a cooperative mode of interaction with the end-labeled MAR fragment. Measurement of relative affinities of MAR subfragments to SP120 showed that the whole region is required for efficient binding. This is consistent with the minimal length for MAR estimated thus far by in situ mapping experiments. The MAR derived from another gene, fushitarazu, also bound specifically to SP120. Immunostaining of whole cells and isolated nuclei with a monoclonal antibody raised against SP120 indicated that the protein is localized in a nuclear skeletal structure. These results suggest the involvement of SP120 in the MAR-mediated anchorage of nuclear DNA to the nuclear scaffold. PMID:8509422
Tsutsui, K; Tsutsui, K; Okada, S; Watarai, S; Seki, S; Yasuda, T; Shohmori, T
The roles of multiple importins for nuclear import of murine aristaless-related homeobox protein.
Nuclear import of proteins with nuclear localization signals (NLSs) is mediated by shuttling carriers, the importins. Some cargoes display more than a single NLS, and among these are homeodomain proteins such as Arx, which is critical for development of multiple tissues. Arx has two functional NLSs. The present studies show that several pathways can import Arx via its NLS2, which is within its DNA binding homeodomain. Using an in vitro nuclear import assay, we show that import of Arx via NLS2 can be mediated by importin beta1, importin 9, or importin 13, with binding being strongest to importin beta1. All binding is sensitive to RanGTP. Experiments based on precise domain deletions indicate that NLS2 binds impbeta1, imp9, and imp13 and includes both an importin binding subdomain and a regulatory subdomain with arginine residues being important for function. Moreover, Arx can be co-precipitated with these importins when NLS2 is present. Although nuclear import of Arx can be mediated by these three importin betas, importin beta1 seems to play the major role judging from in vivo small interfering RNA ablations and the in vitro import assay. This is the first evidence to show the role of importin beta1 in nuclear import of paired-type homeodomain proteins. We propose a novel and possibly quite general mechanism for nuclear import of paired-type homeodomain proteins which is critical for development. PMID:19494118
Lin, Wenbo; Ye, Wenduo; Cai, Lanlan; Meng, Xinyi; Ke, Guifen; Huang, Caoxin; Peng, Zi; Yu, Yinhua; Golden, Jeffrey A; Tartakoff, Alan M; Tao, Tao
Nuclear Import and the Evolution of a Multifunctional RNA-binding Protein
La (SS-B) is a highly expressed protein that is able to bind 3?-oligouridylate and other common RNA sequence/structural motifs. By virtue of these interactions, La is present in a myriad of nuclear and cytoplasmic ribonucleoprotein complexes in vivo where it may function as an RNA-folding protein or RNA chaperone. We have recently characterized the nuclear import pathway of the S. cerevisiae La, Lhp1p. The soluble transport factor, or karyopherin, that mediates the import of Lhp1p is Kap108p/Sxm1p. We have now determined a 113-amino acid domain of Lhp1p that is brought to the nucleus by Kap108p. Unexpectedly, this domain does not coincide with the previously identified nuclear localization signal of human La. Furthermore, when expressed in Saccharomyces cerevisiae, the nuclear localization of Schizosaccharomyces pombe, Drosophila, and human La proteins are independent of Kap108p. We have been able to reconstitute the nuclear import of human La into permeabilized HeLa cells using the recombinant human factors karyopherin ?2, karyopherin ?1, Ran, and p10. As such, the yeast and human La proteins are imported using different sequence motifs and dissimilar karyopherins. Our results are consistent with an intermingling of the nuclear import and evolution of La.
Rosenblum, Jonathan S.; Pemberton, Lucy F.; Bonifaci, Neris; Blobel, Gunter
Chronic cyclophosphamide exposure alters the profile of rat sperm nuclear matrix proteins.
Chronic exposure of male rats to the alkylating agent cyclophosphamide, a well-known male-mediated developmental toxicant, alters gene expression in male germ cells as well as in early preimplantation embryos sired by cyclophosphamide-exposed males. Sperm DNA is organized by the nuclear matrix into loop-domains in a sequence-specific manner. In somatic cells, loop-domain organization is involved in gene regulation. Various structural and functional components of the nuclear matrix are targets for chemotherapeutic agents. Consequently, we hypothesized that cyclophosphamide treatment would alter the expression of sperm nuclear matrix proteins. Adult male rats were treated for 4 wk with saline or cyclophosphamide (6.0 mg kg(-1) day(-1)), and the nuclear matrix was extracted from cauda epididymal sperm. Proteins were analyzed by two-dimensional gel electrophoresis. Identified proteins within the nuclear matrix proteome were mainly involved in cell structure, transcription, translation, DNA binding, protein processing, signal transduction, metabolism, cell defense, or detoxification. Interestingly, cyclophosphamide selectively induced numerous changes in cell defense and detoxification proteins, most notably, in all known forms of the antioxidant enzyme glutathione peroxidase 4, in addition to an uncharacterized 54-kDa form; an overall increase in glutathione peroxidase 4 immunoreactivity was observed in the nuclear matrix extracts from cyclophosphamide-exposed spermatozoa. An increase in glutathione peroxidase 4 expression suggests a role for this enzyme in maintaining nuclear matrix stability and function. These results led us to propose that a change in composition of the nuclear matrix in response to drug exposure was a factor in altered sperm function and embryo development. PMID:17475930
Codrington, Alexis M; Hales, Barbara F; Robaire, Bernard
Nuclear Import and Export of Venezuelan Equine Encephalitis Virus Nonstructural Protein 2?
Many RNA viruses, which replicate predominantly in the cytoplasm, have nuclear components that contribute to their life cycle or pathogenesis. We investigated the intracellular localization of the multifunctional nonstructural protein 2 (nsP2) in mammalian cells infected with Venezuelan equine encephalitis virus (VEE), an important, naturally emerging zoonotic alphavirus. VEE nsP2 localizes to both the cytoplasm and the nucleus of mammalian cells in the context of infection and also when expressed alone. Through the analysis of a series of enhanced green fluorescent protein fusions, a segment of nsP2 that completely localizes to the nucleus of mammalian cells was identified. Within this region, mutation of the putative nuclear localization signal (NLS) PGKMV diminished, but did not obliterate, the ability of the protein to localize to the nucleus, suggesting that this sequence contributes to the nuclear localization of VEE nsP2. Furthermore, VEE nsP2 specifically interacted with the nuclear import protein karyopherin-?1 but not with karyopherin-?2, -3, or -4, suggesting that karyopherin-?1 transports nsP2 to the nucleus during infection. Additionally, a novel nuclear export signal (NES) was identified, which included residues L526 and L528 of VEE nsP2. Leptomycin B treatment resulted in nuclear accumulation of nsP2, demonstrating that nuclear export of nsP2 is mediated via the CRM1 nuclear export pathway. Disruption of either the NLS or the NES in nsP2 compromised essential viral functions. Taken together, these results establish the bidirectional transport of nsP2 across the nuclear membrane, suggesting that a critical function of nsP2 during infection involves its shuttling between the cytoplasm and the nucleus.
Montgomery, Stephanie A.; Johnston, Robert E.
Characterization of the nuclear localization signal of the mouse TET3 protein
Highlights: �Amino acid sequence KKRK is responsible for nuclear localization of TET3. �Amino acid sequence KKRK are capable of targeting the cytoplasmic proteins to the nucleus. �Amino acid sequence KKRK are conserved in TET3 orthologs. -- Abstract: DNA demethylation is associated with gene activation and is mediated by a family of ten-eleven translocation (TET) dioxygenase. The TET3 protein is a 1668-amino-acid DNA demethylase that is predicted to possess five nuclear localization signals (NLSs). In this paper, we used a series of green fluorescent protein-tagged and mutation constructs to identify a conserved NLS (KKRK) embedded between amino acid 1615 and 1618 of mouse TET3. The KKRK sequence facilitates the cytoplasmic protein�s translocation into the nucleus. Additionally TET3 may be imported into the nucleus by importin-? and importin-?.
Xiao, Peng; Zhou, Xiao-long; Zhang, Hong-xiao; Xiong, Kai; Teng, Yun; Huang, Xian-ju; Cao, Rui; Wang, Yi; Liu, Hong-lin, E-mail: email@example.com
Identification of amino acid sequences in the polyomavirus capsid proteins that serve as nuclear localization signals
NASA Technical Reports Server (NTRS)
The molecular mechanism participating in the transport of newly synthesized proteins from the cytoplasm to the nucleus in mammalian cells is poorly understood. Recently, the nuclear localization signal sequences (NLS) of many nuclear proteins have been identified, and most have been found to be composed of a highly basic amino acid stretch. A genetic "subtractive" and a biochemical "additive" approach were used in our studies to identify the NLS's of the polyomavirus structural capsid proteins. An NLS was identified at the N-terminus (Ala1-Pro-Lys-Arg-Lys-Ser-Gly-Val-Ser-Lys-Cys11) of the major capsid protein VP1 and at the C-terminus (Glu307 -Glu-Asp-Gly-Pro-Glu-Lys-Lys-Lys-Arg-Arg-Leu318) of the VP2/VP3 minor capsid proteins.
Chang, D.; Haynes, J. I. Jr; Brady, J. N.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)
Tus, an E. coli protein, contains mammalian nuclear targeting and exporting signals.
Shuttling of proteins between nucleus and cytoplasm in mammalian cells is facilitated by the presence of nuclear localization signals (NLS) and nuclear export signals (NES), respectively. However, we have found that Tus, an E. coli replication fork arresting protein, contains separate sequences that function efficiently as NLS and NES in mammalian cell lines, as judged by cellular location of GFP-fusion proteins. The NLS was localized to a short stretch of 9 amino acids in the carboxy-terminus of Tus protein. Alterations of any of these basic amino acids almost completely abolished the nuclear targeting. The NES comprises a cluster of leucine/hydrophobic residues located within 21 amino acids at the amino terminus of Tus. Finally, we have shown that purified GFP-Tus fusion protein or GFP-Tus NLS fusion protein, when added to the culture media, was internalized very efficiently into mammalian cells. Thus, Tus is perhaps the first reported bacterial protein to possess both NLS and NES, and has the capability to transduce protein into mammalian cells. PMID:20126275
Kaczmarczyk, Stanislaw J; Sitaraman, Kalavathy; Hill, Thomas; Hartley, James L; Chatterjee, Deb K
Tus, an E. coli Protein, Contains Mammalian Nuclear Targeting and Exporting Signals
Shuttling of proteins between nucleus and cytoplasm in mammalian cells is facilitated by the presence of nuclear localization signals (NLS) and nuclear export signals (NES), respectively. However, we have found that Tus, an E. coli replication fork arresting protein, contains separate sequences that function efficiently as NLS and NES in mammalian cell lines, as judged by cellular location of GFP-fusion proteins. The NLS was localized to a short stretch of 9 amino acids in the carboxy-terminus of Tus protein. Alterations of any of these basic amino acids almost completely abolished the nuclear targeting. The NES comprises a cluster of leucine/hydrophobic residues located within 21 amino acids at the amino terminus of Tus. Finally, we have shown that purified GFP-Tus fusion protein or GFP-Tus NLS fusion protein, when added to the culture media, was internalized very efficiently into mammalian cells. Thus, Tus is perhaps the first reported bacterial protein to possess both NLS and NES, and has the capability to transduce protein into mammalian cells.
Kaczmarczyk, Stanislaw J.; Sitaraman, Kalavathy; Hill, Thomas; Hartley, James L.; Chatterjee, Deb K.
Signal- and importin-dependent nuclear targeting of the kidney anion exchanger 1-binding protein kanadaptin.
Kanadaptin (kidney anion exchanger adaptor protein) has recently been identified as a protein with binding activity to the cytoplasmic domain of the kidney Na(+)-independent Cl(-)/HCO(-)(3) anion exchanger 1 (kAE1). Since it is widely expressed in tissues devoid of kAE1, however, kanadaptin is likely to have additional cellular roles. This is supported by its multidomain structure, and possession of three clusters of basic amino acids exhibiting similarity to known nuclear localization sequences (NLSs). In the present study, we use immunofluorescence and subcellular fractionation approaches to demonstrate that kanadaptin is localized within the nuclei of various epithelial and non-epithelial cultured cell types. The role of the different NLSs is examined in transfection studies using plasmids encoding full-length kanadaptin with or without green fluorescent protein (GFP) as a fusion tag, as well as truncation derivatives thereof. Strong nuclear localization of fusion proteins containing amino acids 140-230 of kanadaptin, which include the sequence AVSRKRKA(193) (NLS1) was observed. Substitution of Arg(191) with a threonine residue resulted in a cytoplasmic location of the expressed protein, while NLS1 proved sufficient to target an otherwise cytoplasmically localized beta-galactosidase-GFP fusion protein to the nucleus. Using a direct binding assay we show that a fusion protein containing kanadaptin amino acids 1-231 (but not the Thr(191) substituted derivative) is recognized with nM affinity by the conventional NLS-binding importin alpha/beta heterodimer. Nuclear import studies on microinjected and permeabilized rat hepatoma cells demonstrated functionality of the NLS in nuclear targeting, with inhibition by antibodies demonstrating the requirement of both importin alpha and beta for nuclear import of kanadaptin. That kanadaptin possesses a functional importin-alpha/beta-recognized NLS explains the nuclear localization of kanadaptin in various cultured cell types, and opens up the possibility that kanadaptin may have a signalling role in the nucleus.
Hubner, Stefan; Jans, David A; Xiao, Chong-Yun; John, Anna P; Drenckhahn, Detlev
0610009K11Rik, a testis-specific and germ cell nuclear receptor-interacting protein
Using an in silico approach, a putative nuclear receptor-interacting protein 0610009K11Rik was identified in mouse testis. We named this gene as testis-specific nuclear receptor-interacting protein-1 (Tnrip-1). Tnrip-1 was predominantly expressed in the testis of adult mouse tissues. Expression of Tnrip-1 in the testis was regulated during postnatal development, with robust expression in 14-day-old or older testes. In situ hybridization analyses showed that Tnrip-1 is highly expressed in pachytene spermatocytes and spermatids. Consistent with its mRNA expression, Tnrip-1 protein was detected in adult mouse testes. Immunohistochemical studies showed that Tnrip-1 is a nuclear protein and mainly expressed in pachytene spermatocytes and round spermatids. Moreover, co-immunoprecipitation analyses showed that endogenous Tnrip-1 protein can interact with germ cell nuclear receptor (GCNF) in adult mouse testes. Our results suggest that Tnrip-1 is a testis-specific and GCNF-interacting protein which may be involved in modulation of GCNF-mediated gene transcription in spermatogenic cells within the testis.
Zhang, Heng; Denhard, Leslie A.; Zhou, Huaxin; Liu, Lan-Hsin; Lan, Zi-Jian
KDP-1 is a nuclear envelope KASH protein required for cell-cycle progression
Summary Klarsicht, ANC-1 and Syne homology (KASH) proteins localize to the outer nuclear membrane where they connect the nucleus to the cytoskeleton. KASH proteins interact with Sad1-UNC-84 (SUN) proteins to transfer forces across the nuclear envelope to position nuclei or move chromosomes. A new KASH protein, KDP-1, was identified in a membrane yeast two-hybrid screen of a Caenorhabditis elegans library using the SUN protein UNC-84 as bait. KDP-1 also interacted with SUN-1. KDP-1 was enriched at the nuclear envelope in a variety of tissues and required SUN-1 for nuclear envelope localization in the germline. Genetic analyses showed that kdp-1 was essential for embryonic viability, larval growth and germline development. kdp-1(RNAi) delayed the entry into mitosis in embryos, led to a small mitotic zone in the germline, and caused an endomitotic phenotype. Aspects of these phenotypes were similar to those seen in sun-1(RNAi), suggesting that KDP-1 functions with SUN-1 in the germline and early embryo. The data suggest that KDP-1 is a novel KASH protein that functions to ensure the timely progression of the cell cycle between the end of S phase and the entry into mitosis.
McGee, Matthew D.; Stagljar, Igor; Starr, Daniel A.
Studies in protein dynamics using heteronuclear nuclear magnetic resonance spectroscopy
NASA Astrophysics Data System (ADS)
Dynamic processes in proteins are important for their biological function. Several issues in protein dynamics are addressed by applying existing NMR methodologies to investigate dynamics of several small proteins. Amide H/D exchange rates have been measured for the N-terminal domain of the ribosomal protein L9, residues 1--56. The results suggest that the structure of the domain is preserved in isolation and that the stability of the isolated domain is comparable to the stability of this domain in intact L9. Single domain proteins can fold in vitro at rates in excess of 1 x 104 s-1. Measurement of folding rates of this magnitude poses a considerable technical challenge. Off-resonance 15N R1rho measurements are shown to be capable of measuring such fast protein folding rates. The measurements were performed on a sample of the peripheral subunit-binding domain from the dihydrolopoamide acetyltransferase component of the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus 15N labeled at Ala 11. Fast intramolecular motions (on ps-ns time scale) can be studied by heteronuclear laboratory frame NMR relaxation. The temperature dependence of the backbone dynamics of the 36-resiude subdomain of the F-actin bundling protein villin has been investigated by studying the temperature dependence of order parameters obtained from 15N relaxation measurements. The results support the hypothesis that one of the possible mechanisms of thermostability is to lower the heat capacity difference between the folded and unfolded states by lowering the contribution from the backbone dynamics. A commonly used model-free approach for the interpretation of the relaxation data for macromolecules in solution is modified to correct for the decoupling approximation between the overall and internal motions.
Vugmeyster, Liliya
Xenopus in vitro assays to analyze the function of transmembrane nucleoporins and targeting of inner nuclear membrane proteins.
Xenopus egg extracts have been widely used to study cell cycle regulation and to analyze mitotic or nuclear processes on a biochemical level. Most instrumental, proteins of interest can be immunodepleted by specific antibodies. However, this approach has been restricted to non-membrane proteins, which limits its versatility especially when studying membrane-dependent processes such as nuclear envelope reformation at the end of mitosis or nuclear pore complex assembly. We describe here the methods developed and used in our laboratory to specifically remove transmembrane proteins from endogenous membranes and to insert recombinant integral membrane proteins into endogenous membranes. The latter procedure is important not only for readdition of a depleted protein in rescue experiments but also for introducing artificial membrane proteins such as reporters to investigate the passage of inner nuclear membrane proteins through nuclear pore complexes. PMID:24857731
Eisenhardt, Nathalie; Schooley, Allana; Antonin, Wolfram
Multidimensional profiling of cell surface proteins and nuclear markers
Cell membrane proteins play an important role in tissue architecture and cell-cell communication. We hypothesize that segmentation and multidimensional characterization of the distribution of cell membrane proteins, on a cell-by-cell basis, enable improved classification of treatment groups and identify important characteristics that can otherwise be hidden. We have developed a series of computational steps to (i) delineate cell membrane protein signals and associate them with a specific nucleus; (ii) compute a coupled representation of the multiplexed DNA content with membrane proteins; (iii) rank computed features associated with such a multidimensional representation; (iv) visualize selected features for comparative evaluation through heatmaps; and (v) discriminate between treatment groups in an optimal fashion. The novelty of our method is in the segmentation of the membrane signal and the multidimensional representation of phenotypic signature on a cell-by-cell basis. To test the utility of this method, the proposed computational steps were applied to images of cells that have been irradiated with different radiation qualities in the presence and absence of other small molecules. These samples are labeled for their DNA content and E-cadherin membrane proteins. We demonstrate that multidimensional representations of cell-by-cell phenotypes improve predictive and visualization capabilities among different treatment groups, and identify hidden variables.
Han, Ju; Chang, Hang; Andarawewa, Kumari; Yaswen, Paul; Helen Barcellos-Hoff, Mary; Parvin, Bahram
CGGBP1 is a nuclear and midbody protein regulating abscission
Abscission marks the completion of cell division and its failure is associated with delayed cytokinesis and even tetraploidization. Aberrant abscission and consequential ploidy changes can underlie various diseases including cancer. Midbody, a transient structure formed in the intercellular bridge during telophase, contains several proteins including Aurora kinase B (AURKB), which participate in abscission. We report here an unexpected expression pattern and function of the transcription repressor protein CGG triplet repeat-binding protein 1 (CGGBP1), in normal human fibroblasts. We show that CGGBP1, a chromatin-associated protein, trans-localizes to spindle midzone and midbodies in a manner similar to that of AURKB. CGGBP1 depletion resulted in a cell cycle block at G2, characterized by failure of cells to undergo mitosis and also reduced entry into S phase. Consistent with its presence in the midbodies, live microscopy showed that CGGBP1 deficiency caused mitotic failure at abscission resulting in tetraploidy, which could be rescued by CGGBP1 overexpression. These results show that CGGBP1 is a bona fide midbody protein required for normal abscission and mitosis in general.
Singh, Umashankar, E-mail: firstname.lastname@example.com; Westermark, Bengt
Identification and characterisation of a nuclear localisation signal in the SMN associated protein, Gemin4.
Gemin4 is a ubiquitously expressed multifunctional protein that is involved in U snRNP assembly, apoptosis, nuclear/cytoplasmic transportation, transcription, and RNAi pathways. Gemin4 is one of the core components of the Gemin-complex, which also contains survival motor neuron (SMN), the seven Gemin proteins (Gemin2-8), and Unrip. Mutations in the SMN1 gene cause the autosomal recessive disorder spinal muscular atrophy (SMA). Although the functions assigned to Gemin4 predominantly occur in the nucleus, the mechanisms that mediate the nuclear import of Gemin4 remain unclear. Here, using a novel panel of Gemin4 constructs we identify a canonical nuclear import sequence (NLS) in the N-terminus of Gemin4. The Gemin4 NLS is necessary and independently sufficient to mediate nuclear import of Gemin4. This is the first functional NLS identified within the SMN-Gemin complex. PMID:18675250
Lorson, Monique A; Dickson, Alexa M; Shaw, Debra J; Todd, Adrian G; Young, Elizabeth C; Morse, Robert; Wolstencroft, Catherine; Lorson, Christian L; Young, Philip J
Identification and characterisation of a nuclear localisation signal in the SMN associated protein, Gemin4
Gemin4 is a ubiquitously expressed multifunctional protein that is involved in U snRNP assembly, apoptosis, nuclear/cytoplasmic transportation, transcription, and RNAi pathways. Gemin4 is one of the core components of the Gemin-complex, which also contains survival motor neuron (SMN), the seven Gemin proteins (Gemin2-8), and Unrip. Mutations in the SMN1 gene cause the autosomal recessive disorder spinal muscular atrophy (SMA). Although the functions assigned to Gemin4 predominantly occur in the nucleus, the mechanisms that mediate the nuclear import of Gemin4 remain unclear. Here, using a novel panel of Gemin4 constructs we identify a canonical nuclear import sequence (NLS) in the N-terminus of Gemin4. The Gemin4 NLS is necessary and independently sufficient to mediate nuclear import of Gemin4. This is the first functional NLS identified within the SMN-Gemin complex.
Lorson, Monique A.; Dickson, Alexa M. [Department of Veterinary Pathobiology, Bond Life Sciences Center, 1201 Rollins Road, University of Missouri, Columbia, MO 65211 (United States); Shaw, Debra J.; Todd, Adrian G. [Institute of Biomedical and Clinical Neurobiology, IBCS, Peninsula College of Medicine and Dentistry, St. Luke's Campus, Exeter, EX1 2LU (United Kingdom); Young, Elizabeth C. [Department of Veterinary Pathobiology, Bond Life Sciences Center, 1201 Rollins Road, University of Missouri, Columbia, MO 65211 (United States); Morse, Robert; Wolstencroft, Catherine [Institute of Biomedical and Clinical Neurobiology, IBCS, Peninsula College of Medicine and Dentistry, St. Luke's Campus, Exeter, EX1 2LU (United Kingdom); Lorson, Christian L. [Department of Veterinary Pathobiology, Bond Life Sciences Center, 1201 Rollins Road, University of Missouri, Columbia, MO 65211 (United States); Young, Philip J. [Institute of Biomedical and Clinical Neurobiology, IBCS, Peninsula College of Medicine and Dentistry, St. Luke's Campus, Exeter, EX1 2LU (United Kingdom)], E-mail: firstname.lastname@example.com
Interactions and three-dimensional localization of a group of nuclear pore complex proteins
We have used antibodies directed against a number of nuclear pore complex (NPC) proteins to determine their mutual interactions and location within the three-dimensional structure of the NPC. A mono- clonal antibody, termed QE5, recognized three NPC polypeptides, p250, NUP153, and p62 on Western blots, and labeled the nuclear envelope of several cul- tured cell lines by immunofluorescence microscopy. These
N. Pante; Ricardo Bastos; Isabel McMorrow; Brian Burke; Ueli Aebi
A viral movement protein as a nuclear shuttle. The geminivirus BR1 movement protein contains domains essential for interaction with BL1 and nuclear localization.
For the nuclear replicating bipartite geminiviruses such as squash leaf curl to systemically infect the host requires the active participation of two virus-encoded movement proteins, BR1 and BL1. These act in a cooperative manner to transport the viral single-stranded DNA genome from its site of replication in the nucleus to the cell periphery (A.A. Sanderfoot, S.G. Lazarowitz [1995] Plant Cell 7: 1185-1194). We have proposed that BR1 functions as a nuclear shuttle protein, transporting the viral single-stranded DNA to and from the nucleus as a complex that is recognized by BL1 for movement to adjacent cells. To further investigate this, we expressed BR1 mutants known to affect viral infectivity in Spodoptera frugiperda insect cells and Nicotiana tabacum L. cv Xanthi protoplasts and found these to be defective in either their nuclear targeting or their ability to be redirected to the cell periphery when co-expressed with BL1. Translational fusions to beta-glucuronidase and alanine-scanning mutagenesis further demonstrated that the C-terminal 86 amino acids of BR1 contains a domain(s) essential for its interaction with BL1 and identified two nuclear localization signals within the N-terminal 113 residues of BR1. These nuclear localization signals were precisely located within distinct 16- and 22-peptide segments of BR1. These studies support and extend our model for squash leaf curl virus movement, showing that BR1 has a domain structure, with an N-terminal region required for nuclear targeting and a C-terminal region required for its interaction with BL1.
Sanderfoot, A A; Ingham, D J; Lazarowitz, S G
A novel heterogeneous nuclear RNP protein with a unique distribution on nascent transcripts
Immediately after the initiation of transcription in eukaryotes, nascent RNA polymerase II transcripts are bound by nuclear proteins resulting in the formation of heterogeneous nuclear ribonucleoprotein (hnRNP) complexes. hnRNP complexes from HeLa cell nuclei contain greater than 20 major proteins in the molecular mass range of 34,000- 120,000 D. Among these are the previously described A, B, and C groups of proteins (34,000-43,000 D) and several larger, and as yet uncharacterized, proteins. Here we describe the isolation and characterization of a novel hnRNP protein termed the L protein (64-68 kD by mobility in SDS-polyacrylamide gels). Although L is a bona fide component of hnRNP complexes, it also appears to be a different type of hnRNP protein from those previously characterized. A considerable amount of L is found outside hnRNP complexes, and monoclonal antibodies to the L protein also strongly stain unidentified discrete nonnucleolar structures, in addition to nucleoplasm, in HeLa cell nuclei. Interestingly, the same antibodies stain the majority of nonnucleolar nascent transcripts from the loops of lampbrush chromosomes in the newt, but the most intense staining is localized to the landmark giant loops. The L protein is the first protein of giant loops identified so far, and antibodies to it thus provide a useful tool with which to study these unique RNAs. In addition, isolation and sequencing of cDNA clones for the L protein from human cells predicts a glycine- and proline-rich protein of 60,187 D, which contains two 80 amino acid segments only distantly related to the RNP consensus sequence-type RNA- binding domain. The L protein, therefore, is a new type of hnRNP protein.
Expression of a novel nuclear protein in activated and in tat-I expressing T cells.
The intracellular events that occur in T lymphoid cells after activation or after infection with HIV-1 are not well defined. In the case of HIV-1 infection, it is unknown whether the tat-I gene, an essential gene for viral replication, affects host cell nuclear factors. Using two-dimensional PAGE, we have identified a novel nuclear protein, designated nuclear protein-28,000 (NP-28), which is induced in Jurkat T cells by stimulation with PMA and/or PHA or ionomycin. This nuclear protein has an apparent molecular mass of 28,000 Da and an isoelectric point of 4.6. Interestingly, Jurkat cells transfected with tat-I express higher levels of NP-28 constitutively, without added stimulation. Incubation of Jurkat cells expressing tat-I with PMA and/or PHA or ionomycin causes superinduction of NP-28. We have therefore identified a novel lymphoid nuclear protein induced by T cell activation that occurs in tat-I expressing cells in the absence of activating agents. PMID:1988491
Bielinska, A; Baier, L; Hailat, N; Strahler, J R; Nabel, G J; Hanash, S
An improved genetic system for detection and analysis of protein nuclear import signals
Background Nuclear import of proteins is typically mediated by their physical interaction with soluble cytosolic receptor proteins via a nuclear localization signal (NLS). A simple genetic assay to detect active NLSs based on their function in the yeast Saccharomyces cerevisiae has been previously described. In that system, a chimera consisting of a modified bacterial LexA DNA binding domain and the transcriptional activation domain of the yeast Gal4 protein is fused to a candidate NLS. A functional NLS will redirect the chimeric fusion to the yeast cell nucleus and activate transcription of a reporter gene. Results We have reengineered this nuclear import system to expand its utility and tested it using known NLS sequences from adenovirus E1A. Firstly, the vector has been reconstructed to reduce the level of chimera expression. Secondly, an irrelevant "stuffer" sequence from the E. coli maltose binding protein was used to increase the size of the chimera above the passive diffusion limit of the nuclear pore complex. The improved vector also contains an expanded multiple cloning site and a hemagglutinin epitope tag to allow confirmation of expression. Conclusion The alterations in expression level and composition of the fusions used in this nuclear import system greatly reduce background activity in ?-galactosidase assays, improving sensitivity and allowing more quantitative analysis of NLS bearing sequences.
Marshall, Kris S; Zhang, Zhiying; Curran, Jennifer; Derbyshire, Stephanie; Mymryk, Joe S
Functional Activity of the Fanconi Anemia Protein FAA Requires FAC Binding and Nuclear Localization
Fanconi anemia (FA) is an autosomal recessive disease characterized by genomic instability, cancer susceptibility, and cellular hypersensitivity to DNA-cross-linking agents. Eight complementation groups of FA (FA-A through FA-H) have been identified. Two FA genes, corresponding to complementation groups FA-A and FA-C, have been cloned, but the functions of the encoded FAA and FAC proteins remain unknown. We have recently demonstrated that FAA and FAC interact to form a nuclear complex. In this study, we have analyzed a series of mutant forms of the FAA protein with respect to functional activity, FAC binding, and nuclear localization. Mutation or deletion of the amino-terminal nuclear localization signal (NLS) of FAA results in loss of functional activity, loss of FAC binding, and cytoplasmic retention of FAA. Replacement of the NLS sequence with a heterologous NLS sequence, derived from the simian virus 40 T antigen, results in nuclear localization but does not rescue functional activity or FAC binding. Nuclear localization of the FAA protein is therefore necessary but not sufficient for FAA function. Mutant forms of FAA which fail to bind to FAC also fail to promote the nuclear accumulation of FAC. In addition, wild-type FAC promotes the accumulation of wild-type FAA in the nucleus. Our results suggest that FAA and FAC perform a concerted function in the cell nucleus, required for the maintenance of chromosomal stability.
Naf, Dieter; Kupfer, Gary M.; Suliman, Ahmed; Lambert, Kathleen; D'Andrea, Alan D.
Identification of unique SUN-interacting nuclear envelope proteins with diverse functions in plants.
Although a plethora of nuclear envelope (NE) transmembrane proteins (NETs) have been identified in opisthokonts, plant NETs are largely unknown. The only known NET homologues in plants are Sad1/UNC-84 (SUN) proteins, which bind Klarsicht/ANC-1/Syne-1 homology (KASH) proteins. Therefore, de novo identification of plant NETs is necessary. Based on similarities between opisthokont KASH proteins and the only known plant KASH proteins, WPP domain-interacting proteins, we used a computational method to identify the KASH subset of plant NETs. Ten potential plant KASH protein families were identified, and five candidates from four of these families were verified for their NE localization, depending on SUN domain interaction. Of those, Arabidopsis thaliana SINE1 is involved in actin-dependent nuclear positioning in guard cells, whereas its paralogue SINE2 contributes to innate immunity against an oomycete pathogen. This study dramatically expands our knowledge of plant KASH proteins and suggests that plants and opisthokonts have recruited different KASH proteins to perform NE regulatory functions. PMID:24891605
Zhou, Xiao; Graumann, Katja; Wirthmueller, Lennart; Jones, Jonathan D G; Meier, Iris
NP-40 reduces contamination by endogenous biotinylated carboxylases during purification of biotin tagged nuclear proteins.
We describe here a simple procedure for greatly reducing contamination of nuclear extracts by naturally biotinylated cytoplasmic carboxylases, which represent a major source of non-specific background when employing BirA-mediated biotinylation tagging for the purification and characterization of nuclear protein complexes by mass spectrometry. We show that the use of 0.5% of the non-ionic detergent Nonidet-40 (NP-40) during cell lysis and nuclei isolation is sufficient to practically eliminate contamination of nuclear extracts by carboxylases and to greatly reduce background signals in downstream mass spectrometric analyses. PMID:23500724
Papageorgiou, Dimitris N; Demmers, Jeroen; Strouboulis, John
RNF38 encodes a nuclear ubiquitin protein ligase that modifies p53
Highlights: �RNF38 is shown to be a nuclear protein with a bipartite nuclear localization signal. �RNF38 protein is purified and shown to have ubiquitin protein ligase (E3) activity. �We show that RNF38 binds p53 and can ubiquitinate p53 in vitro. �Overexpression of RNF38 increases p53 ubiquitination in HEK293T cells. �Overexpression of RNF38 in HEK293T cells alters p53 localization. -- Abstract: The RNF38 gene encodes a RING finger protein of unknown function. Here we demonstrate that RNF38 is a functional ubiquitin protein ligase (E3). We show that RNF38 isoform 1 is localized to the nucleus by a bipartite nuclear localization sequence (NLS). We confirm that RNF38 is a binding partner of p53 and demonstrate that RNF38 can ubiquitinate p53 in vitro and in vivo. Finally, we show that overexpression of RNF38 in HEK293T cells results in relocalization of p53 to discrete foci associated with PML nuclear bodies. These results suggest RNF38 is an E3 ubiquitin ligase that may play a role in regulating p53.
Sheren, Jamie E. [Department of Pathology, The University of Colorado Anschutz Medical Campus, Aurora, CO 80045 (United States)] [Department of Pathology, The University of Colorado Anschutz Medical Campus, Aurora, CO 80045 (United States); Kassenbrock, C. Kenneth, E-mail: email@example.com [Department of Pathology, The University of Colorado Anschutz Medical Campus, Aurora, CO 80045 (United States); Department of Biology, Colorado State University, Fort Collins, CO 80523-1878 (United States)
Nuclear transport requires freely diffusing nuclear transport proteins to facilitate movement of cargo molecules through the nuclear pore. We analyzed dynamic properties of importin ?, importin ?, Ran and NTF2 in nucleus, cytoplasm and at the nuclear pore of neuroblastoma cells using fluorescence correlation spectroscopy. Mobile components were quantified by global fitting of autocorrelation data from multiple cells. Immobile components were quantified by analysis of photobleaching kinetics. Wild type Ran was compared to various mutant Ran proteins to identify components representing GTP or GDP forms of Ran. Untreated cells were compared to cells treated with nocodazole or latrunculin to identify components associated with cytoskeletal elements. The results indicate that freely diffusing importin ?, importin ?, Ran and NTF2 are in dynamic equilibrium with larger pools associated with immobile binding partners such as microtubules in the cytoplasm. These findings suggest that formation of freely diffusing nuclear transport intermediates is in competition with binding to immobile partners. Variation in concentrations of freely diffusing nuclear transport intermediates among cells indicates that the nuclear transport system is sufficiently robust to function over a wide range of conditions.
Quantitative phosphoproteomic profiling of PINK1-deficient cells identifies phosphorylation changes in nuclear proteins.
The Parkinson's disease (PD) associated gene PINK1 encodes a protein kinase that mediates the phosphorylation of multiple proteins involved in mitochondrial homeostasis. The broader downstream signaling events mediated by PINK1 kinase activity have not been well documented. We combine quantitative phosphoproteomic strategies with siRNA mediated PINK1 knock down in mammalian cells to identify alterations of phosphorylation events downstream of PINK1. Although down-regulation of PINK1 has no major effect on the proteome expression in these cells, phosphorylation of over one hundred proteins was reduced reflecting basal levels of phosphorylation signaling events downstream of PINK1. Motif analysis of the residues flanking the phosphorylation sites indicates proline-directed kinase specificity. Surprisingly, we found that the downstream signaling nodes included many transcription factors, as well as nuclear proteins involved in DNA and RNA metabolism. Thus, PINK1 dependent phosphorylation signaling may regulate nuclear activities. PMID:24626860
Qin, Xiaoyan; Zheng, Chaoya; Yates Iii, John R; Liao, Lujian
The Drosophila nuclear lamina protein Otefin is required for germline stem cell survival
Summary LEM domain (LEM-D) proteins are components of an extensive protein network that assembles beneath the inner nuclear envelope. Defects in LEM-D proteins cause tissue-restricted human diseases associated with altered stem cell homeostasis. Otefin (Ote) is a Drosophila LEM-D protein that is intrinsically required for female germline stem cells (GSCs) maintenance. Previous studies linked Ote loss with transcriptional activation of the key differentiation gene, bag-of-marbles (bam), leading to the model that Ote tethers the bam gene to the nuclear periphery for gene silencing. Using genetic and phenotypic analyses of multiple ote?/? backgrounds, we obtained evidence that is inconsistent with this model. We show that bam repression is maintained in ote?/? GSCs and that germ cell loss persists in ote?/?, bam?/? mutants, together demonstrating that GSC loss is independent of bam transcription. We show the primary defect in ote?/? GSCs is a block of differentiation, which ultimately leads to germ cell death.
Barton, Lacy J.; Pinto, Belinda S.; Wallrath, Lori L.; Geyer, Pamela K.
One hour after the injection of 5 mu c of L-lysine-U-C¹⁴ into ; each of a group of rats bearing the Walker 256 carcinosarcoma, the acid-soluble ; proteins were extracted from nuclear preparations of the tumor. The proteins of ; these extracts were chromatographed on carboxymethylcellulose, with formic acid ; as the eluting agent. Rechromatography of 150 mg of RP2-L¹
H. Busch; L. S. Hnilica; S. Chien; J. R. Davis; C. W. Taylor
Identification and characterisation of a nuclear localisation signal in the SMN associated protein, Gemin4
Gemin4 is a ubiquitously expressed multifunctional protein that is involved in U snRNP assembly, apoptosis, nuclear\\/cytoplasmic transportation, transcription, and RNAi pathways. Gemin4 is one of the core components of the Gemin-complex, which also contains survival motor neuron (SMN), the seven Gemin proteins (Gemin2�8), and Unrip. Mutations in the SMN1 gene cause the autosomal recessive disorder spinal muscular atrophy (SMA). Although
Monique A. Lorson; Alexa M. Dickson; Debra J. Shaw; Adrian G. Todd; Elizabeth C. Young; Robert Morse; Catherine Wolstencroft; Christian L. Lorson; Philip J. Young
Identification of the Proteins of the Yeast U1 Small Nuclear Ribonucleoprotein Complex by Mass Spectrometry
Here we report the rapid identification of the proteins of the spliceosomal U1 small nuclear ribonucleoprotein (snRNP) from the yeast Saccharomyces cerevisiae by searching mass spectrometric data in genomic sequence databases. The U1 snRNP, containing a histidine-tagged 70K protein, was isolated from cell extracts by anti m3G-cap immunoaffinity and subsequent nickel nitrilotriacetic acid chromatography. A U1 snRNP fraction containing 20
Gitte Neubauer; Alexander Gottschalk; Patrizia Fabrizio; Bertrand Seraphin; Reinhard Luhrmann; Matthias Mann
Evidence for two-step processing of nuclear-encoded chloroplast proteins during membrane assembly
A plastome (chloroplast genome) mutant of tobacco, lutescens-1, displays abnormal degradation of the chloroplast-encoded polypeptides which form the core complex of photosystem II (PSI)). Two nuclear- encoded proteins (present in polymorphic forms), which normally function in the water oxidation pro- cess of PSII, accumulate as larger size-class polypep- tides in mutant thylakoid membranes. These accumu- lated proteins are intermediate in
Catherine E Chia; Charles J. Amtzen
Expanded Polyglutamine Protein Forms Nuclear Inclusions and Causes Neural Degeneration in Drosophila
Spinocerebellar ataxia type 3 (SCA3\\/MJD) is one of at least eight human neurodegenerative diseases caused by glutamine-repeat expansion. We have recreated glutamine-repeat disease in Drosophila using a segment of the SCA3\\/MJD protein. Targeted expression of the protein with an expanded polyglutamine repeat led to nuclear inclusion (NI) formation and late-onset cell degeneration. Differential sensitivity to the mutant transgene was observed
John M. Warrick; Henry L. Paulson; Gladys L. Gray-Board; Quang T. Bui; Kenneth H. Fischbeck; Randall N. Pittman; Nancy M. Bonini
Identification of Nuclear Protein Targets for Six Leukemogenic Tyrosine Kinases Governed by Post-Translational Regulation
Mutated tyrosine kinases are associated with a number of different haematological malignancies including myeloproliferative disorders, lymphoma and acute myeloid leukaemia. The potential commonalities in the action of six of these leukemogenic proteins on nuclear proteins were investigated using systematic proteomic analysis. The effects on over 3600 nuclear proteins and 1500 phosphopeptide sites were relatively quantified in seven isogenic cell lines. The effects of the kinases were diverse although some commonalities were found. Comparison of the nuclear proteomic data with transcriptome data and cytoplasmic proteomic data indicated that the major changes are due to post-translational mechanisms rather than changes in mRNA or protein distribution. Analysis of the promoter regions of genes whose protein levels changed in response to the kinases showed the most common binding site found was that for NF?B whilst other sites such as those for the glucocorticoid receptor were also found. Glucocorticoid receptor levels and phosphorylation were decreased by all 6 PTKs. Whilst Glucocorticoid receptor action can potentiate NF?B action those proteins where genes have NF?B binding sites were in often regulated post-translationally. However all 6 PTKs showed evidence of NFkB pathway modulation via activation via altered IkB and NFKB levels. Validation of a common change was also undertaken with PMS2, a DNA mismatch repair protein. PMS2 nuclear levels were decreased in response to the expression of all 6 kinases, with no concomitant change in mRNA level or cytosolic protein level. Response to thioguanine, that requires the mismatch repair pathway, was modulated by all 6 oncogenic kinases. In summary common targets for 6 oncogenic PTKs have been found that are regulated by post-translational mechanisms. They represent potential new avenues for therapies but also demonstrate the post-translational regulation is a key target of leukaemogenic kinases.
Pierce, Andrew; Williamson, Andrew; Jaworska, Ewa; Griffiths, John R.; Taylor, Sam; Walker, Michael; O'Dea, Mark Aspinall; Spooncer, Elaine; Unwin, Richard D.; Poolman, Toryn; Ray, David; Whetton, Anthony D.
Identification of a Classical Bipartite Nuclear Localization Signal in the Drosophila TEA/ATTS Protein Scalloped
Drosophila melanogaster wing development has been shown to rely on the activity of a complex of two proteins, Scalloped (Sd) and Vestigial (Vg). Within this complex, Sd is known to provide DNA binding though its TEA/ATTS domain, while Vg modulates this binding and provides transcriptional activation through N- and C-terminal activation domains. There is also evidence that Sd is required for the nuclear translocation of Vg. Indeed, a candidate sequence which shows consensus to the bipartite family of nuclear localization signals (NLSs) has been identified within Sd previously, though it is not known if it is functional, or if additional unpredicted signals that mediate nuclear transport exist within the protein. By expressing various enhanced green fluorescent protein (eGFP) tagged constructs within Drosophila S2 cells, we demonstrate that this NLS is indeed functional and necessary for the proper nuclear localization of Sd. Additionally, the region containing the NLS is critical for the wildtype function of ectopically expressed Sd, in the context of wing development. Using site-directed mutagenesis, we have identified a group of five amino acids within this NLS which is critical for its function, as well as another group of two which is of lesser importance. Together with data that suggests that this sequence mediates interactions with Importin-?3, we conclude that the identified NLS is likely a classical bipartite signal. Further dissection of Sd has also revealed that a large portion of the C-terminal domain of the protein is required its proper nuclear localization. Finally, a Leptomycin B (LB) sensitive signal which appears to facilitate nuclear export is identified, raising the possibility that Sd also contains a nuclear export signal (NES).
Magico, Adam C.; Bell, John B.
R7BP: A Surprising New Link Between G Proteins, RGS Proteins, and Nuclear Signaling in the Brain
NSDL National Science Digital Library
The regulators of G protein signaling (RGS proteins) bind directly to G protein alpha (Gα) subunits in brain and other tissues to determine the strength, duration, and fidelity of neurotransmitter receptor signaling. A recent study shows, quite unexpectedly, that one class of RGS proteins [the R7 subfamily bound to Gβ5 (R7-Gβ5)] shuttles between the plasma membrane and the nucleus with assistance from a novel shuttle protein, R7BP. R7BP binds directly to R7-Gβ5 and the protein complex is tethered to the plasma membrane by addition of a lipid, palmitate, on R7BP. Removal of palmitate results in the translocation of the R7BP–R7-Gβ5 complex to the nucleus, presumably for nontraditional signaling functions. These findings suggest an entirely novel mechanism for regulating neurotransmitter signaling. That is, R7BP transduces signals directly from receptors and G proteins at the plasma membrane to the nucleus, and this plasma membrane–nuclear shuttling is controlled by reversible palmitoylation of R7BP.
John R. Hepler (Emory University School of Medicine;Department of Pharmacology REV)
Changes in the nuclear protein kinase activities in the regenerating liver of partially irradiated rat
X rays (4.8 Gy) inhibit both DNA synthesis and phosphorylation of histone H1 in the regenerating liver of the rat. To determine the cause of the inhibition of histone H1 phosphorylation, changes in the nuclear protein kinase activities during the prereplicative phase of regeneration were measured. The cAMP-dependent protein kinase activity was low during regeneration, and the changes in the activity were not statistically significant. The cAMP-independent protein kinase activity increased at 15 h, decreased at 18 h, and increased again at 24 h after partial hepatectomy. X irradiation prior to partial hepatectomy did not inhibit the increase at 15 h, but it did inhibit the increase at 24 h. The activity was not inhibited by isoquinolinesulfonamide inhibitors such as H-7, and it was activated by a commercial preparation of an inhibitor protein of the cAMP-dependent kinase. It was also inhibited by quercetin. The possibility that the radiation-sensitive nuclear protein kinase is a nuclear cAMP-independent protein kinase specific for histone H1 is considered.
Asami, K.; Kobayashi, H.; Fujiwara, A.; Yasumasu, I. (National Institute of Radiological Sciences, Tokyo (Japan))
Ribosome biogenesis factors bind a nuclear envelope SUN domain protein to cluster yeast telomeres
Two interacting ribosome biogenesis factors, Ebp2 and Rrs1, associate with Mps3, an essential inner nuclear membrane protein. Both are found in foci along the nuclear periphery, like Mps3, as well as in the nucleolus. Temperature-sensitive ebp2 and rrs1 mutations that compromise ribosome biogenesis displace the mutant proteins from the nuclear rim and lead to a distorted nuclear shape. Mps3 is known to contribute to the S-phase anchoring of telomeres through its interaction with the silent information regulator Sir4 and yKu. Intriguingly, we find that both Ebp2 and Rrs1 interact with the C-terminal domain of Sir4, and that conditional inactivation of either ebp2 or rrs1 interferes with both the clustering and silencing of yeast telomeres, while telomere tethering to the nuclear periphery remains intact. Importantly, expression of an Ebp2�Mps3 fusion protein in the ebp2 mutant suppresses the defect in telomere clustering, but not its defects in growth or ribosome biogenesis. Our results suggest that the ribosome biogenesis factors Ebp2 and Rrs1 cooperate with Mps3 to mediate telomere clustering, but not telomere tethering, by binding Sir4.
Horigome, Chihiro; Okada, Takafumi; Shimazu, Kyoko; Gasser, Susan M; Mizuta, Keiko
Beta-Like Importins Mediate the Nuclear Translocation of Mitogen-Activated Protein Kinases
The rapid nuclear translocation of signaling proteins upon stimulation is important for the regulation of de novo gene expression. We have studied the stimulated nuclear shuttling of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases (MAPKs) and found that they translocate into the nucleus in a Ran-dependent, but NLS- or NTS-independent, manner, unrelated to their catalytic activity. We show that this translocation involves three ?-like importins, importins 3, 7, and 9 (Imp3/7/9). Knockdown of these importins inhibits the nuclear translocation of the MAPKs and, thereby, activation of their transcription factor targets. We further demonstrate that the translocation requires the stimulated formation of heterotrimers composed of Imp3/Imp7/MAPK or Imp3/Imp9/MAPK. JNK1/2 and p38?/? bind to either Imp7 or Imp9 upon stimulated posttranslational modification of the two Imps, while Imp3 joins the complex after its stimulation-induced phosphorylation. Once formed, these heterotrimers move to the nuclear envelope, where importin 3 remains, while importins 7 and 9 escort the MAPKs into the nucleus. These results suggest that ?-like importins are central mediators of stimulated nuclear translocation of signaling proteins and therefore add a central level of regulation to stimulated transcription.
Zehorai, Eldar
Beta-like importins mediate the nuclear translocation of mitogen-activated protein kinases.
The rapid nuclear translocation of signaling proteins upon stimulation is important for the regulation of de novo gene expression. We have studied the stimulated nuclear shuttling of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases (MAPKs) and found that they translocate into the nucleus in a Ran-dependent, but NLS- or NTS-independent, manner, unrelated to their catalytic activity. We show that this translocation involves three ?-like importins, importins 3, 7, and 9 (Imp3/7/9). Knockdown of these importins inhibits the nuclear translocation of the MAPKs and, thereby, activation of their transcription factor targets. We further demonstrate that the translocation requires the stimulated formation of heterotrimers composed of Imp3/Imp7/MAPK or Imp3/Imp9/MAPK. JNK1/2 and p38?/? bind to either Imp7 or Imp9 upon stimulated posttranslational modification of the two Imps, while Imp3 joins the complex after its stimulation-induced phosphorylation. Once formed, these heterotrimers move to the nuclear envelope, where importin 3 remains, while importins 7 and 9 escort the MAPKs into the nucleus. These results suggest that ?-like importins are central mediators of stimulated nuclear translocation of signaling proteins and therefore add a central level of regulation to stimulated transcription. PMID:24216760
Zehorai, Eldar; Seger, Rony
The tight junction protein Z O-2 has several functional nuclear export signals
The tight junction (TJ) protein ZO-2 changes its subcellular distribution according to the state of confluency of the culture. Thus in confluent monolayers, it localizes at the TJ region whereas in sparse cultures it concentrates at the nucleus. The canine sequence of ZO-2 displays four putative nuclear export signals (NES), two at the second PDZ domain (NES-0 and NES-1) and the rest at the GK region (NES-2 and NES-3). The functionality of NES-0 and NES-3 was unknown, hence here we have explored it with a nuclear export assay, injecting into the nucleus of MDCK cells peptides corresponding to the ZO-2 NES sequences chemically coupled to ovalbumin. We show that both NES-0 and NES-3 are functional and sensitive to leptomycin B. We also demonstrate that NES-1, previously characterized as a non functional NES, is rendered capable of nuclear export upon the acquisition of a negative charge at its Ser369 residue. Experiments performed injecting at the nucleus WT and mutated ZO-2-GST fusion proteins revealed the need of both NES-0 and NES-1, and NES-2 and NES-3 for attaining an efficient nuclear exit of the respective amino and middle segments of ZO-2. Moreover, the transfection of MDCK cells with full-length ZO-2 revealed that the mutation of any of the NES present in the molecule was sufficient to induce nuclear accumulation of the protein.
Gonzalez-Mariscal, Lorenza [Department of Physiology, Biophysics and Neuroscience, Center for Research and Advanced Studies (CINVESTAV), Ave. Instituto Politecnico Nacional 2508, Mexico, D.F., 07360 (Mexico)]. E-mail: email@example.com; Ponce, Arturo [Department of Physiology, Biophysics and Neuroscience, Center for Research and Advanced Studies (CINVESTAV), Ave. Instituto Politecnico Nacional 2508, Mexico, D.F., 07360 (Mexico); Alarcon, Lourdes [Department of Physiology, Biophysics and Neuroscience, Center for Research and Advanced Studies (CINVESTAV), Ave. Instituto Politecnico Nacional 2508, Mexico, D.F., 07360 (Mexico); Jaramillo, Blanca Estela [Department of Physiology, Biophysics and Neuroscience, Center for Research and Advanced Studies (CINVESTAV), Ave. Instituto Politecnico Nacional 2508, Mexico, D.F., 07360 (Mexico)
Protein dynamics in living cells: Fundamental developments of fluorescence correlation spectroscopy and investigations of protein nuclear import mechanisms
NASA Astrophysics Data System (ADS)
Fluorescence correlation spectroscopy (FCS) and fluorescence imaging have become very important tools for investigating molecular dynamics and molecular interactions in biological systems, especially in the intracellular environment. This dissertation focuses on both the development of FCS theory and methods and the application of FCS in studying protein dynamics in living cells. Fundamental theoretical treatments are presented regarding the effects of excitation saturation on FCS observation volume profiles, as well as detailed consideration of how objective lens fill factors influence volume profiles. We also consider theoretical models for anomalous diffusion in FCS, providing important clarification to the existing literature. We then discuss the use of FCS to study protein dynamics in living cells and demonstrate that the average mobility of biological molecules can be reliably characterized. The effects of different probe preparation methods on intracellular dynamics are also considered. The capability to apply FCS to investigate protein dynamics and interactions within living cells was exploited to investigate protein trafficking into the nucleus of eukaryotic cells. We present a comprehensive characterization of the intracellular mobility of nuclear import cargoes and receptors, and we show that these measurements have led to new insight into nuclear trafficking mechanisms in vivo.
Wu, Jianrong
A RNA helix-destabilizing protein is a major component of Artemia salina nuclear ribonucleoproteins.
A major component of 30S heterogeneous nuclear ribonucleoprotein (hnRNP) particles from Artemia salina is HD40, a protein that has been characterized as a RNA helix-destabilizing protein [Marvil, D. K., Nowak, L. & Szer, W. (1980) J. Biol. Chem. 255, 6466-6472; Nowak, L., Marvil, D. K., Thomas, J. O., Boublik, M. & Szer, W. (1980) J. Biol. Chem. 255, 6473-6478]. HD40 binds to and disrupts the secondary structure of nuclear RNA fragments isolated from 30S hnRNP with a stoichiometry of one protein per 10-12 nucleotides. The addition of HD40 in excess of this ratio results in the formation of bead-like HD4-nuclear RNA complexes that are similar in properties and appearance to native 30S hnRNP particles. The heterogeneous nuclear RNA (hnRNA) in the HD40-hnRNA complexes is unstacked and unfolded to about the same extent as the RNA in the native 30S hnRNP particles. HD40 is strikingly similar in molecular weight (40,000) and amino acid composition (no cysteine, high glycine, presence of dimethylarginine, and blocked NH2 terminus) to eukaryotic hnRNP proteins isolated from many cell types. HD40 can be separated into three isoelectric species with basic pIs, which appears to be posttranslational modifications of a single polypeptide chain. Images
Thomas, J O; Raziuddin; Sobota, A; Boublik, M; Szer, W
Fanconi Anemia Proteins FANCA, FANCC, and FANCG\\/XRCC9 Interact in a Functional Nuclear Complex
Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with at least eight comple- mentation groups (A to H). Three FA genes, corresponding to complementation groups A, C, and G, have been cloned, but their cellular function remains unknown. We have previously demonstrated that the FANCA and FANCC proteins interact and form a nuclear complex in normal cells, suggesting
Chromosomal clustering of nuclear genes encoding mitochondrial and chloroplast proteins in Arabidopsis
We present a statistical analysis of chromosomal clustering among nuclear genes encoding mitochondrial or chloroplast proteins in Arabidopsis. For both orga- nelles, the clustering was significantly increased above the expectation, but the clustering effect was weak, and most clusters were small and dispersed. Clustered genes showed coexpression but not more than expected, and no substantial synteny was detected in other
Andrey Alexeyenko; A. Harvey Millar; James Whelan; Erik L. L. Sonnhammer
High-mobility group box 1 protein (HMGB1): nuclear weapon in the immune arsenal
High-mobility group box 1 protein (HMGB1), which previously was thought to function only as a nuclear factor that enhances transcription, was recently discovered to be a crucial cytokine that mediates the response to infection, injury and inflammation. These observations have led to the emergence of a new field in immunology that is focused on understanding the mechanisms of HMGB1 release,
Michael T. Lotze; Kevin J. Tracey
Nuclear localization of the major vault protein in U373 cells
The major vault protein (MVP) is the predominant member of a large ribonucleoprotein particle, named vault. Vaults are abundant in the cytosol of mammalian cells. Mammalian MVP has previously been reported to be associated with the nucleus, particularly its cytosolic surface on which vaults are thought to dock at or near the nuclear pore complex. To date the presence of
Marco Slesina; Elisabeth M. Inman; Leonard H. Rome; Walter Volknandt
Immunoreactivity for calcium-binding proteins defines subregions of the vestibular nuclear complex of the cat
The vestibular nuclear complex (VNC) is classically divided into four nuclei on the basis of cytoarchitectonics. However, anatomical data on the distribution of afferents to the VNC and the distribution of cells of origin of different efferent pathways suggest a more complex internal organization. Immunoreactivity for calcium-binding proteins has proven useful in many areas of the brain for revealing structure
Joan S. Baizer; James F. Baker
Nuclear Localization Signal(s) Required for Nuclear Targeting of the Maize Regulatory Protein Opaque2
The maize regulatory protein Opaque-2 (02) localizes to the nucleus in both maize and tobacco cells. Here we show that in-frame carboxy- and amino-terminal fusions of 02 to reporter protein p-glucuronidase (GUS) were sufficient to direct GUS to the nucleus in transgenic tobacco plants and in transiently transformed onion cells. Two independent regions of 02 containing 135 and 149 amino
Marguerite J. Varagona; Robert J. Schmidt; Natasha V. Raikhelai
An N-terminal nuclear localization sequence but not the calmodulin-binding domain mediates nuclear localization of nucleomorphin, a protein that regulates nuclear number in Dictyostelium
Nucleomorphin is a novel nuclear calmodulin (CaM)-binding protein (CaMBP) containing an extensive DEED (glu/asp repeat) domain that regulates nuclear number. GFP-constructs of the 38 kDa NumA1 isoform localize as intranuclear patches adjacent to the inner nuclear membrane. The translocation of CaMBPs into nuclei has previously been shown by others to be mediated by both classic nuclear localization sequences (NLSs) and CaM-binding domains (CaMBDs). Here we show that NumA1 possesses a CaMBD ({sup 171}EDVSRFIKGKLLQKQQKIYKDLERF{sup 195}) containing both calcium-dependent-binding motifs and an IQ-like motif for calcium-independent binding. GFP-constructs containing only NumA1 residues 1-129, lacking the DEED and CaMBDs, still localized as patches at the internal periphery of nuclei thus ruling out a direct role for the CaMBD in nuclear import. These constructs contained the amino acid residues {sup 48}KKSYQDPEIIAHSRPRK{sup 64} that include both a putative bipartite and classical NLS. GFP-bipartite NLS constructs localized uniformly within nuclei but not as patches. As with previous work, removal of the DEED domain resulted in highly multinucleate cells. However as shown here, multinuclearity only occurred when the NLS was present allowing the protein to enter nuclei. Site-directed mutation analysis in which the NLS was changed to {sup 48}EF{sup 49} abolished the stability of the GFP fusion at the protein but not RNA level preventing subcellular analyses. Cells transfected with the {sup 48}EF{sup 49} construct exhibited slowed growth when compared to parental AX3 cells and other GFP-NumA1 deletion mutants. In addition to identifying an NLS that is sufficient for nuclear translocation of nucleomorphin and ruling out CaM-binding in this event, this work shows that the nuclear localization of NumA1 is crucial to its ability to regulate nuclear number in Dictyostelium.
Myre, Michael A. [Department of Biology, University of Toronto at Mississauga, Mississauga, Ont. (Canada); O'Day, Danton H. [Department of Biology, University of Toronto at Mississauga, Mississauga, Ont. (Canada)]. E-mail: firstname.lastname@example.com
Mammalian sperm translate nuclear-encoded proteins by mitochondrial-type ribosomes
It is widely accepted that spermatozoa are translationally silent. The present study demonstrates, for the first time, incorporation of labeled amino acids into polypeptides during sperm capacitation, which was completely inhibited by mitochondrial translation inhibitors but not by the cytoplasmic translation inhibitor. Unlike 80S cytoplasmic ribosomes, 55S mitochondrial ribosomes were present in polysomal fractions, indicating that these ribosomes are actively involved in protein translation in spermatozoa. Inhibition of protein translation significantly reduced sperm motility, capacitation and in vitro fertilization rate. Thus, contrary to the accepted dogma, nuclear genes are expressed as proteins in sperm during their residence in the female reproductive tract until fertilization.
Gur, Yael; Breitbart, Haim
SUMOylation regulates the nuclear mobility of CREB binding protein and its association with nuclear bodies in live cells
The lysine acetyltransferase CREB binding protein (CBP) is required for chromatin modification and transcription at many gene promoters. In fixed cells, a large proportion of CBP colocalises to PML or nuclear bodies. Using live cell imaging, we show here that YFP-tagged CBP expressed in HEK293 cells undergoes gradual accumulation in nuclear bodies, some of which are mobile and migrate towards the nuclear envelope. Deletion of a short lysine-rich domain that contains the major SUMO acceptor sites of CBP abrogated its ability to be SUMO modified, and prevented its association with endogenous SUMO-1/PML speckles in vivo. This SUMO-defective CBP showed enhanced ability to co-activate AML1-mediated transcription. Deletion mapping revealed that the SUMO-modified region was not sufficient for targeting CBP to PML bodies, as C-terminally truncated mutants containing this domain showed a strong reduction in accumulation at PML bodies. Fluorescence recovery after photo-bleaching (FRAP) experiments revealed that YFP-CBP{Delta}998-1087 had a retarded recovery time in the nucleus, as compared to YFP-CBP. These results indicate that SUMOylation regulates CBP function by influencing its shuttling between nuclear bodies and chromatin microenvironments.
Ryan, Colm M.; Kindle, Karin B.; Collins, Hilary M. [Gene Regulation Group, Centre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD (United Kingdom)] [Gene Regulation Group, Centre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD (United Kingdom); Heery, David M., E-mail: firstname.lastname@example.com [Gene Regulation Group, Centre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD (United Kingdom)
Interferon gamma regulates binding of two nuclear protein complexes in a macrophage cell line.
Interferon gamma (IFN-gamma) is a potent inducer of major histocompatibility complex (MHC) antigens during normal immune responses and in abnormal responses in autoimmune disease. In this report we identify two nuclear factors whose binding to the murine E beta class II MHC beta-chain gene is regulated by this cytokine. IFN-gamma stimulation of murine macrophages results in the appearance of increased binding of one protein complex, complex A, and decreased binding of a second, faster migrating protein complex, complex B. Although the contact residues for both of these proteins lie within the highly conserved Y-box transcriptional element, their binding specificity differs. The protein in complex B is a CCAAT-box-binding protein that may be similar or identical to NF-Y or YB1, previously identified class II Y-box-binding proteins. The DNA sequence requirements for the binding of the slower migrating complex, complex A, are not limited to CCAAT-box sequences but include sequences upstream of the Y box. These upstream sequences are required both for IFN-gamma-induced gene transcription and for IFN-gamma-induced modulation of binding activity. These data suggest a model in which upstream sequences contribute to formation of a lymphokine-regulated complex downstream. The IFN-gamma-induced binding protein described as complex A in this report differs from the IFN-gamma, -alpha, or -beta-induced nuclear factors previously identified. Images
Finn, P W; Kara, C J; Douhan, J; Van, T T; Folsom, V; Glimcher, L H
Protein carbon-13 spin systems by a single two-dimensional nuclear magnetic resonance experiment
By applying a two-dimensional double-quantum carbon-13 nuclear magnetic resonance experiment to a protein uniformly enriched to 26% carbon-13, networks of directly bonded carbon atoms were identified by virtue of their one-bond spin-spin couplings and were classified by amino acid type according to their particular single- and double-quantum chemical shift patterns. Spin systems of 75 of the 98 amino acid residues in a protein, oxidized Anabaena 7120 ferredoxin (molecular weight 11,000), were identified by this approach, which represents a key step in an improved methodology for assigning protein nuclear magnetic resonance spectra. Missing spin systems corresponded primarily to residues located adjacent to the paramagnetic iron-sulfur cluster. 25 references, 2 figures.
Oh, B.H.; Westler, W.M.; Darba, P.; Markley, J.L.
Strategies to inhibit viral protein nuclear import: HIV-1 as a target
Nuclear import is a critical step in the life cycle of HIV-1. During the early (pre-integration) stages of infection, HIV-1 has to transport its pre-integration complex into the nucleus for integration into the host cell chromatin, while at the later (post-integration) stages viral regulatory proteins Tat and Rev need to get into the nucleus to stimulate transcription and regulate splicing and nuclear export of subgenomic and genomic RNAs. Given such important role of nuclear import in HIV-1 life cycle, this step presents an attractive target for anti-viral therapeutic intervention. In this review, we describe the current state of our understanding of the interactions regulating nuclear import of the HIV-1 pre-integration complex and describe current approaches to inhibit it.
Levin, Aviad; Loyter, Abraham; Bukrinsky, Michael
A Crowdsourced nucleus: understanding nuclear organization in terms of dynamically networked protein function.
The spatial organization of the nucleus results in a compartmentalized structure that affects all aspects of nuclear function. This compartmentalization involves genome organization as well as the formation of nuclear bodies and plays a role in many functions, including gene regulation, genome stability, replication, and RNA processing. Here we review the recent findings associated with the spatial organization of the nucleus and reveal that a common theme for nuclear proteins is their ability to participate in a variety of functions and pathways. We consider this multiplicity of function in terms of Crowdsourcing, a recent phenomenon in the world of information technology, and suggest that this model provides a novel way to synthesize the many intersections between nuclear organization and function. This article is part of a Special Issue entitled: Chromatin and epigenetic regulation of animal development. PMID:24412853
Wood, Ashley M; Garza-Gongora, Arturo G; Kosak, Steven T
Evidence for an inhibitory feedback loop regulating simian virus 40 large T-antigen fusion protein nuclear transport.
Nuclear protein import is central to eukaryotic cell function. It is dependent on ATP, temperature and cytosolic factors, and requires specific targeting sequences called nuclear localization signals (NLSs). Nuclear import kinetics was studied in vitro using digitonin-permeabilized cells of the HTC rat hepatoma cell line and a fluorescently labelled beta-galactosidase fusion protein carrying amino acids 111-135 of the simian virus 40 large T-antigen (T-ag), including the NLS. Nuclear accumulation was rapid, reaching steady-state after about 80 min at 37 degrees C (t1/2 at about 17 min). Surprisingly, maximal nuclear concentration was found to be directly proportional to the concentration of the cytosolic extract and of cytoplasmic T-ag protein. Neither preincubation of cells for 1 h at 37 degrees C before the addition of T-ag protein nor the addition of fresh transport medium after 1 h and continuation of the incubation for another hour affected the maximal nuclear concentration. If cells were allowed to accumulate T-ag protein for 1 h before the addition of fresh transport medium containing different concentrations of T-ag protein and incubated for a further hour, the maximal nuclear concentration did not change unless the concentration of T-ag protein in the second transport mixture exceeded that in the first, in which case the nuclear concentration increased. Nuclear import of T-ag thus appeared (i) to be strictly unidirectional over 2 h at 37 degrees C and (ii) to be regulated by an inhibitory feedback loop, whereby the cytosolic concentration of protein appears to determine directly the precise end point of nuclear accumulation. This study represents the first characterization of this previously undescribed mechanism of regulation of nuclear protein import.
Seydel, U; Jans, D A
Nuclear PRP20 protein is required for mRNA export.
The yeast PRP20 protein is highly homologous in structure and function to the RCC1 protein of higher eukaryotes. The RCC1 protein is involved in the regulation of the onset of mitosis, whereas the PRP20 protein was shown to be required for accurate and efficient mRNA metabolism. The first observable phenotype in mutant prp20 cells when shifted from permissive to non-permissive temperature is a loss of nuclear PRP20 protein. Concomitantly, an accumulation of poly(A)+ RNA in the nucleus is observed. The temperature-sensitive RCC1 allele in the mutant hamster cell line tsBN2 leads to a similar accumulation of mRNA in the nucleus. Images
Amberg, D C; Fleischmann, M; Stagljar, I; Cole, C N; Aebi, M
A protein recycling system for nuclear magnetic resonance-based screening of drug candidates.
A sample-treating system for nuclear magnetic resonance (NMR)-based interaction screening between drug candidates (small molecules) and a protein of interest was developed by applying high-performance liquid chromatography (HPLC) technology. The system prepares a test solution by mixing a (15)N-labeled protein solution and a solution of each candidate compound, loads it to a flow cell-type NMR probe, and recycles the protein after the data acquisition. The system was designed to behave differently according to the information obtained in NMR measurements. In a test operation with a 100-compound library, the system could single out known interacting substances properly. Recovery values of the protein and one representative compound were 75 and 71%, respectively, and the recovered protein was found intact as intended. PMID:16626618
Hirayama, Aya; Shirota, Osamu; Nomura, Mitsuru; Nagadoi, Aritaka; Nishimura, Yoshifumi
A nuclear export signal within the structural Gag protein is required for prototype foamy virus replication
Background The Gag polyproteins play distinct roles during the replication cycle of retroviruses, hijacking many cellular machineries to fulfill them. In the case of the prototype foamy virus (PFV), Gag structural proteins undergo transient nuclear trafficking after their synthesis, returning back to the cytoplasm for capsid assembly and virus egress. The functional role of this nuclear stage as well as the molecular mechanism(s) responsible for Gag nuclear export are not understood. Results We have identified a leptomycin B (LMB)-sensitive nuclear export sequence (NES) within the N-terminus of PFV Gag that is absolutely required for the completion of late stages of virus replication. Point mutations of conserved residues within this motif lead to nuclear redistribution of Gag, preventing subsequent virus egress. We have shown that a NES-defective PFV Gag acts as a dominant negative mutant by sequestrating its wild-type counterpart in the nucleus. Trans-complementation experiments with the heterologous NES of HIV-1 Rev allow the cytoplasmic redistribution of FV Gag, but fail to restore infectivity. Conclusions PFV Gag-Gag interactions are finely tuned in the cytoplasm to regulate their functions, capsid assembly, and virus release. In the nucleus, we have shown Gag-Gag interactions which could be involved in the nuclear export of Gag and viral RNA. We propose that nuclear export of unspliced and partially spliced PFV RNAs relies on two complementary mechanisms, which take place successively during the replication cycle.
Sequence motif in control regions of the H+/K+ ATPase alpha and beta subunit genes recognized by gastric specific nuclear protein(s).
A nuclear protein(s) from rat or pig stomach recognized a conserved sequence in the 5'-upstream regions of the rat and human H+/K(+)-ATPase alpha subunit genes. A gel retardation assay suggested that part of the binding site was located in the TAATCAGCTG sequence. No nuclear proteins capable of the binding could be detected in other tissues of rat (liver, brain, kidney, spleen and lung) or pig liver. The sequence motif (GATAGC) located 5'-upstream of the beta-subunit gene also seemed to be recognized by the same protein, because the binding of nuclear protein to the sequence motifs in the alpha and beta subunits was mutually competitive. Considering the sense-strand sequence of the binding motif in the alpha-subunit gene, we conclude that (G/C)PuPu(G/C)NGAT(A/T)PuPy is a core sequence motif for the gastric specific DNA binding protein (PCSF, parietal cell specific factor). PMID:1312019
Tamura, S; Oshiman, K; Nishi, T; Mori, M; Maeda, M; Futai, M
Cell density-dependent nuclear/cytoplasmic localization of NORPEG (RAI14) protein
NORPEG (RAI14), a developmentally regulated gene induced by retinoic acid, encodes a 980 amino acid (aa) residue protein containing six ankyrin repeats and a long coiled-coil domain [Kutty et al., J. Biol. Chem. 276 (2001), pp. 2831-2840]. We have expressed aa residues 1-287 of NORPEG and used the recombinant protein to produce an anti-NORPEG polyclonal antibody. Confocal immunofluorescence analysis showed that the subcellular localization of NORPEG in retinal pigment epithelial (ARPE-19) cells varies with cell density, with predominantly nuclear localization in nonconfluent cells, but a cytoplasmic localization, reminiscent of cytoskeleton, in confluent cultures. Interestingly, an evolutionarily conserved putative monopartite nuclear localization signal (P{sup 27}KKRKAP{sup 276}) was identified by analyzing the sequences of NORPEG and its orthologs. GFP-NORPEG (2-287 aa), a fusion protein containing this signal, was indeed localized to nuclei when expressed in ARPE-19 or COS-7 cells. Deletion and mutation analysis indicated that the identified nuclear localization sequence is indispensable for nuclear targeting.
Kutty, R. Krishnan [Section on Biochemistry, Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892 (United States)]. E-mail: firstname.lastname@example.com; Chen, Shanyi [Section on Biochemistry, Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Samuel, William [Section on Biochemistry, Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Vijayasarathy, Camasamudram [National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD 20892 (United States); Duncan, Todd [Section on Biochemistry, Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Tsai, Jen-Yue [Biological Imaging Core, National Eye Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Fariss, Robert N. [Biological Imaging Core, National Eye Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Carper, Deborah [Section on Molecular Therapeutics, National Eye Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Jaworski, Cynthia [Section on Molecular Therapeutics, National Eye Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Wiggert, Barbara [Section on Biochemistry, Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892 (United States)
SUN proteins facilitate the removal of membranes from chromatin during nuclear envelope breakdown.
SUN proteins reside in the inner nuclear membrane and form complexes with KASH proteins of the outer nuclear membrane that connect the nuclear envelope (NE) to the cytoskeleton. These complexes have well-established functions in nuclear anchorage and migration in interphase, but little is known about their involvement in mitotic processes. Our analysis demonstrates that simultaneous depletion of human SUN1 and SUN2 delayed removal of membranes from chromatin during NE breakdown (NEBD) and impaired the formation of prophase NE invaginations (PNEIs), similar to microtubule depolymerization or down-regulation of the dynein cofactors NudE/EL. In addition, overexpression of dominant-negative SUN and KASH constructs reduced the occurrence of PNEI, indicating a requirement for functional SUN-KASH complexes in NE remodeling. Codepletion of SUN1/2 slowed cell proliferation and resulted in an accumulation of morphologically defective and disoriented mitotic spindles. Quantification of mitotic timing revealed a delay between NEBD and chromatin separation, indicating a role of SUN proteins in bipolar spindle assembly and mitotic progression. PMID:24662567
Turgay, Yagmur; Champion, Lysie; Balazs, Csaba; Held, Michael; Toso, Alberto; Gerlich, Daniel W; Meraldi, Patrick; Kutay, Ulrike
MLV based viral-like-particles for delivery of toxic proteins and nuclear transcription factors.
We have developed nanoparticles based on Murine Leukemia Virus virus-like-particles (VLPs) that efficiently deliver therapeutic bioactive proteins in their native state into target cells. Nuclear transcription factors and toxic proteins were incorporated into the VLPs from stable producer cells without transducing viral-encoded genetic material. Delivery of nuclear transcription factors required incorporation of nuclear export signals (NESs) into the vector backbone for the efficient formation of VLPs. In the presence of an appropriate targeting Env glycoprotein, transcription factors delivered and activated nuclear transcription in the target cells. Additionally, we show delivery of the bacterial toxin, MazF, which is an ACA-specific mRNA interferase resulted in the induction of cell death. The stable producer cells are protected from the toxin through co-expression of the anti-toxin MazE and continuously released MazF incorporating VLPs. This highly adaptable platform can be harnessed to alter and regulate cellular processes by bioactive protein delivery. PMID:24997480
Wu, Dai-Tze; Roth, Monica J
C-reactive protein reacts with the U1 small nuclear ribonucleoprotein.
C-reactive protein (CRP) was found to produce a small, discrete, speckled fluorescence pattern in the nucleus of HEp-2 cells. Double staining with anti-RNP serum and CRP produced very similar staining patterns. By counterimmunoelectrophoresis CRP was bound to extractable nuclear antigens found in rabbit thymus extract. The reactive components of the extract were only partially sensitive to treatment with RNase. CRP immunoprecipitated the U1 RNA species from [32P]labeled HeLa cells and the protein bands of the Sm/RNP complex from [35S]-methionine-labeled HeLa cells. By blotting, CRP bound to several discrete bands in a calcium-dependent, PC-inhibitable manner. Two of the bands comigrated with the 70K protein band associated with the U1 snRNP, and its major breakdown product. Binding to these bands was inhibited by both EDTA and PC indicating that CRP binds these proteins through the PC-binding site. Binding to the 70K protein of the U1 snRNP was confirmed by reactivity with the recombinant 70K protein in a dot blot. These findings indicate the CRP binds to the U1-RNP snRNP particle. Considering the ability of CRP to inhibit antibody responses to its ligands and its ability to activate C and promote phagocytosis it is suggested that CRP may play a role in the regulation of autoantibody responses to nuclear Ag. PMID:2477447
Du Clos, T W
Importin alpha2-interacting proteins with nuclear roles during mammalian spermatogenesis.
Spermatogenesis, the process of generating haploid sperm capable of fertilizing the female gamete, requires the timely transport into the nucleus of transcription and chromatin-remodeling factors, mediated by members of the importin (IMP) superfamily. Previous IMP expression profiling implies a role for IMPalpha2 in testicular germ cells late in spermatogenesis. To identify interacting proteins of IMPalpha2 that are potential drivers of germ cell development, we performed yeast two-hybrid screening of an adult mouse testis library. IMPalpha2 interactions were verified by coimmunoprecipitation approaches, whereas immunohistochemical staining of testis sections confirmed their coexpression with IMPalpha2 in specific testicular cell types. Key interactors identified were a novel isoform of a cysteine and histidine rich protein (Chrp), a protein inhibitor of activated STAT (PIAS) family member involved in transcriptional regulation and sumoylation, Androgen receptor interacting protein 3 (Arip3), and Homologous protein 2 (Hop2), known to be involved in homologous chromosome pairing and recombination, all of which are highly expressed in the testis and show mRNA expression profiles similar to that of IMPalpha2 throughout testicular development. This is the first study to identify binding partners of IMPalpha2 in the developmental context of germ line development, and we propose that the regulated expression and timely IMPalpha2-mediated nuclear transport of these proteins may coordinate events during spermatogenesis, with IMPalpha2-mediated nuclear localization representing a potentially critical developmental switch in the testis. PMID:21900684
Ly-Huynh, Jennifer D; Lieu, Kim G; Major, Andrew T; Whiley, Penelope A F; Holt, Janet E; Loveland, Kate L; Jans, David A
Human papillomavirus 16 E7 protein is associated with the nuclear matrix.
The cellular localization of the human papillomavirus (HPV)-16 E7 gene product in the cell lines CaSki and SiHa has been determined by both biochemical and immunocytochemical methods. These measurements show E7 to be localized in the cell nucleus, specifically with the nonchromatin nuclear structure or nuclear matrix. This localization of E7 required an unambiguous fractionation of the nuclear constituents. This was achieved by using a gentle sequential fractionation procedure to prepare the scaffold consisting of the nuclear matrix and intermediate filaments (NM-IF). Chromatin was cleaved with nuclease and the resulting nucleosomes eluted with 0.25 M ammonium sulfate. Immunostaining of cells after this extraction procedure with monoclonal antibodies (mAbs) to E7 revealed a fine grained, punctate nuclear fluorescence in CaSki and SiHa, which was absent in normal cervical keratinocytes and the HPV-negative cell line C33.1. Western blots of cell fractions with these mAbs showed that E7 was localized in the NM-IF fraction in SiHa and CaSki but was not detected in HPV-negative cells. A second protein of slightly higher mobility is identified by these antisera in HPV-16-containing cells. The data suggest that the previous inability to directly visualize E7 by immunocytology is due to the masking of epitopes by cellular components and not to low levels of protein. Images
Greenfield, I; Nickerson, J; Penman, S; Stanley, M
Regulated nuclear trafficking of the homeodomain protein otx1 in cortical neurons.
Otx1 is a homeodomain protein required for axon refinement by layer 5 neurons in developing cerebral cortex. Otx1 localizes to the cytoplasm of progenitor cells in the rat ventricular zone, and remains cytoplasmic as neurons migrate and begin to differentiate. Nuclear translocation occurs during the first week of postnatal life, when layer 5 neurons begin pruning their long-distance axonal projections. Deletion analysis reveals that Otx1 is imported actively into cell nuclei, that the N-terminus of Otx1 is necessary for nuclear import, and that a putative nuclear localization sequence within this domain is sufficient to direct nuclear import in a variety of cell lines. In contrast, GFP-Otx1 fusion proteins that contain the N-terminus are retained in the cytoplasm of cortical progenitor cells, mimicking the distribution of Otx1 in vivo. These results suggest that ventricular cells actively sequester Otx1 in the cytoplasm, either by preventing nuclear import or by promoting a balance of export over import signals. PMID:11906214
Zhang, Y Alex; Okada, Ami; Lew, Chuen Hong; McConnell, Susan K
Nuclear translocation of adeno-associated virus type 2 capsid proteins for virion assembly.
Adeno-associated virus (AAV) capsid assembly occurs in the nucleus. Newly synthesized capsid proteins VP1, VP2 and VP3 contain several basic regions (BRs), which may act as nuclear localization signals (NLSs). Mutation of BR2 and BR3, located at the VP1 and VP2 N termini, marginally reduced nuclear uptake of VP1 or VP2, but not of VP3, when expressed in the context of the whole AAV type 2 (AAV2) genome. Combined mutation of BR1, BR2 and BR3 resulted in capsids with slightly reduced amounts of VP1. Expression of isolated VP1/2 N termini revealed an influence of BR3 on nuclear transport, whilst BR1 or BR2 had no effect. However, deletion of an N-terminal fragment in front of the BR elements strongly reduced nuclear uptake of VP1/2 N termini. Mutation of BR4, present in all three capsid proteins, led to their retention in the cytoplasm and to the formation of speckles, resulting in a lack of capsid formation and a significant reduction in VP levels. In a VP fragment comprising BR2, BR3 and BR4, the BR4 element was not necessary for nuclear localization. Mutation of BR5 in the C-terminal part of the VPs resulted in a speckled protein distribution in the nucleus, strongly reduced capsid assembly, and low VP1 and VP2 levels. Taken together, these results showed that BR2 and BR3 have a weak influence on nuclear transport of VP1 and VP2, whilst combined mutation of BR1, BR2 and BR3 influences the stoichiometry of VPs in assembled capsids. BR4 and BR5 play a crucial role in capsid assembly but have no NLS activity. PMID:22694902
Popa-Wagner, Ruth; Sonntag, Florian; Schmidt, Kristin; King, Jason; Kleinschmidt, J�rgen A
Molecular cloning and characterization of Mustang, a novel nuclear protein expressed during skeletal development and regeneration.
Bone regeneration occurs as a series of events that requires temporal and spatial orchestration of numerous cell types guided by the transcriptional activity of thousands of genes, as recently demonstrated by our laboratory. Using the rat femoral fracture model, bioinformatics, cloning, expression assays, fusion proteins, and transfection, we report on the identification and characterization of one such differentially expressed gene, termed Mustang (musculoskeletal temporally activated novel gene). Mustang encodes for an 82 amino acid nuclear protein with no homology to any known protein family. However, other species homologues (mouse, human, cow) were identified within EST (expressed sequence tag) databases. Nuclear localization was confirmed using a GFP-Mustang fusion protein. Using in situ hybridization, Mustang expression was localized to differentiating periosteal osteogenic cells, proliferating chondrocytes, and osteoblasts of the fracture callus. Unlike adult tissues, developing embryos abundantly express Mustang, especially in mesenchymal condensations of limbs, vertebral perichondrium, and mesenchymal cells of the intervertebral discs. Although the precise function of Mustang is unknown, its unique pattern of expression during bone development and regeneration, absence in adult tissues (except skeletal muscle and tendon), and nuclear localization suggest that Mustang is involved in the development and regeneration of the mammalian musculoskeletal system. PMID:14718386
Lombardo, Frank; Komatsu, David; Hadjiargyrou, Michael
The putative nuclear localization signal of the human RAD52 protein is a potential sumoylation site.
RAD52, a key factor in homologous recombination (HR), plays important roles in both RAD51-dependent and -independent HR pathways. Several studies have suggested a link between the functional regulation of RAD52 and the protein modification by a small ubiquitin-like modifier (SUMO). However, the molecular mechanism underlying the regulation of RAD52 by SUMO is unknown. To begin investigating this mechanism, we identified possible target sites for sumoylation in the human RAD52 protein by preparing a RAD52-SUMO complex using an established Escherichia coli sumoylation system. Mass spectrometry and amino acid sequencing of the enzymatically digested fragments of the purified complex revealed that the putative nuclear localization signal located near the C terminus of RAD52 was sumoylated. Biochemical studies of the RAD52-SUMO complex suggested that sumoylation at the identified site has no apparent effect on the DNA binding, D-loop formation, ssDNA annealing and RAD51-binding activities of RAD52. On the other hand, visualization of the GFP-fused RAD52 protein in the human cell that contained mutations at the identified sumoylation sites showed clear differences in the cytosolic and nuclear distributions of the protein. These results suggest the possibility of sumoylation playing an important role in the nuclear transport of RAD52. PMID:20190268
Saito, Kengo; Kagawa, Wataru; Suzuki, Takehiro; Suzuki, Hidekazu; Yokoyama, Shigeyuki; Saitoh, Hisato; Tashiro, Satoshi; Dohmae, Naoshi; Kurumizaka, Hitoshi
Nuclear targeting determinants of the far upstream element binding protein, a c-myc transcription factor.
FUSE binding protein (FBP) binds in vivo and in vitro with the single-stranded far upstream element (FUSE) upstream of the c-myc gene. In addition to its transcriptional role, FBP and its closely related siblings FBP2 (KSRP) and FBP3 have been reported to bind RNA and participate in various steps of RNA processing, transport or catabolism. To perform these diverse functions, FBP must traffic to different nuclear sites. To identify determinants of nuclear localization, full-length FBP or fragments thereof were fused to green fluorescent protein. Fluorescent-FBP localized in the nucleus in three patterns, diffuse, dots and spots. Each pattern was conferred by a distinct nuclear localization signal (NLS): a classical bipartite NLS in the N-terminal and two non-canonical signals, an alpha-helix in the third KH-motif of the nucleic acid binding domain and a tyrosine-rich motif in the C-terminal transcription activation domain. Upon treatment with the transcription inhibitor actinomycin D, FBP completely re-localized into dots, but did not exit from the nucleus. This is in contrast to many general RNA-binding proteins, which shuttle from the nucleus upon treatment with actinomycin D. Furthermore, FBP co-localized with transcription sites and with the general transcription factor TFIIH, but not with the splicing factor SC-35. Taken together, these data reveal complex intranuclear trafficking of FBP and support a transcriptional role for this protein. PMID:11071946
He, L; Weber, A; Levens, D
Nuclear localization of mouse Mx1 protein is necessary for inhibition of influenza virus.
The interferon-induced Mx1 protein of mice confers selective resistance to influenza virus. It inhibits viral mRNA synthesis in the nucleus of influenza virus-infected cells. The related human MxA protein is localized in the cytoplasm and can inhibit influenza virus and vesicular stomatitis virus but not other viruses. MxA blocks a poorly defined cytoplasmic multiplication step of influenza virus that follows primary transcription of the viral genome. We previously showed that nuclear variants of MxA that carry an artificial nuclear translocation signal were also active against influenza virus. However, these variants blocked primary transcription of influenza virus. In the present study, we addressed the question of whether cytoplasmic forms of Mx1 were capable of mimicking the antiviral action of MxA by determining the antiviral activities of mutant mouse Mx1 protein. Cytoplasmic Mx1(E614), which differs from wild-type Mx1 by a single amino acid substitution in its nuclear transport signal, failed to inhibit the multiplication of influenza virus and vesicular stomatitis virus. Relocation of Mx1(E614) to the nucleus with the help of the simian virus 40 large T nuclear translocation signal attached to its amino terminus restored the influenza virus-inhibiting activity. Other changes in the carboxy-terminal region of Mx1 also abolished transport to the nucleus and simultaneously abolished antiviral activity. One of these variants, Mx1/A, gained activity against influenza virus upon relocation to the nucleus. These results demonstrate that unlike human MxA, the mouse Mx1 protein can function only in the nucleus. This finding has important implications regarding the mechanistic details of Mx protein action. Images
Zurcher, T; Pavlovic, J; Staeheli, P
Nuclear localization of DMP1 proteins suggests a role in intracellular signaling
Highlights: Black-Right-Pointing-Pointer Nuclear localization of DMP1 in various cell lines. Black-Right-Pointing-Pointer Non-synchronized cells show either nuclear or cytoplasmic localization of DMP1. Black-Right-Pointing-Pointer Nuclear DMP1 is restricted to the nucleoplasm but absent in the nucleolus. -- Abstract: Dentin matrix protein 1 (DMP1) is highly expressed in odontoblasts and osteoblasts/osteocytes and plays an essential role in tooth and bone mineralization and phosphate homeostasis. It is debatable whether DMP1, in addition to its function in the extracellular matrix, can enter the nucleus and function as a transcription factor. To better understand its function, we examined the nuclear localization of endogenous and exogenous DMP1 in C3H10T1/2 mesenchymal cells, MC3T3-E1 preosteoblast cells and 17IIA11 odontoblast-like cells. RT-PCR analyses showed the expression of endogenous Dmp1 in all three cell lines, while Western-blot analysis detected a major DMP1 protein band corresponding to the 57 kDa C-terminal fragment generated by proteolytic processing of the secreted full-length DMP1. Immunofluorescent staining demonstrated that non-synchronized cells presented two subpopulations with either nuclear or cytoplasmic localization of endogenous DMP1. In addition, cells transfected with a construct expressing HA-tagged full-length DMP1 also showed either nuclear or cytoplasmic localization of the exogenous DMP1 when examined with an antibody against the HA tag. Furthermore, nuclear DMP1 was restricted to the nucleoplasm but was absent in the nucleolus. In conclusion, these findings suggest that, apart from its role as a constituent of dentin and bone matrix, DMP1 might play a regulatory role in the nucleus.
Siyam, Arwa [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A and M Health Science Center, 3302 Gaston Ave., Dallas, TX 75246-2013 (United States) [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A and M Health Science Center, 3302 Gaston Ave., Dallas, TX 75246-2013 (United States); Department of Endodontology, Kornberg School of Dentistry, Temple University, 3223 North Broad Street, Philadelphia, PA 19140-5007 (United States); Wang, Suzhen; Qin, Chunlin; Mues, Gabriele [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A and M Health Science Center, 3302 Gaston Ave., Dallas, TX 75246-2013 (United States)] [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A and M Health Science Center, 3302 Gaston Ave., Dallas, TX 75246-2013 (United States); Stevens, Roy [Department of Endodontology, Kornberg School of Dentistry, Temple University, 3223 North Broad Street, Philadelphia, PA 19140-5007 (United States)] [Department of Endodontology, Kornberg School of Dentistry, Temple University, 3223 North Broad Street, Philadelphia, PA 19140-5007 (United States); D'Souza, Rena N. [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A and M Health Science Center, 3302 Gaston Ave., Dallas, TX 75246-2013 (United States)] [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A and M Health Science Center, 3302 Gaston Ave., Dallas, TX 75246-2013 (United States); Lu, Yongbo, E-mail: email@example.com [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A and M Health Science Center, 3302 Gaston Ave., Dallas, TX 75246-2013 (United States)] [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A and M Health Science Center, 3302 Gaston Ave., Dallas, TX 75246-2013 (United States)
Cloning and characterisation of a nuclear, site specific ssDNA binding protein.
Estradiol inducible, liver-specific expression of the apoVLDL II gene is mediated through the estrogen receptor and a variety of other DNA-binding proteins. In the present study we report the cloning and characterisation of a single-strand DNA binding protein that interacts with the lower strand of a complex regulatory site, which includes the major estrogen responsive element and a site that resembles the rat albumin site D (apoVLDL II site D). Based on its binding specificity determined with electro-mobility shift assays, the protein is named single-strand D-box binding factor (ssDBF). Analysis of the deduced 302 amino acid sequence revealed that the protein belongs to the heteronuclear ribonucleoprotein A/B family (hnRNP A/B) and resembles other known eukaryotic single-strand DNA binding proteins. Transient transfection experiments in a chicken liver cell-line showed that the protein represses estrogen-induced transcription. A protein with similar binding characteristics is present in liver nuclear extract. The relevance of the occurrence of this protein to the expression of the apoVLDL II gene is discussed. Images
Smidt, M P; Russchen, B; Snippe, L; Wijnholds, J; Ab, G
Expression of nuclear lamin A and muscle-specific proteins in differentiating muscle cells in ovo and in vitro
Primary cultures and tissue samples of chicken embryonic muscle were immunologically probed for the expression of muscle-specific proteins, such as myosin heavy chain and the tropomyosins, as well as for the nuclear lamina protein, lamin A. As determined by quantitative immunoblotting, the expres- sion of lamin A and the muscle-specific proteins were at low levels or absent in predifferentiation myoblasts
David Lourim; Jim Jung-Ching Lin
TIM Family Proteins Promote the Lysosomal Degradation of the Nuclear Receptor NUR77*
T cell immunoglobulin and mucin domain (TIM) proteins are cell-surface signaling receptors in T cells and scavenger receptors in antigen-presenting cells and kidney tubular epithelia. Here, we demonstrated a function for TIM proteins in mediating the degradation of NUR77, a nuclear receptor implicated in the cellular response to injury. TIM proteins interacted with and mediated the lysosomal degradation of NUR77 in a phosphoinositide 3-kinase�dependent pathway. We also showed dynamic cycling of TIM-1 to and from the cell surface through clathrin-dependent constitutive endocytosis. Blocking this process or mutating the phosphatidylserine-binding pocket in TIM-1 abrogated TIM-1�mediated degradation of NUR77. In an in vitro model of kidney injury, silencing TIM-1 increased NUR77 abundance and decreased epithelial cell survival. These results show that TIM proteins may affect immune cell function and the response of the kidney to injury.
Balasubramanian, Savithri; Kota, Satya Keerthi; Kuchroo, Vijay K; Humphreys, Benjamin D; Strom, Terry B
Identification of a functional nuclear export signal in the green fluorescent protein asFP499
The green fluorescent protein (GFP) asFP499 from Anemonia sulcata is a distant homologue of the GFP from Aequorea victoria. We cloned the asFP499 gene into a mammalian expression vector and showed that this protein was expressed in the human lymphoblast cell line Ramos RA1 and in the embryonic kidney 293T cell line (HEK 293T). In HEK 293T cells, asFP499 was localized mainly in the cytoplasm, suggesting that the protein was excluded from the nucleus. We identified {sub 194}LRMEKLNI{sub 201} as a candidate nuclear export signal in asFP499 and mutated the isoleucine at position 201 to an alanine. Unlike the wildtype form, the mutant protein was distributed throughout the cytoplasm and nucleus. This is First report of a GFP that contains a functional NES.
Mustafa, Huseyin [Cooperative Research Centre for Diagnostics at CSIRO Molecular and Health Technologies, 343 Royal Parade, Parkville, Vic. 3052 (Australia)]. E-mail: email@example.com; Strasser, Bernd [Cooperative Research Centre for Diagnostics at CSIRO Molecular and Health Technologies, 343 Royal Parade, Parkville, Vic. 3052 (Australia); Rauth, Sabine [Cooperative Research Centre for Diagnostics at CSIRO Molecular and Health Technologies, 343 Royal Parade, Parkville, Vic. 3052 (Australia); Irving, Robert A. [Cooperative Research Centre for Diagnostics at CSIRO Molecular and Health Technologies, 343 Royal Parade, Parkville, Vic. 3052 (Australia); Wark, Kim L. [Cooperative Research Centre for Diagnostics at CSIRO Molecular and Health Technologies, 343 Royal Parade, Parkville, Vic. 3052 (Australia)
A novel mechanism for regulating the activity of proliferating cell nuclear antigen by a small protein
Proliferating cell nuclear antigen (PCNA) forms a trimeric ring that associates with and influences the activity of many proteins participating in DNA metabolic processes and cell cycle progression. Previously, an uncharacterized small protein, encoded by TK0808 in the archaeon Thermococcus kodakarensis, was shown to stably interact with PCNA in vivo. Here, we show that this protein, designated Thermococcales inhibitor of PCNA (TIP), binds to PCNA in vitro and inhibits PCNA-dependent activities likely by preventing PCNA trimerization. Using hydrogen/deuterium exchange mass spectrometry and site-directed mutagenesis, the interacting regions of PCNA and TIP were identified. Most proteins bind to PCNA via a PCNA-interacting peptide (PIP) motif that interacts with the inter domain connecting loop (IDCL) on PCNA. TIP, however, lacks any known PCNA-interacting motif, suggesting a new mechanism for PCNA binding and regulation of PCNA-dependent activities, which may support the development of a new subclass of therapeutic biomolecules for inhibiting PCNA.
Li, Zhuo; Huang, Richard Y.-C.; Yopp, Daniel C.; Hileman, Travis H.; Santangelo, Thomas J.; Hurwitz, Jerard; Hudgens, Jeffrey W.; Kelman, Zvi
Improved method to retain cytosolic reporter protein fluorescence while staining for nuclear proteins.
Staining of transcription factors (TFs) together with retention of fluorescent reporter proteins is hindered by loss of fluorescence using current available methods. In this study, it is shown that current TF staining protocols do not destroy fluorescent proteins (FPs) but rather that fixation is not sufficient to retain FPs in the cytosol of the permeabilized cells. In this article, a simple and reliable protocol is elaborated, which allows efficient TF and cytokine staining while retaining FPs inside fixed cells. � 2014 International Society for Advancement of Cytometry. PMID:24616430
Heinen, Andr� P; Wanke, Florian; Moos, Sonja; Attig, Sebastian; Luche, Herv�; Pal, Prajna Paramita; Budisa, Nediljko; Fehling, Hans J�rg; Waisman, Ari; Kurschus, Florian C
Importin 7 and Nup358 Promote Nuclear Import of the Protein Component of Human Telomerase
In actively dividing eukaryotic cells, chromosome ends (telomeres) are subject to progressive shortening, unless they are maintained by the action of telomerase, a dedicated enzyme that adds DNA sequence repeats to chromosomal 3?end. For its enzymatic function on telomeres, telomerase requires nuclear import of its protein component (hTERT in human cells) and assembly with the RNA component, TERC. We now confirm a major nuclear localization signal (NLS) in the N-terminal region of hTERT and describe a novel one in the C-terminal part. Using an siRNA approach to deplete several import receptors, we identify importin 7 as a soluble nuclear transport factor that is required for efficient import. At the level of the nuclear pore complex (NPC), Nup358, a nucleoporin that forms the cytoplasmic filaments of the NPC, plays an important role in nuclear import of hTERT. A structure-function analysis of Nup358 revealed that the zinc finger region of the nucleoporin is of particular importance for transport of hTERT. Together, our study sheds light on the nuclear import pathway of hTERT.
Frohnert, Cornelia; Hutten, Saskia; Walde, Sarah; Nath, Annegret; Kehlenbach, Ralph H.
Importin 7 and Nup358 promote nuclear import of the protein component of human telomerase.
In actively dividing eukaryotic cells, chromosome ends (telomeres) are subject to progressive shortening, unless they are maintained by the action of telomerase, a dedicated enzyme that adds DNA sequence repeats to chromosomal 3'end. For its enzymatic function on telomeres, telomerase requires nuclear import of its protein component (hTERT in human cells) and assembly with the RNA component, TERC. We now confirm a major nuclear localization signal (NLS) in the N-terminal region of hTERT and describe a novel one in the C-terminal part. Using an siRNA approach to deplete several import receptors, we identify importin 7 as a soluble nuclear transport factor that is required for efficient import. At the level of the nuclear pore complex (NPC), Nup358, a nucleoporin that forms the cytoplasmic filaments of the NPC, plays an important role in nuclear import of hTERT. A structure-function analysis of Nup358 revealed that the zinc finger region of the nucleoporin is of particular importance for transport of hTERT. Together, our study sheds light on the nuclear import pathway of hTERT. PMID:24586428
Frohnert, Cornelia; Hutten, Saskia; W�lde, Sarah; Nath, Annegret; Kehlenbach, Ralph H
Muscovy duck reovirus p10.8 protein localizes to the nucleus via a nonconventional nuclear localization signal
Background It was previously report that the first open reading frame of Muscovy duck reocvirus S4 gene encodes a 95-amino-acid protein, designed p10.8, which has no sequence similarity to other known proteins. Its amino acid sequence offers no clues about its function. Results Subcellular localization and nuclear import signal of p10.8 were characterized. We found that p10.8 protein localizes to the nucleus of infected and transfected cells, suggesting that p10.8 nuclear localization is not facilitated by viral infection or any other viral protein. A functional non-canonical nuclear localization signal (NLS) for p10.8 was identified and mapped to N-terminus residues 1�40. The NLS has the ability to retarget a large cytoplasmic protein to the nucleus. Conclusions p10.8 imported into the nucleus might via a nonconventional signal nuclear signal.
Tristetraprolin Inhibits Poly(A)-Tail Synthesis in Nuclear mRNA that Contains AU-Rich Elements by Interacting with Poly(A)-Binding Protein Nuclear 1
Background Tristetraprolin binds mRNA AU-rich elements and thereby facilitates the destabilization of mature mRNA in the cytosol. Methodology/Principal Findings To understand how tristetraprolin mechanistically functions, we biopanned with a phage-display library for proteins that interact with tristetraprolin and retrieved, among others, a fragment of poly(A)-binding protein nuclear 1, which assists in the 3'-polyadenylation of mRNA by binding to immature poly(A) tails and thereby increases the activity of poly(A) polymerase, which is directly responsible for polyadenylation. The tristetraprolin/poly(A)-binding protein nuclear 1 interaction was characterized using tristetraprolin and poly(A)-binding protein nuclear 1 deletion mutants in pull-down and co-immunoprecipitation assays. Tristetraprolin interacted with the carboxyl-terminal region of poly(A)-binding protein nuclear 1 via its tandem zinc finger domain and another region. Although tristetraprolin and poly(A)-binding protein nuclear 1 are located in both the cytoplasm and the nucleus, they interacted in vivo in only the nucleus. In vitro, tristetraprolin bound both poly(A)-binding protein nuclear 1 and poly(A) polymerase and thereby inhibited polyadenylation of AU-rich element�containing mRNAs encoding tumor necrosis factor ?, GM-CSF, and interleukin-10. A tandem zinc finger domain�deleted tristetraprolin mutant was a less effective inhibitor. Expression of a tristetraprolin mutant restricted to the nucleus resulted in downregulation of an AU-rich element�containing tumor necrosis factor ?/luciferase mRNA construct. Conclusion/Significance In addition to its known cytosolic mRNA�degrading function, tristetraprolin inhibits poly(A) tail synthesis by interacting with poly(A)-binding protein nuclear 1 in the nucleus to regulate expression of AU-rich element�containing mRNA.
Chiang, Pei-Yu; Lin, Nien-Yi; Shen, Yu-Fang; Chang, Geen-Dong; Chang, Ching-Jin
Nuclear Localization of Japanese Encephalitis Virus Core Protein Enhances Viral Replication�
Japanese encephalitis virus (JEV) core protein was detected in both the nucleoli and cytoplasm of mammalian and insect cell lines infected with JEV or transfected with the expression plasmid of the core protein. Mutation analysis revealed that Gly42 and Pro43 in the core protein are essential for the nuclear and nucleolar localization. A mutant M4243 virus in which both Gly42 and Pro43 were replaced by Ala was recovered by plasmid-based reverse genetics. In C6/36 mosquito cells, the M4243 virus exhibited RNA replication and protein synthesis comparable to wild-type JEV, whereas propagation in Vero cells was impaired. The mutant core protein was detected in the cytoplasm but not in the nucleus of either C6/36 or Vero cell lines infected with the M4243 virus. The impaired propagation of M4243 in mammalian cells was recovered by the expression of wild-type core protein in trans but not by that of the mutant core protein. Although M4243 mutant virus exhibited a high level of neurovirulence comparable to wild-type JEV in spite of the approximately 100-fold-lower viral propagation after intracerebral inoculation to 3-week-old mice of strain Jcl:ICR, no virus was recovered from the brain after intraperitoneal inoculation of the mutant. These results indicate that nuclear localization of JEV core protein plays crucial roles not only in the replication in mammalian cells in vitro but also in the pathogenesis of encephalitis induced by JEV in vivo.
Mori, Yoshio; Okabayashi, Tamaki; Yamashita, Tetsuo; Zhao, Zijiang; Wakita, Takaji; Yasui, Kotaro; Hasebe, Futoshi; Tadano, Masayuki; Konishi, Eiji; Moriishi, Kohji; Matsuura, Yoshiharu
Temporal association of protamine 1 with the inner nuclear membrane protein lamin B receptor during spermiogenesis.
During mammalian spermiogenesis, histones are replaced by transition proteins, which are in turn replaced by protamines P1 and P2. P1 protamine contains a short arginine/serine-rich (RS) domain that is highly phosphorylated before being deposited into sperm chromatin and almost completely dephosphorylated during sperm maturation. We now demonstrate that, in elongating spermatids, this phosphorylation is required for the temporal association of P1 protamine with lamin B receptor (LBR), an inner nuclear membrane protein that also possesses a stretch of RS dipeptides at its nucleoplasmic NH(2)-terminal domain. Previous studies have shown that the cellular protein p32 also binds tightly to the unmodified RS domain of LBR. Extending those findings, we now present evidence that p32 prevents phosphorylation of LBR and furthermore that dissociation of this protein precedes P1 protamine association. Our data suggest that docking of protamine 1 to the nuclear envelope is an important intermediate step in spermiogenesis and reveal a novel role for SR protein kinases and p32. PMID:14701833
Mylonis, Ilias; Drosou, Victoria; Brancorsini, Stefano; Nikolakaki, Eleni; Sassone-Corsi, Paolo; Giannakouros, Thomas
LaRbp38: A Leishmania amazonensis protein that binds nuclear and kinetoplast DNAs
Leishmania amazonensis causes a wide spectrum of leishmaniasis. There are no vaccines or adequate treatment for leishmaniasis, therefore there is considerable interest in the identification of new targets for anti-leishmania drugs. The central role of telomere-binding proteins in cell maintenance makes these proteins potential targets for new drugs. In this work, we used a combination of purification chromatographies to screen L. amazonensis proteins for molecules capable of binding double-stranded telomeric DNA. This approach resulted in the purification of a 38 kDa polypeptide that was identified by mass spectrometry as Rbp38, a trypanosomatid protein previously shown to stabilize mitochondrial RNA and to associate with nuclear and kinetoplast DNAs. Western blotting and supershift assays confirmed the identity of the protein as LaRbp38. Competition and chromatin immunoprecipitation assays confirmed that LaRbp38 interacted with kinetoplast and nuclear DNAs in vivo and suggested that LaRbp38 may have dual cellular localization and more than one function.
Lira, C.B.B. [Departamento de Genetica, IB, Universidade Estadual de Sao Paulo, UNESP, 18618-000, Botucatu, SP (Brazil); Instituto de Biologia, UNICAMP, Campinas, SP (Brazil); Siqueira Neto, J.L. [Departamento de Genetica, IB, Universidade Estadual de Sao Paulo, UNESP, 18618-000, Botucatu, SP (Brazil); Instituto de Biologia, UNICAMP, Campinas, SP (Brazil); Giardini, M.A. [Departamento de Genetica, IB, Universidade Estadual de Sao Paulo, UNESP, 18618-000, Botucatu, SP (Brazil); Instituto de Biologia, UNICAMP, Campinas, SP (Brazil); Winck, F.V. [Instituto de Biologia, UNICAMP, Campinas, SP (Brazil); Ramos, C.H.I. [Instituto de Quimica, UNICAMP, Campinas, SP (Brazil); Cano, M.I.N. [Departamento de Genetica, IB, Universidade Estadual de Sao Paulo, UNESP, 18618-000, Botucatu, SP (Brazil)]. E-mail: firstname.lastname@example.com
Molecular basis for the redox control of nuclear transport of the structural chromatin protein Hmgb1
Oxidative stress can induce a covalent disulfide bond between protein and peptide thiols that is reversible through enzymatic catalysis. This process provides a post-translational mechanism for control of protein function and may also protect thiol groups from irreversible oxidation. High mobility group protein B1 (Hmgb1), a DNA-binding structural chromosomal protein and transcriptional co-activator was identified as a substrate of glutaredoxin. Hmgb1 contains 3 cysteines, Cys23, 45, and 106. In mild oxidative conditions, Cys23 and Cys45 readily form an intramolecular disulfide bridge, whereas Cys106 remains in the reduced form. The disulfide bond between Cys23 and Cys45 is a target of glutathione-dependent reduction by glutaredoxin. Endogenous Hmgb1 as well as GFP-tagged wild-type Hmgb1 co-localize in the nucleus of CHO cells. While replacement of Hmgb1 Cys23 and/or 45 with serines did not affect the nuclear distribution of the mutant proteins, Cys106-to-Ser and triple cysteine mutations impaired nuclear localization of Hmgb1. Our cysteine targeted mutational analysis suggests that Cys23 and 45 induce conformational changes in response to oxidative stress, whereas Cys106 appears to be critical for the nucleocytoplasmic shuttling of Hmgb1.
Hoppe, George [Cole Eye Institute, Cleveland Clinic, Cleveland, OH 44195 (United States)]. E-mail: email@example.com; Talcott, Katherine E. [Cole Eye Institute, Cleveland Clinic, Cleveland, OH 44195 (United States); Bhattacharya, Sanjoy K. [Cole Eye Institute, Cleveland Clinic, Cleveland, OH 44195 (United States); Crabb, John W. [Cole Eye Institute, Cleveland Clinic, Cleveland, OH 44195 (United States); Sears, Jonathan E. [Cole Eye Institute, Cleveland Clinic, Cleveland, OH 44195 (United States)
Nuclear Microinjection to Assess How Heterologously Expressed Proteins Impact Ca2+ Signals in Xenopus Oocytes
The Xenopus oocyte is frequently used for heterologous expression and for studying the spatiotemporal patterning of Ca2+ signals. Here, we outline a protocol for nuclear microinjection of the Xenopus oocyte for the purpose of studying how subsequently expressed proteins impact intracellular Ca2+ signals evoked by inositol trisphosphate (InsP3). Injected oocytes can easily be identified by reporter technologies and the impact of heterologously expressed proteins on the generation and properties of InsP3-evoked Ca2+ signals can be resolved using caged InsP3 and fluorescent Ca2+ indicators.
Lin-Moshier, Yaping; Marchant, Jonathan S.
NUP98 fusion in human leukemia: dysregulation of the nuclear pore and homeodomain proteins.
NUP98 is fused to a variety of partner genes, including abdominal B-like HOX, in human myeloid and T-cell malignancies via chromosomal translocation involving 11p15. NUP98 encodes a 98-kd nucleoporin that is a component of the nuclear pore complex and functions in nucleocytoplasmic transport, with its N-terminal GLFG repeats used as a docking site for karyopherins. Disruption of NUP98 may affect the nuclear pore function, and the abnormal expression and altered function of fusion partners may also be critical for leukemia development. Recent studies using mouse models expressing NUP98-HOX have confirmed its leukemogenic potential, and cooperative genes for NUP98-HOXA9 in leukemogenesis have been identified in these studies.Thus, the NUP98 chimera is a unique molecule that provides valuable information regarding nuclear pore function and the role of the homeobox protein in leukemogenesis/carcinogenesis. PMID:16105755
Nakamura, Takuro
A nuclear FK506-binding protein is a histone chaperone regulating rDNA silencing
We report a novel chromatin-modulating factor, nuclear FK506-binding protein (FKBP). It is a member of the peptidyl prolyl cis-trans isomerase (PPIase) family, whose members were originally identified as enzymes that assist in the proper folding of polypeptides. The endogenous FKBP gene is required for the in vivo silencing of gene expression at the rDNA locus and FKBP has histone chaperone
Takashi Kuzuhara; Masami Horikoshi
Transcriptional activation of NAD +-dependent protein deacetylase SIRT1 by nuclear receptor TLX
An orphan nuclear receptor TLX is a transcriptional repressor that promotes the proliferation and self-renewal of neural precursor cells (NPCs). SIRT1, an NAD+-dependent protein deacetylase, is highly expressed in the NPCs and participates in neurogenesis. Here, we found that TLX colocalized with SIRT1 and knockdown of TLX by small interfering RNAs decreased SIRT1 levels in NPCs. TLX increased the SIRT1
Naotoshi Iwahara; Shin Hisahara; Takashi Hayashi; Yoshiyuki Horio
Utilization of nuclear structural proteins for targeted therapy and detection of proliferative and differentiation disorders
The localization of nuclear apparatus proteins (NUMA) is used to identify tumor cells and different stages in the tumor progression and differentiation processes. There is a characteristic organization of NuMA in tumor cells and in phenotypically normal cells. NuMA distribution patterns are significantly less diffuse in proliferating non-malignant cells compared to malignant cells. The technique encompasses cell immunostaining using a NuMA specific antibody, and microscopic analysis of NuMA distribution within each nucleus.
Lelievre, Sophie (Berkeley, CA); Bissell, Mina (Berkeley, CA)
Identification and characterisation of two trypanosome TFIIS proteins exhibiting particular domain architectures and differential nuclear localisations
Summary Nuclear transcription of Trypanosoma brucei displays unusual features. Most protein-coding genes are orga- nized in large directional gene clusters, which are transcribed polycistronically by RNA polymerase II (pol II) with subsequent processing to generate mature mRNA. Here, we describe the identification and char- acterization of two trypanosome homologues of tran- scription elongation factor TFIIS (TbTFIIS1 and TbTFIIS2-1). TFIIS has
Pierrick Uzureau; Jan-Peter Daniels; David Walgraffe; Bill Wickstead; Etienne Pays; Keith Gull; Luc Vanhamme
Adenovirus Hexon Protein Enhances Nuclear Delivery and Increases Transgene Expression of Polyethylenimine\\/Plasmid DNA Vectors
Inefficient nuclear delivery restricts transgene expression using polyelectrolyte DNA vectors. To increase transfer from the cytoplasm to the nucleus, we have covalently linked adenovirus hexon protein to polyethylenimine (PEI, 800 kDa). Activity of the conjugate was compared with PEI and PEI linked to albumin. Hexon-containing complexes gave 10-fold greater transgene expression in HepG2 cells than PEI\\/DNA or complexes containing albumin,
Robert C. Carlisle; Thierry Bettinger; Manfred Ogris; Sarah Hale; Vivien Mautner; Leonard W. Seymour
UNCL, the mammalian homologue of UNC-50, is an inner nuclear membrane RNA-binding protein.
We isolated a mammalian homologue of the C. elegans gene unc-50 that we have named UNCL. The 777 kb rat UNCL cDNA encodes a 259 amino acid protein that is expressed in a wide variety of tissues with highest mRNA levels in brain, kidney and testis. Hydropathy plot analysis and in vitro translation experiments with microsomal membranes indicate that UNCL is a transmembrane protein. Hemagglutinin tagged UNCL was stably transfected into SaOS-2 osteosarcoma cells and exhibited a nuclear rim staining pattern which was retained following extraction with 1% Triton X-100, suggesting that UNCL localizes to the inner nuclear membrane. UNCL-HA was extractable in 350 mM NaCl, suggesting that UNCL is not associated with the nuclear matrix. Homopolymer RNA-binding assays performed on in vitro translated UNCL protein and 'structural modeling by homology' suggest that UNCL binds RNA via an amino-terminal RNA Recognition-like Motif. Since unc-50 is required for expression of assembled muscle-type nicotinic receptors in the nematode we investigated whether UNCL had a similar function for mammalian nicotinic receptors. When UNCL was co-expressed with neural nicotinic receptors in Xenopus oocytes or COS cells it increased expression of functional cell surface receptors up to 1. 6-fold. We conclude that UNCL is a novel inner nuclear membrane protein that associates with RNA and is involved in the cell-surface expression of neuronal nicotinic receptors. UNCL plays a broader role because UNCL homologues are present in two yeast and a plant species, none of which express nicotinic receptors and it is also found in tissues that lack nicotinic receptors. PMID:10980252
Fitzgerald, J; Kennedy, D; Viseshakul, N; Cohen, B N; Mattick, J; Bateman, J F; Forsayeth, J R
A cancer-associated RING finger protein, RNF43, is a ubiquitin ligase that interacts with a nuclear protein, HAP95
RNF43 is a recently discovered RING finger protein that is implicated in colon cancer pathogenesis. This protein possesses growth-promoting activity but its mechanism remains unknown. In this study, to gain insight into the biological action of RNF43 we characterized it biochemically and intracellularly. A combination of indirect immunofluorescence analysis and biochemical fractionation experiments suggests that RNF43 resides in the endoplasmic reticulum (ER) as well as in the nuclear envelope. Sucrose density gradient fractionation demonstrates that RNF43 co-exists with emerin, a representative inner nuclear membrane protein in the nuclear subcompartment. The cell-free system with pure components reveals that recombinant RNF43 fused with maltose-binding protein has autoubiquitylation activity. By the yeast two-hybrid screening we identified HAP95, a chromatin-associated protein interfacing the nuclear envelope, as an RNF43-interacting protein and substantiated this interaction in intact cells by the co-immunoprecipitation experiments. HAP95 is ubiquitylated and subjected to a proteasome-dependent degradation pathway, however, the experiments in which 293 cells expressing both RNF43 and HAP95 were treated with a proteasome inhibitor, MG132, show that HAP95 is unlikely to serve as a substrate of RNF43 ubiquitin ligase. These results infer that RNF43 is a resident protein of the ER and, at least partially, the nuclear membrane, with ubiquitin ligase activity and may be involved in cell growth control potentially through the interaction with HAP95.
Sugiura, Takeyuki [Discovery Research Laboratory, Tokyo R and D Center, Daiichi Pharmaceutical Co. Ltd., Daiichi-Sankyo group, 16-13, Kitakasai 1-Chome, Edogawa-ku, Tokyo, 134-8630 (Japan)], E-mail: firstname.lastname@example.com; Yamaguchi, Aya; Miyamoto, Kentaro [Discovery Research Laboratory, Tokyo R and D Center, Daiichi Pharmaceutical Co. Ltd., Daiichi-Sankyo group, 16-13, Kitakasai 1-Chome, Edogawa-ku, Tokyo, 134-8630 (Japan)
A cancer-associated RING finger protein, RNF43, is a ubiquitin ligase that interacts with a nuclear protein, HAP95.
RNF43 is a recently discovered RING finger protein that is implicated in colon cancer pathogenesis. This protein possesses growth-promoting activity but its mechanism remains unknown. In this study, to gain insight into the biological action of RNF43 we characterized it biochemically and intracellularly. A combination of indirect immunofluorescence analysis and biochemical fractionation experiments suggests that RNF43 resides in the endoplasmic reticulum (ER) as well as in the nuclear envelope. Sucrose density gradient fractionation demonstrates that RNF43 co-exists with emerin, a representative inner nuclear membrane protein in the nuclear subcompartment. The cell-free system with pure components reveals that recombinant RNF43 fused with maltose-binding protein has autoubiquitylation activity. By the yeast two-hybrid screening we identified HAP95, a chromatin-associated protein interfacing the nuclear envelope, as an RNF43-interacting protein and substantiated this interaction in intact cells by the co-immunoprecipitation experiments. HAP95 is ubiquitylated and subjected to a proteasome-dependent degradation pathway, however, the experiments in which 293 cells expressing both RNF43 and HAP95 were treated with a proteasome inhibitor, MG132, show that HAP95 is unlikely to serve as a substrate of RNF43 ubiquitin ligase. These results infer that RNF43 is a resident protein of the ER and, at least partially, the nuclear membrane, with ubiquitin ligase activity and may be involved in cell growth control potentially through the interaction with HAP95. PMID:18313049
Sugiura, Takeyuki; Yamaguchi, Aya; Miyamoto, Kentaro
Characterization of the cassava geminivirus transcription activation protein putative nuclear localization signal.
The geminivirus transcription activation protein (TrAP) localizes to the nucleus and contains a putative nuclear localization signal (NLS) ((28)PRRRR(32)) on the N-terminus. The role of individual residues of this putative NLS on nuclear localization and symptom induction was investigated using TrAP of East African cassava mosaic Cameroon virus (EACMCV). Subcellular localization was conducted using the green fluorescent protein (GFP). Results showed that the proline residue at position 28 (Pro-28) is essential for nuclear localization whereas individually, none of the four contiguous arginines is necessary for nuclear targeting. The role of each of the five NLS amino acid residues on TrAP-mediated disease phenotype and gene silencing suppression was investigated by expressing these mutants in Nicotiana benthamiana from the PVX vector and under the control of the Cauliflower mosaic virus 35S promoter. Results showed that all five residues of the NLS play a role on disease phenotype production in N. benthamiana plants. Furthermore, each of the NLS residues appeared to be required for suppression of VIGS but appeared not to be required for the ability of TrAP to transactivate transcription and interact with adenosine kinase (ADK). PMID:19665038
Chowda-Reddy, R V; Dong, Wubei; Felton, Christian; Ryman, Danielle; Ballard, Keith; Fondong, Vincent N
Heterogenous nuclear ribonucleoprotein Q increases protein expression from HIV-1 Rev-dependent transcripts
Background Heterogenous nuclear ribonucleoproteins (hnRNPs) control many processes of the gene expression machinery including mRNA transcription, splicing, export, stability and translation. Recent data show interaction of the HIV-1 Rev regulatory protein with a subset of hnRNP proteins, that includes hnRNP Q, suggesting that hnRNPs can contribute to regulation of HIV-1 gene expression by Rev. Findings In this work we address the effect of hnRNP Q on Rev-dependent gene expression. We show that hnRNP Q overexpression increased levels of proteins produced from a Rev-dependent reporter gene in the presence of Rev. Increased protein levels did not correlate with changes in either the levels or the nucleocytoplasmic distribution of Rev-dependent reporter mRNAs. Similar observations were made in persistently HIV-1 infected HeLa cells. In these cells, hnRNP Q overexpression increased levels of the HIV-1 Gag-p24 protein, while levels of viral Rev-dependent mRNAs were not affected. Conclusion Our data indicate that hnRNP Q can stimulate the protein production of Rev-dependent mRNAs without changing mRNA levels and mRNA export, respectively. This suggests that hnRNP Q can boost HIV gene expression at the level of protein production.
Ubinuclein, a novel nuclear protein interacting with cellular and viral transcription factors.
The major target tissues for Epstein-Barr virus (EBV) infection are B lymphocytes and epithelial cells of the oropharyngeal zone. The product of the EBV BZLF1 early gene, EB1, a member of the basic leucine-zipper family of transcription factors, interacts with both viral and cellular promoters and transcription factors, modulating the reactivation of latent EBV infection. Here, we characterize a novel cellular protein interacting with the basic domains of EB1 and c-Jun, and competing of their binding to the AP1 consensus site. The transcript is present in a wide variety of human adult, fetal, and tumor tissues, and the protein is detected in the nuclei throughout the human epidermis and as either grainy or punctuate nuclear staining in the cultured keratinocytes. The overexpression of tagged cDNA constructs in keratinocytes revealed that the NH(2) terminus is essential for the nuclear localization, while the central domain is responsible for the interaction with EB1 and for the phenotype of transfected keratinocytes similar to terminal differentiation. The gene was identified in tail-to-tail orientation with the periplakin gene (PPL) in human chromosome 16p13.3 and in a syntenic region in mouse chromosome 16. We designated this novel ubiquitously expressed nuclear protein as ubinuclein and the corresponding gene as UBN1. PMID:10725330
Aho, S; Buisson, M; Pajunen, T; Ryoo, Y W; Giot, J F; Gruffat, H; Sergeant, A; Uitto, J
Oxidized LDL enhances stretch-induced smooth muscle cell proliferation through alterations in nuclear protein import.
Mechanical stress contributes to hypertension and atherosclerosis partly through the stimulation of vascular smooth muscle cell (VSMC) proliferation. Oxidized low density lipoprotein (oxLDL) is another important atherogenic factor that can increase VSMC proliferation. The purpose of this study was to investigate whether oxLDL could further enhance the proliferative action of mechanical stretch on VSMC, and to determine the mechanism responsible for this interaction. Because nuclear protein import is critical in regulating gene expression, transcription, and cell proliferation, its involvement in the mitogenic effects of oxLDL and mechanical stress was studied. OxLDL enhanced the proliferative effects of mechanical stretch on its own in rabbit aortic VSMC, and induced increases in the expression of HSP60 in an additive manner. Adenoviral-mediated overexpression of HSP60 induced increases in cell proliferation compared with uninfected VSMC. Mechanical stretch and oxLDL stimulated the rate of nuclear protein import in VSMC and increased the expression of nucleoporins. These effects were sensitive to inhibition of the MAPK pathway. We conclude that oxLDL and mechanical stretch have a synergistic effect on VSMC proliferation. This synergistic effect is induced through a stimulation of nuclear protein import via HSP60 and an activation of the MAPK pathway. PMID:23210434
Chahine, Mirna N; Dibrov, Elena; Blackwood, David P; Pierce, Grant N
The inner-nuclear-envelope protein emerin regulates HIV-1 infectivity.
Primate lentiviruses such as human immunodeficiency type 1 (HIV-1) have the capacity to infect non-dividing cells such as tissue macrophages. In the process, viral complementary DNA traverses the nuclear envelope to integrate within chromatin. Given the intimate association between chromatin and the nuclear envelope, we examined whether HIV-1 appropriates nuclear envelope components during infection. Here we show that emerin, an integral inner-nuclear-envelope protein, is necessary for HIV-1 infection. Infection of primary macrophages lacking emerin was abortive in that viral cDNA localized to the nucleus but integration into chromatin was inefficient, and conversion of viral cDNA to non-functional episomal cDNA increased. HIV-1 cDNA associated with emerin in vivo, and the interaction of viral cDNA with chromatin was dependent on emerin. Barrier-to-autointegration factor (BAF), the LEM (LAP, emerin, MAN) binding partner of emerin, was required for the association of viral cDNA with emerin and for the ability of emerin to support virus infection. Therefore emerin, which bridges the interface between the inner nuclear envelope and chromatin, may be necessary for chromatin engagement by viral cDNA before integration. PMID:16680152
Jacque, Jean-Marc; Stevenson, Mario
Unichrom, a novel nuclear matrix protein, binds to the Ars insulator and canonical MARs.
Eukaryotic genomic DNA is organized into loop structures by attachments to the nuclear matrix. These attachments to the nuclear matrix have been supposed to form the boundaries of chromosomal DNA. Insulators or boundary elements are defined by two characteristics: they interrupt promoter-enhancer communications when inserted between them, and they suppress the silencing of transgenes stably integrated into inactive chromosomal domains. We recently identified an insulator element in the upstream region of the sea urchin arylsulfatase (HpArs) gene that shows both enhancer blocking and suppression of position effects. Here, we report that Unichrom, originally identified by its G-stretch DNA binding capability, is a nuclear matrix protein that binds to the Ars insulator and canonical nuclear matrix attachment regions (MARs). We also show that Unichrom recognizes the minor groove of the AT-rich region within the Ars insulator, which may have a base-unpairing property, as well as the G-stretch DNA. Furthermore, Unichrom selectively interacts with poly(dG).poly(dC), poly(dA).poly(dT) and poly(dAT).poly(dAT), but not with poly(dGC).poly(dGC). Unichrom also shows high affinity for single-stranded G- and C-stretches. We discuss the DNA binding motif of Unichrom and the function of Unichrom in the nuclear matrix. PMID:16547401
Tagashira, Hideki; Shimotori, Taishin; Sakamoto, Naoaki; Katahira, Masato; Miyanoiri, Yohei; Yamamoto, Takashi; Mitsunaga-Nakatsubo, Keiko; Shimada, Hiraku; Kusunoki, Shinichiro; Akasaka, Koji
Protein kinase A-dependent phosphorylation modulates DNA-binding activity of hepatocyte nuclear factor 4.
Hepatocyte nuclear factor 4 (HNF4), a liver-enriched transcription factor of the nuclear receptor superfamily, is critical for development and liver-specific gene expression. Here, we demonstrate that its DNA-binding activity is modulated posttranslationally by phosphorylation in vivo, ex vivo, and in vitro. In vivo, HNF4 DNA-binding activity is reduced by fasting and by inducers of intracellular cyclic AMP (cAMP) accumulation. A consensus protein kinase A (PKA) phosphorylation site located within the A box of its DNA-binding domain has been identified, and its role in phosphorylation-dependent inhibition of HNF4 DNA-binding activity has been investigated. Mutants of HNF4 in which two potentially phosphorylatable serines have been replaced by either neutral or charged amino acids were able to bind DNA in vitro with affinity similar to that of the wild-type protein. However, phosphorylation by PKA strongly repressed the binding affinity of the wild-type factor but not that of HNF4 mutants. Accordingly, in transfection assays, expression vectors for the mutated HNF4 proteins activated transcription more efficiently than that for the wild-type protein-when cotransfected with the PKA catalytic subunit expression vector. Therefore, HNF4 is a direct target of PKA which might be involved in the transcriptional inhibition of liver genes by cAMP inducers.
Viollet, B; Kahn, A; Raymondjean, M
Covalent modification of nuclear and cytoplasmic proteins of hamster embryo cells during PAH metabolism
It is well known that polynuclear aromatic hydrocarbons (PAH) are metabolized by mammalian cells to derivatives which covalently modify cellular macromolecules. Much effort has been devoted to defining the molecular structure of the major adducts formed with DNA in cells treated with benzo(a)pyrene (B(a)P). However, despite elucidation of the major pathways involved it has not been possible to unequivocally link the formation of specific adducts with the steps leading to malignant tranaformation. During the metabolism of B(a)P by hamster embryo cells (HECs), over 70% of the binding to nuclear macromolecules involves protein. Since nuclear proteins are known to play important roles in the structure of chromatin and are thought to be involved in the regulation of gene expression, the modification of these proteins by the introduction of PAH-adducts might reasonably be expected to perturb the structure and function of chromatin. Such perturbations could be involved in the processes which cause malignant transformation. Therefore, it seemed important to us to begin to define the pathways leading to protein-PAH binding in HECs treated with B(a)P.
MacLeod, M.C.; Kootstra, A.; Slaga, T.J.; Selkirk, J.K.
Dynamic force-induced direct dissociation of protein complexes in a nuclear body in living cells.
Despite past progress in understanding mechanisms of cellular mechanotransduction, it is unclear whether a local surface force can directly alter nuclear functions without intermediate biochemical cascades. Here we show that a local dynamic force via integrins results in direct displacements of coilin and SMN proteins in Cajal bodies and direct dissociation of coilin-SMN associated complexes. Spontaneous movements of coilin increase more than those of SMN in the same Cajal body after dynamic force application. Fluorescence resonance energy transfer changes of coilin-SMN depend on force magnitude, an intact F-actin, cytoskeletal tension, Lamin A/C, or substrate rigidity. Other protein pairs in Cajal bodies exhibit different magnitudes of fluorescence resonance energy transfer. Dynamic cyclic force induces tiny phase lags between various protein pairs in Cajal bodies, suggesting viscoelastic interactions between them. These findings demonstrate that dynamic force-induced direct structural changes of protein complexes in Cajal bodies may represent a unique mechanism of mechanotransduction that impacts on nuclear functions involved in gene expression. PMID:22643893
Poh, Yeh-Chuin; Shevtsov, Sergey P; Chowdhury, Farhan; Wu, Douglas C; Na, Sungsoo; Dundr, Miroslav; Wang, Ning
Induction of apoptosis is driven by nuclear retention of protein kinase C delta.
Protein kinase C delta (PKC delta) mediates apoptosis downstream of many apoptotic stimuli. Because of its ubiquitous expression, tight regulation of the proapoptotic function of PKC delta is critical for cell survival. Full-length PKC delta is found in all cells, whereas the catalytic fragment of PKC delta, generated by caspase cleavage, is only present in cells undergoing apoptosis. Here we show that full-length PKC delta transiently accumulates in the nucleus in response to etoposide and that nuclear translocation precedes caspase cleavage of PKC delta. Nuclear PKC delta is either cleaved by caspase 3, resulting in accumulation of the catalytic fragment in the nucleus, or rapidly exported by a Crm1-sensitive pathway, thereby assuring that sustained nuclear accumulation of PKC delta is coupled to caspase activation. Nuclear accumulation of PKC delta is necessary for caspase cleavage, as mutants of PKC delta that do not translocate to the nucleus are not cleaved. However, caspase cleavage of PKC delta per se is not required for apoptosis, as an uncleavable form of PKC delta induces apoptosis when retained in the nucleus by the addition of an SV-40 nuclear localization signal. Finally, we show that kinase negative full-length PKC delta does not translocate to the nucleus in apoptotic cells but instead inhibits apoptosis by blocking nuclear import of endogenous PKC delta. These studies demonstrate that generation of the PKC delta catalytic fragment is a critical step for commitment to apoptosis and that nuclear import and export of PKC delta plays a key role in regulating the survival/death pathway. PMID:17562707
DeVries-Seimon, Tracie A; Ohm, Angela M; Humphries, Michael J; Reyland, Mary E
A discrete 3' region of U6 small nuclear RNA modulates the phosphorylation cycle of the C1 heterogeneous nuclear ribonucleoprotein particle protein.
The C heterogeneous ribonucleoprotein particle (hnRNP) protein bind to nascent pre-mRNA and may participate in assembly of the early prespliceosome. Ser/Thr phosphorylation of the C1 hnRNP protein in HeLa nuclear extracts regulates its binding to pre-mRNA (S. H. Mayrand, P. Dwen, and T. Pederson, Proc. Natl. Acad. Sci. USA 90:7764-7768, 1993). We have now further investigated the phosphorylation cycle of the C1 hnRNP protein, with emphasis on its regulation. Pretreatment of nuclear extracts with micrococcal nuclease eliminated the phosphorylation of C1 hnRNP protein, but pretreatment with DNase did not, suggesting a dependence on RNA. Oligodeoxynucleotide-targeted RNase H cleavage of U1, U2, and U4 small nuclear RNAs did not affect the phosphorylation of C1 hnRNP protein. However, cleavage of nucleotides 78 to 95, but not other regions, of U6 small nuclear RNA resulted in an inhibition of the dephosphorylation step of the C1 hnRNP protein phosphorylation cycle. This inhibition was as pronounced as that seen with the serine/threonine protein phosphatase inhibitor okadaic acid. C1 hnRNP protein dephosphorylation could be completely restored by the addition of intact U6 RNA. Add-back experiments with mutant RNAs further delineated the minimal region essential for C1 protein dephosphorylation as residing in nucleotides 85 to 92 of U6 RNA. These results illuminate a hitherto unanticipated function of U6 RNA: the modulation of a phosphorylation-dephosphorylation cycle of C1 hnRNP protein that influences the binding affinity of this protein for pre-mRNA. This newly revealed function of U6 RNA is likely to play a very early role in the prespliceosome assembly pathway, prior to U6 RNA's entry into the mature spliceosome's active center.
Mayrand, S H; Fung, P A; Pederson, T
Nuclear Import of UBL-Domain Protein Mdy2 Is Required for Heat-Induced Stress Response in Saccharomyces cerevisiae
Ubiquitin (Ub) and ubiquitin-like (UBL) proteins regulate a diverse array of cellular pathways through covalent as well as non-covalent interactions with target proteins. Yeast protein Mdy2 (Get5) and its human homolog GdX (Ubl4a) belong to the class of UBL proteins which do not form conjugates with other proteins. Mdy2 is required for cell survival under heat stress and for efficient mating. As part of a complex with Sgt2 and Get4 it has been implicated in the biogenesis of tail-anchored proteins. Interestingly, in response to heat stress, Mdy2 protein that is predominantly localized in the nucleus co-localized with poly(A)-binding protein Pab1 to cytoplasmic stress granules suggesting that nucleocytoplasmic shuttling is of functional importance. Here we investigate the nuclear import of Mdy2, a process that is independent of the Get4/Sgt2 complex but required for stress response. Nuclear import is mediated by an N-terminal nuclear localization signal (NLS) and this process is essential for the heat stress response. In contrast, cells expressing Mdy2 lacking a nuclear export signal (NES) behave like wild type. Importantly, both Mdy2 and Mdy2-?NES, but not Mdy2-?NLS, physically interact with Pab1 and this interaction correlates with the accumulation in cytoplasmic stress granules. Thus, the nuclear history of the UBL Mdy2 appears to be essential for its function in cytoplasmic stress granules during the rapid cellular response to heat stress.
Arhzaouy, Khalid; Ramezani-Rad, Massoud
Extremely Long-lived Nuclear Pore Proteins in the Rat Brain+
To combat the functional decline of the proteome, cells use the process of protein turnover to replace potentially impaired polypeptides with new functional copies. Here we found that extremely long-lived proteins (ELLPs) did not turn over in post-mitotic cells of the rat central nervous system. These ELLPs were associated with chromatin and the nuclear pore complex, the central transport channels that mediate all molecular trafficking in and out of the nucleus. The longevity of these proteins would be expected to expose them to potentially harmful metabolites putting them at risk of accumulating damage over extended periods of time. Thus, it is possible that failure to maintain proper levels and functional integrity of ELLPs in non-proliferative cells might contribute to age-related deterioration in cell and tissue function.
Savas, Jeffrey N.; Toyama, Brandon H.; Xu, Tao; Yates, John R.; Hetzer, Martin W.
Deuterium nuclear magnetic resonance study of protein dynamics and protein-lipid interactions in model and biological membranes
The dynamics of the Halobacterium halobium purple membrane protein, bacteriorhodopsin; and the motional order of the fluorescent lipid probe, diphenylhexatriene, in various model membrane systems, have been investigated by deuterium nuclear magnetic resonance (NMR) spectroscopy. /sup 2/H NMR spectra and spin-lattice relaxation times of selectively-deuterated amino acids in the crystalline solid state and in biosynthetically enriched bacteriorhodopsin, have been obtained. Analysis of the data has led to the determination of the type and rate of amino acid motion in these systems. The motions of amino acid residues, are shown to be very sensitive to packing considerations. Large differences in both the type and rate of motion are observed for a particular amino acid in various crystal lattice forms and in the membrane protein. Moreover, motional heterogeneity is found to occur in some crystal lattice forms and especially in bacteriorhodopsin. The major types of motion that are observed include methyl group rotation (by three-fold hops), phenyl ring flips (180/sup 0/ hops), ring libration, chain libration, side-chain hops, and isotropic reorientation. The last two motions are seen only in the membrane protein. All of these motions can occur at widely different rates depending on the packing environment, but in bacteriorhodopsin, these rates are generally greater than 10/sup 6/ sec/sup -1/. /sup 2/H NMR spectra of selectively-deuterated diphenylhexatriene in dimyristoylphosphatidylcholine multilamellar bilayers in the liquid crystalline phase, as a function of temperature and membrane composition, have also been obtained.
Kintanar, A.B.
Quantitative regulation of nuclear pore complex proteins by O-GlcNAcylation.
The nuclear pore complex (NPC) is a macromolecular assembly consisting of approximately 30 different proteins called nucleoporins. Several nucleoporins are O-GlcNAcylated, which is a post-translational modification in which the monosaccharide ?-N-acetylglucosamine (GlcNAc) is attached to serine or threonine residues within proteins. However, the biological significance of this modification on nucleoporins remains obscure. Here we found that Nup62 and Nup88 protein levels were significantly decreased upon knockdown of O-GlcNAc transferase (OGT), which catalyzes the O-GlcNAcylation of intracellular proteins. Although Nup88, unlike Nup62, was not recognized by an anti-O-GlcNAc antibody or WGA-HRP, knockdown of Nup62 caused a reduction in Nup88 protein levels, suggesting that the observed decrease in Nup88 in OGT knocked-down cells is due to a decrease in Nup62. Furthermore, we found that Nup88 was preferentially associated with O-GlcNAcylated Nup62 compared with non-O-GlcNAcylated Nup62. These results indicate that Nup62 protein levels are primarily maintained by O-GlcNAcylation and that Nup88 is quantitatively regulated through its interaction with O-GlcNAcylated Nup62. PMID:23777819
Mizuguchi-Hata, Chiaki; Ogawa, Yutaka; Oka, Masahiro; Yoneda, Yoshihiro
Reverse micelles as a platform for dynamic nuclear polarization in solution NMR of proteins.
Despite tremendous advances in recent years, solution NMR remains fundamentally restricted due to its inherent insensitivity. Dynamic nuclear polarization (DNP) potentially offers significant improvements in this respect. The basic DNP strategy is to irradiate the EPR transitions of a stable radical and transfer this nonequilibrium polarization to the hydrogen spins of water, which will in turn transfer polarization to the hydrogens of the macromolecule. Unfortunately, these EPR transitions lie in the microwave range of the electromagnetic spectrum where bulk water absorbs strongly, often resulting in catastrophic heating. Furthermore, the residence times of water on the surface of the protein in bulk solution are generally too short for efficient transfer of polarization. Here we take advantage of the properties of solutions of encapsulated proteins dissolved in low viscosity solvents to implement DNP in liquids. Such samples are largely transparent to the microwave frequencies required and thereby avoid significant heating. Nitroxide radicals are introduced into the reverse micelle system in three ways: attached to the protein, embedded in the reverse micelle shell, and free in the aqueous core. Significant enhancements of the water resonance ranging up to ?-93 at 0.35 T were observed. We also find that the hydration properties of encapsulated proteins allow for efficient polarization transfer from water to the protein. These and other observations suggest that merging reverse micelle encapsulation technology with DNP offers a route to a significant increase in the sensitivity of solution NMR spectroscopy of proteins and other biomolecules. PMID:24456213
Valentine, Kathleen G; Mathies, Guinevere; B�dard, Sabrina; Nucci, Nathaniel V; Dodevski, Igor; Stetz, Matthew A; Can, Thach V; Griffin, Robert G; Wand, A Joshua
cGMP-DEPENDENT PROTEIN KINASE ANCHORING BY IRAG REGULATES ITS NUCLEAR TRANSLOCATION AND TRANSCRIPTIONAL ACTIVITY
Type I cGMP-dependent protein kinases (PKGs) translocate to the nucleus to regulate gene expression in some, but not all cell types; we hypothesized that nuclear translocation of PKG may be regulated by extra-nuclear anchoring proteins. The inositol 1,4,5-triphosphate (IP3) receptor-associated cGMP kinase substrate (IRAG) binds to the N-terminus of PKG I?, but not PKG I?, and in smooth muscle cells, IRAG and PKG I? are in a complex with the IP3 receptor at endoplasmatic reticulum membranes, where the complex regulates calcium release [Schlossmann et al., Nature, 404 (2000) 197]. We found that co-expression of IRAG and PKG I? in baby hamster kidney cells prevented cGMP-induced PKG I? translocation to the nucleus, and decreased cGMP/PKG I? transactivation of a cAMP-response element-dependent reporter gene. These effects required the PKG I?/IRAG association, as demonstrated by a binding-incompetent IRAG mutant, and were specific for PKG I?, as nuclear translocation and reporter gene activation by PKG I? was not affected by IRAG. A phosphorylation-deficient IRAG mutant that is no longer functionally regulated by PKG phosphorylation suppressed cGMP/PKG I? transcriptional activity, indicating that IRAG�s effect was not explained by changes in intracellular calcium, and was not related to competition of IRAG with other PKG substrates. These results demonstrate that PKG anchoring to a specific binding protein is sufficient to dictate subcellular localization of the kinase and affect cGMP signaling in the nucleus, and may explain why nuclear translocation of PKG I does not occur in all cell types.
Casteel, Darren E.; Zhang, Tong; Zhuang, Shunhui; Pilz, Renate B.
Vaccinia virus protein N2 is a nuclear IRF3 inhibitor that promotes virulence.
Vaccinia virus (VACV) expresses many proteins that are non-essential for virus replication but promote virulence by inhibiting components of the host immune response to infection. These immunomodulators include a family of proteins that have, or are predicted to have, a structure related to the B-cell lymphoma (Bcl)-2 protein. Five members of the VACV Bcl-2 family (N1, B14, A52, F1 and K7) have had their crystal structure solved, others have been characterized and a function assigned (C6, A46), and others are predicted to be Bcl-2 proteins but are uncharacterized hitherto (N2, B22, C1). Data presented here show that N2 is a nuclear protein that is expressed early during infection and inhibits the activation of interferon regulatory factor (IRF)3. Consistent with its nuclear localization, N2 inhibits IRF3 downstream of the TANK-binding kinase (TBK)-1 and after IRF3 translocation into the nucleus. A mutant VACV strain Western Reserve lacking the N2L gene (v?N2) showed normal replication and spread in cultured cells compared to wild-type parental (vN2) and revertant (vN2-rev) viruses, but was attenuated in two murine models of infection. After intranasal infection, the v?N2 mutant induced lower weight loss and signs of illness, and virus was cleared more rapidly from the infected tissue. In the intradermal model of infection, v?N2 induced smaller lesions that were resolved more rapidly. In summary, the N2 protein is an intracellular virulence factor that inhibits IRF3 activity in the nucleus. PMID:23761407
Ferguson, Brian J; Benfield, Camilla T O; Ren, Hongwei; Lee, Vivian H; Frazer, Gordon L; Strnadova, Pavla; Sumner, Rebecca P; Smith, Geoffrey L
Identification of nuclear genes encoding chloroplast-localized proteins required for embryo development in Arabidopsis.
We describe here the diversity of chloroplast proteins required for embryo development in Arabidopsis (Arabidopsis thaliana). Interfering with certain chloroplast functions has long been known to result in embryo lethality. What has not been reported before is a comprehensive screen for embryo-defective (emb) mutants altered in chloroplast proteins. From a collection of transposon and T-DNA insertion lines at the RIKEN chloroplast function database (http://rarge.psc.riken.jp/chloroplast/) that initially appeared to lack homozygotes and segregate for defective seeds, we identified 23 additional examples of EMB genes that likely encode chloroplast-localized proteins. Fourteen gene identities were confirmed with allelism tests involving duplicate mutant alleles. We then queried journal publications and the SeedGenes database (www.seedgenes.org) to establish a comprehensive dataset of 381 nuclear genes encoding chloroplast proteins of Arabidopsis associated with embryo-defective (119 genes), plant pigment (121 genes), gametophyte (three genes), and alternate (138 genes) phenotypes. Loci were ranked based on the level of certainty that the gene responsible for the phenotype had been identified and the protein product localized to chloroplasts. Embryo development is frequently arrested when amino acid, vitamin, or nucleotide biosynthesis is disrupted but proceeds when photosynthesis is compromised and when levels of chlorophyll, carotenoids, or terpenoids are reduced. Chloroplast translation is also required for embryo development, with genes encoding chloroplast ribosomal and pentatricopeptide repeat proteins well represented among EMB datasets. The chloroplast accD locus, which is necessary for fatty acid biosynthesis, is essential in Arabidopsis but not in Brassica napus or maize (Zea mays), where duplicated nuclear genes compensate for its absence or loss of function. PMID:21139083
Bryant, Nicole; Lloyd, Johnny; Sweeney, Colleen; Myouga, Fumiyoshi; Meinke, David
The serum response factor nuclear localization signal: general implications for cyclic AMP-dependent protein kinase activity in control of nuclear translocation.
We have identified a basic sequence in the N-terminal region of the 67-kDa serum response factor (p67SRF or SRF) responsible for its nuclear localization. A peptide containing this nuclear localization signal (NLS) translocates rabbit immunoglobulin G (IgG) into the nucleus as efficiently as a peptide encoding the simian virus 40 NLS. This effect is abolished by substituting any two of the four basic residues in this NLS. Overexpression of a modified form of SRF in which these basic residues have been mutated confirms the absolute requirement for this sequence, and not the other basic amino acid sequences adjacent to it, in the nuclear localization of SRF. Since this NLS is in close proximity to potential phosphorylation sites for the cAMP-dependent protein kinase (A-kinase), we further investigated if A-kinase plays a role in the nuclear location of SRF. The nuclear transport of SRF proteins requires basal A-kinase activity, since inhibition of A-kinase by using either the specific inhibitory peptide PKIm or type II regulatory subunits (RII) completely prevents the nuclear localization of plasmid-expressed tagged SRF or an SRF-NLS-IgG conjugate. Direct phosphorylation of SRF by A-kinase can be discounted in this effect, since mutation of the putative phosphorylation sites in either the NLS peptide or the encoded full-length SRF protein had no effect on nuclear transport of the mutants. Finally, in support of an implication of A-kinase-dependent phosphorylation in a more general mechanism affecting nuclear import, we show that the nuclear transport of a simian virus 40-NLS-conjugated IgG or purified cyclin A protein is also blocked by inhibition of A-kinase, even though neither contains any potential sites for phosphorylation by A-kinase or can be phosphorylated by A-kinase in vitro.
Gauthier-Rouviere, C; Vandromme, M; Lautredou, N; Cai, Q Q; Girard, F; Fernandez, A; Lamb, N
Long isoform of ErbB3 binding protein, p48, mediates protein kinase B/Akt-dependent HDM2 stabilization and nuclear localization
p48 is a long isoform of the ErbB3 binding protein that has oncogenic functions including promotion of carcinogenesis and induction of malignant transformation through negative regulation of tumor suppressor p53. Here, we show that high level of p48 protein expression leads to enhance HDM2 phosphorylation by Akt and inhibits the self-ubiquitination of HDM2 by up-regulation of Akt activity, thereby promoting its protein stability. Moreover, p48 expression leads to accumulated nuclear localization of HDM2, whereas p48 depletion disturbs its nuclear localization. Hence, higher expression of p48 in cancer cells reduces p53 levels through modulation of HDM2 nuclear localization and protein stability via regulation of its Akt-mediated phosphorylation.
Kim, Chung Kwon; Lee, Sang Bae; Nguyen, Truong L.X. [Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon, 440-746 (Korea, Republic of)] [Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon, 440-746 (Korea, Republic of); Lee, Kyung-Hoon [Department of Anatomy, Sungkyunkwan University School of Medicine, Suwon, 440-746 (Korea, Republic of) [Department of Anatomy, Sungkyunkwan University School of Medicine, Suwon, 440-746 (Korea, Republic of); Center for Molecular Medicine, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, 440-746 (Korea, Republic of); Um, Sung Hee [Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon, 440-746 (Korea, Republic of) [Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon, 440-746 (Korea, Republic of); Center for Molecular Medicine, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, 440-746 (Korea, Republic of); Kim, Jihoe [School of Biotechnology, Yeungnam University, Gyeongsan 712-749 (Korea, Republic of)] [School of Biotechnology, Yeungnam University, Gyeongsan 712-749 (Korea, Republic of); Ahn, Jee-Yin, E-mail: firstname.lastname@example.com [Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon, 440-746 (Korea, Republic of) [Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon, 440-746 (Korea, Republic of); Center for Molecular Medicine, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, 440-746 (Korea, Republic of)
The PHD Finger of the Chromatin-Associated Protein ING2 Functions as a Nuclear Phosphoinositide Receptor
Phosphoinositides (PtdInsPs) play critical roles in cytoplasmic signal transduction pathways. However, their functions in the nucleus are unclear, as specific nuclear receptors for PtdInsPs have not been identified. Here, we show that ING2, a candidate tumor suppressor protein, is a nuclear PtdInsP receptor. ING2 contains a plant homeodomain (PHD) finger, a motif common to many chromatin-regulatory proteins. We find that
Gozani; Philip Karuman; David R. Jones; Dmitri Ivanov; James Cha; Alexey A. Lugovskoy; Cheryl L. Baird; Hong Zhu; Seth J. Field; Stephen L. Lessnick; Jennifer Villasenor; Bharat Mehrotra; Jian Chen; Vikram R. Rao; Joan S. Brugge; Colin G. Ferguson; Bernard Payrastre; David G. Myszka; Lewis C. Cantley; Gerhard Wagner; Nullin Divecha; Glenn D. Prestwich; Junying Yuan
Modulation of CREB Binding Protein Function by the Promyelocytic (PML) Oncoprotein Suggests a Role for Nuclear Bodies in Hormone Signaling
Disaggregation of the spherical nuclear bodies termed promyelocytic (PML) oncogenic domains (PODs) is a characteristic of acute promyelocytic leukemia. Here, we demonstrate that the cAMP enhancer binding protein (CREB)-binding protein (CBP) associates with PML in vitro and is recruited to the PODs in vivo. Through its association with CBP, wild-type PML dramatically stimulates nuclear receptor transcriptional activity. These results demonstrate
Vassilis Doucas; Marc Tini; David A. Egan; Ronald M. Evans
The Yeast Nucleoporin Nup53p Specifically Interacts with Nic96p and Is Directly Involved in Nuclear Protein Import
The bidirectional nucleocytoplasmic transport of macromolecules is mediated by the nuclear pore complex (NPC) which, in yeast, is composed of ?30 different proteins (nucleoporins). Pre-embedding immunogold-electron microscopy revealed that Nic96p, an essential yeast nucleoporin, is located about the cytoplasmic and the nuclear periphery of the central channel, and near or at the distal ring of the yeast NPC. Genetic approaches further implicated Nic96p in nuclear protein import. To more specifically explore the potential role of Nic96p in nuclear protein import, we performed a two-hybrid screen with NIC96 as the bait against a yeast genomic library to identify transport factors and/or nucleoporins involved in nuclear protein import interacting with Nic96p. By doing so, we identified the yeast nucleoporin Nup53p, which also exhibits multiple locations within the yeast NPC and colocalizes with Nic96p in all its locations. Whereas Nup53p is directly involved in NLS-mediated protein import by its interaction with the yeast nuclear import receptor Kap95p, it appears not to participate in NES-dependent nuclear export.
Fahrenkrog, Birthe; Hubner, Wolfgang; Mandinova, Anna; Pante, Nelly; Keller, Walter; Aebi, Ueli
Proposed mechanism for the stabilization of nuclear receptor DNA binding via protein dimerization.
Hepatocyte nuclear factor 4 (HNF-4) defines a new subgroup of nuclear receptors that exist in solution and bind DNA exclusively as homodimers. We recently showed that the putative ligand binding domain (LBD) of HNF-4 is responsible for dimerization in solution and prevents heterodimerization with other receptors. In this report, the role of the LBD in DNA binding by HNF-4 is further investigated by using electrophoretic mobility shift analysis. A comparison of constructs containing either the DNA binding domain (DBD) alone or the DBD plus the LBD of HNF-4 showed that dimerization via the DBD was sufficient to provide nearly the full DNA binding affinity of the full-length HNF-4. In contrast, dimerization via the DBD was not sufficient to produce a stable protein-DNA complex, whereas dimerization via the LBD increased the half-life of the complex by at least 100-fold. Circular permutation analysis showed that full-length HNF-4 bent DNA by approximately 80 degrees while the DBD bent DNA by only 24 degrees. Nonetheless, analysis of other constructs indicated that the increase in stability afforded by the LBD could be explained only partially by an increased ability to bend DNA. Coimmunoprecipitation studies, on the other hand, showed that dimerization via the LBD produced a protein-protein complex that was much more stable than the corresponding protein-DNA complex. These results led us to propose a model in which dimerization via the LBD stabilizes the receptor on DNA by converting an energetically favorable two-step dissociation event into an energetically unfavorable single-step event. Implications of this one-step model for other nuclear receptors are discussed.
Jiang, G; Lee, U; Sladek, F M
Centrin 2 Localizes to the Vertebrate Nuclear Pore and Plays a Role in mRNA and Protein Export? �
Centrins in vertebrates have traditionally been associated with microtubule-nucleating centers such as the centrosome. Unexpectedly, we found centrin 2 to associate biochemically with nucleoporins, including the Xenopus laevis Nup107-160 complex, a critical subunit of the vertebrate nuclear pore in interphase and of the kinetochores and spindle poles in mitosis. Immunofluorescence of Xenopus cells and in vitro reconstituted nuclei indeed revealed centrin 2 localized at the nuclear pores. Use of the mild detergent digitonin in immunofluorescence also allowed centrin 2 to be clearly visualized at the nuclear pores of human cells. Disruption of nuclear pores using RNA interference of the pore assembly protein ELYS/MEL-28 resulted in a specific decrease of centrin 2 at the nuclear rim of HeLa cells. Functionally, excess expression of either the N- or C-terminal calcium-binding domains of human centrin 2 caused a dominant-negative effect on both mRNA and protein export, leaving protein import intact. The mRNA effect mirrors that found for the Saccharomyes cerevisiae centrin Cdc31p at the yeast nuclear pore, a role until now thought to be unique to yeast. We conclude that in vertebrates, centrin 2 interacts with major subunits of the nuclear pore, exhibits nuclear pore localization, and plays a functional role in multiple nuclear export pathways.
Resendes, Karen K.; Rasala, Beth A.; Forbes, Douglass J.
Interaction of nuclear factors with upstream sequences of a lipid body membrane protein gene from carrot.
To study the regulation of gene expression during embryo development, we isolated a gene, DC 59, expressed in embryos but not in mature carrot plants. Sequence and S1 analysis showed that the gene was composed of one exon encoding a polypeptide of 19 kilodaltons and was highly homologous to the lipid body membrane protein gene L3 from maize. The plant hormone abscisic acid regulated the accumulation of DC 59 mRNA. To understand the mechanism of embryo-specific and hormonal regulation of DC 59, 5' DNA fragments were incubated with nuclear proteins. Two adjacent regions (from -706 to -235) interacted with nuclear extracts from embryos, resulting in the formation of four complexes (C1, C2, C3, and C4). Factors involved in the formation of the C3 and C4 complexes could be competed with sequences upstream of DC 8, a gene that is coordinately expressed with DC 59 during embryo development. DNase I footprinting analysis revealed that nuclear extracts from embryos bound to four AT-rich sequences, and the protected motifs within fragment V were located in the highly homologous upstream regions of DC 59 and DC 8 genes.
Hatzopoulos, P; Franz, G; Choy, L; Sung, R Z
Arginine methylation facilitates the nuclear export of hnRNP proteins
Eukaryotic mRNA processing and export is mediated by various heterogeneous nuclear ribonucleoproteins (hnRNPs). Many of these hnRNPs are methylated on arginine residues. In the yeast, Saccharomyces cerevisiae, the predominant enzyme responsible for arginine methylation is Hmt1p. Hmt1p methylates both Npl3p and Hrp1p, which are shuttling hnRNPs involved in mRNA processing and export. Here, we employ an in vivo nuclear export assay to show that arginine methylation is important for the nuclear export of these hnRNPs. Both Npl3p and Hrp1p fail to exit the nucleus in cells lacking Hmt1p, and overexpression of Hmt1p enhances Npl3p export. The export of a novel hnRNP-like protein, Hrb1p, which does not bind poly(A)+ RNA, however, is not affected by the lack of methylation. Furthermore, we find a genetic relationship between Hmt1p and cap-binding protein 80 (CBP80). Together, these findings establish that one biological role for arginine methylation is in facilitating the export of certain hnRNPs out of the nucleus.
Shen, Elisa C.; Henry, Michael F.; Weiss, Valerie H.; Valentini, Sandro R.; Silver, Pamela A.; Lee, Margaret S.
Nuclear translocation of mouse polycomb m33 protein in regenerating liver.
Immunoblots probed with an antibody to M33 protein, a homolog of Drosophila Polycomb, revealed that most M33 in adult mouse liver had a higher electrophoretic mobility than that in F9 embryonal carcinoma cells. High-mobility 60-kDa M33 localized in the cytoplasmic fraction of liver homogenates, and two less abundant 66- and 70-kDa species were detected in the nuclear fraction. Immunocytochemistry of freeze-substituted tissues showed a punctate pattern of immunofluorescence in the cytoplasm of hepatic parenchymal cells. Nuclear M33 isoforms treated with alkaline phosphatase had increased mobilities corresponding to cytoplasmic M33. In partially hepatectomized mice, nuclear M33 isoforms appeared after 48 h, near the time of maximum DNA synthesis as measured by bromodeoxyuridine incorporation. By 60 h, most M33 was in the form of these low-mobility species, and the pattern of immunofluorescence suggested the existence of chromatin-bound and free states of the protein in the nucleus. Thereafter, high-mobility 60-kDa M33 reappeared. The data are consistent with a phosphorylation-associated translocation mechanism that is a cell cycle-dependent. PMID:11855817
Noguchi, Kousei; Shiurba, Robert; Higashinakagawa, Toru
The interferon alpha induced protein ISG12 is localized to the nuclear membrane.
Interferons exert their biological function mainly through the activation of interferon-stimulated genes (ISGs). ISG12 (originally designated p27) belongs to a family of small, interferon alpha inducible genes of unknown function. We have determined the 5' end sequence of ISG12 cDNA from the human cell lines HeLa and AMA by RACE. Comparing this sequence to ISG12 sequences in the expressed sequence tag (EST) database revealed the presence of two alternative splice variants of ISG12 in human cells exhibiting the same open reading frame. We have sequenced the promoter region of the ISG12 gene and found ISRE, IRF1/IRF2, and STAT elements correlating to the interferon alpha inducibility of the gene. Subsequently, we have expressed human ISG12, a 12-kDa hydrophobic protein in the baculovirus expression system and with a C-terminal FLAG-tag in the human cell line 293. Recombinant ISG12 sediments in the nuclear envelope in both cell types. Finally, we have been able to demonstrate the prevalence of the ISG12 gene product in the nuclear envelope of HeLa cells treated with interferon alpha by immunocytochemical analyses. ISG12 is the first interferon induced protein found localizing to the nuclear envelope. PMID:11722583
Martensen, P M; S�gaard, T M; Gjermandsen, I M; Buttensch�n, H N; Rossing, A B; Bonnevie-Nielsen, V; Rosada, C; Simonsen, J L; Justesen, J
Heterodimerization with small Maf proteins enhances nuclear retention of Nrf2 via masking the NESzip motif.
Nrf2 is the key transcription factor regulating the antioxidant response. When exposed to oxidative stress, Nrf2 translocates to cell nucleus and forms heterodimer with small Maf proteins (sMaf). Nrf2/sMaf heterodimer binds specifically to a cis-acting enhancer called antioxidant response element and initiates transcription of a battery of antioxidant and detoxification genes. Nrf2 possesses a NESzip motif (nuclear export signal co-localized with the leucine zipper (ZIP) domain). Heterodimerization with MafG via ZIP-ZIP binding enhanced Nrf2 nuclear retention, which could be abrogated by the deletion of the ZIP domain or site-directed mutations targeting at the ZIP domain. In addition, dimerization with MafG precluded Nrf2zip/CRM1 binding, suggesting that Nrf2/MafG heterodimerization may simultaneously mask the NESzip motif. MafG-mediated nuclear retention may enable Nrf2 proteins to evade cytosolic proteasomal degradation and consequently stabilize Nrf2 signaling. For the first time, we show that under the physiological condition, the NESzip motif can be switched-off by heterodimerization. PMID:18585411
Li, Wenge; Yu, Siwang; Liu, Tong; Kim, Jung-Hwan; Blank, Volker; Li, Hong; Kong, A-N Tony
The Nuclear Factor-kB Pathway Regulates Cytochrome P450 3A4 Protein Stability
We have previously observed that CYP3A4 protein levels are suppressed by inhibition of the proteasome in primary cultured hepatocytes. Because this result is opposite of what would be expected if CYP3A4 is degraded by the proteasome, it seems likely that there is another protein that is susceptible to proteasomal degradation that regulates CYP3A4 expression. In this study, we evaluate whether the nuclear factor kappa B (NF-kB) pathway is involved in that process. Our model system uses an adenovirus system to express CYP3A4 protein in HepG2 cells, which are derived from human cancer cells. Similar to results in primary hepatocytes, we found that inhibition of the proteasome with MG132 suppresses CYP3A4. Consistent with reports that proteasome inhibition suppresses the NF-kB pathway, we also observe a suppression of inhibitory kB kinase protein levels after treatment with MG132. Treatment of the HepG2 cells with NK-kB Activation Inhibitor also suppresses CYP3A4 proteins levels. In contrast, inhibition of either the proteasome or NF-kB pathways increases CYP3A4 mRNA levels. When the HepG2 cells are treated with cycloheximide, a general inhibitor of translation, the loss of CYP3A4 protein is accelerated by co-treatment with an NF-kB Activation Inhibitor. These results indicate that NF-kB activity regulates CYP3A4 protein stability and suggest that the NF-kB pathway is responsible for the decrease in CYP3A4 protein levels that results from the inhibition of proteasomal activity.
Zangar, Richard C.; Bollinger, Nikki; Verma, Seema; Karin, Norm J.; Lu, Yi
Role of the nuclear migration protein Lis1 in cell morphogenesis in Ustilago maydis
Ustilago maydis is a basidiomycete fungus that exhibits a yeast-like and a filamentous form. Growth of the fungus in the host leads to additional morphological transitions. The different morphologies are characterized by distinct nuclear movements. Dynein and ?-tubulin are required for nuclear movements and for cell morphogenesis of the yeast-like form. Lis1 is a microtubule plus-end tracking protein (+TIPs) conserved in eukaryotes and required for nuclear migration and spindle positioning. Defects in nuclear migration result in altered cell fate and aberrant development in metazoans, slow growth in fungi and disease in humans (e.g. lissencephaly). Here we investigate the role of the human LIS1 homolog in U. maydis and demonstrate that it is essential for cell viability, not previously seen in other fungi. With a conditional null mutation we show that lis1 is necessary for nuclear migration in the yeast-like cell and during the dimorphic transition. Studies of asynchronous exponentially growing cells and time-lapse microscopy uncovered novel functions of lis1: It is necessary for cell morphogenesis, positioning of the septum and cell wall integrity. lis1-depleted cells exhibit altered axes of growth and loss of cell polarity leading to grossly aberrant cells with clusters of nuclei and morphologically altered buds devoid of nuclei. Altered septum positioning and cell wall deposition contribute to the aberrant morphology. lis1-depleted cells lyse, indicative of altered cell wall properties or composition. We also demonstrate, with indirect immunofluorescence to visualize tubulin, that lis1 is necessary for the normal organization of the microtubule cytoskeleton: lis1-depleted cells contain more and longer microtubules that can form coils perpendicular to the long axis of the cell. We propose that lis1 controls microtubule dynamics and thus the regulated delivery of vesicles to growth sites and other cell domains that govern nuclear movements.
Valinluck, Michael; Ahlgren, Sara; Sawada, Mizuho; Locken, Kristopher; Banuett, Flora
Conservation of a Masked Nuclear Export Activity of La Proteins and Its Effects on tRNA Maturation?
La is an RNA-processing-associated phosphoprotein so highly conserved that the human La protein (hLa) can replace the tRNA-processing function of the fission yeast La protein (Sla1p) in vivo. La proteins contain multiple trafficking elements that support interactions with RNAs in different subcellular locations. Prior data indicate that deletion of a nuclear retention element (NRE) causes nuclear export of La and dysfunctional processing of associated pre-tRNAs that are spliced but 5? and 3? unprocessed, with an accompanying decrease in tRNA-mediated suppression, in fission yeast. To further pursue these observations, we first identified conserved residues in the NREs of hLa and Sla1p that when substituted mimic the NRE deletion phenotype. NRE-defective La proteins then deleted of other motifs indicated that RNA recognition motif 1 (RRM1) is required for nuclear export. Mutations of conserved RRM1 residues restored nuclear accumulation of NRE-defective La proteins. Some RRM1 mutations restored nuclear accumulation, prevented disordered pre-tRNA processing, and restored suppression, indicating that the tRNA-related activity of RRM1 and its nuclear export activity could be functionally separated. When mapped onto an hLa structure, the export-sensitive residues comprised surfaces distinct from the RNA-binding surface of RRM1. The data indicate that the NRE has been conserved to mask or functionally override an equally conserved nuclear export activity of RRM1. The data suggest that conserved elements mediate nuclear retention, nuclear export, and RNA-binding activities of the multifunctional La protein and that their interrelationship contributes to the ability of La to engage its different classes of RNA ligands in different cellular locations.
Bayfield, Mark A.; Kaiser, Trish E.; Intine, Robert V.; Maraia, Richard J.
Characterizing RNA-protein interaction using cross-linking and metabolite supplemented nuclear RNA-immunoprecipitation.
RNA-immunoprecipitation (RNA-IP) is a method used to isolate and identify RNA molecules specifically associated with an RNA-binding protein. Non-coding RNAs are emerging as key regulators of many biological and developmental pathways and RNA-IP has become an important tool in studying their function(s). While RNA-IP is successfully used to determine protein-RNA interaction, specific details regarding the level of this association and the metabolic requirement of this interaction which can influence the success of RNA-IP remain unclear. Here, we investigate the conditions required for efficient nuclear RNA-IP using Arabidopsis AGO4 (Argonaute 4) and siRNA binding as the study model. We showed that formaldehyde cross-linking, but not UV cross-linking, allowed for efficient pull-down of 24-nt siRNAs, suggesting that AGO4-siRNA interaction involves other protein(s). We also showed that, while formaldehyde cross-linking could also be performed on purified nuclei, ATP supplementation to the nuclei isolation buffer was needed to efficiently pull down 24-nt siRNAs. This result indicates that ATP is required for efficient siRNA loading onto AGO4. As most of the known RNA-mediated regulatory processes occur in the nucleus, our findings on cross-linking conditions and metabolite requirement for successful AGO4 nuclear RNA-IP provide a valuable insight and future consideration when studying the function of protein-RNA interactions in plants. PMID:24493449
Au, Phil Chi Khang; Helliwell, Chris; Wang, Ming-Bo
Concentration-dependent lamin assembly and its roles in the localization of other nuclear proteins
The nuclear lamina (NL) consists of lamin polymers and proteins that bind to the polymers. Disruption of NL proteins such as lamin and emerin leads to developmental defects and human diseases. However, the expression of multiple lamins, including lamin-A/C, lamin-B1, and lamin-B2, in mammals has made it difficult to study the assembly and function of the NL. Consequently, it has been unclear whether different lamins depend on one another for proper NL assembly and which NL functions are shared by all lamins or are specific to one lamin. Using mouse cells deleted of all or different combinations of lamins, we demonstrate that the assembly of each lamin into the NL depends primarily on the lamin concentration present in the nucleus. When expressed at sufficiently high levels, each lamin alone can assemble into an evenly organized NL, which is in turn sufficient to ensure the even distribution of the nuclear pore complexes. By contrast, only lamin-A can ensure the localization of emerin within the NL. Thus, when investigating the role of the NL in development and disease, it is critical to determine the protein levels of relevant lamins and the intricate shared or specific lamin functions in the tissue of interest.
Guo, Yuxuan; Kim, Youngjo; Shimi, Takeshi; Goldman, Robert D.; Zheng, Yixian
Nuclear pore complex protein sequences determine overall copolymer brush structure and function.
The transport of cargo across the nuclear membrane is highly selective and accomplished by a poorly understood mechanism involving hundreds of nucleoporins lining the inside of the nuclear pore complex (NPC). Currently, there is no clear picture of the overall structure formed by this collection of proteins within the pore, primarily due to their disordered nature. We perform coarse-grained simulations of both individual nucleoporins and grafted rings of nups mimicking the in�vivo geometry of the NPC and supplement this with polymer brush modeling. Our results indicate that different regions or blocks of an individual NPC protein can have distinctly different forms of disorder and that this property appears to be a conserved functional feature. Furthermore, this block structure at the individual protein level is critical to the formation of a unique higher-order polymer brush architecture that can exist in distinct morphologies depending on the effective interaction energy between the phenylalanine glycine (FG) domains of different nups. Because the interactions between FG domains may be modulated by certain forms of transport factors, our results indicate that transitions between brush morphologies could play an important role in regulating transport across the NPC, suggesting novel forms of gated transport across membrane pores with wide biomimetic applicability. PMID:24806932
Ando, David; Zandi, Roya; Kim, Yong Woon; Colvin, Michael; Rexach, Michael; Gopinathan, Ajay
Interaction of nucleosome assembly proteins abolishes nuclear localization of DGK? by attenuating its association with importins.
Diacylglycerol kinase (DGK) is involved in the regulation of lipid-mediated signal transduction through the metabolism of a second messenger diacylglycerol. Of the DGK family, DGK?, which contains a nuclear localization signal, localizes mainly to the nucleus but translocates to the cytoplasm under pathological conditions. However, the detailed mechanism of translocation and its functional significance remain unclear. To elucidate these issues, we used a proteomic approach to search for protein targets that interact with DGK?. Results show that nucleosome assembly protein (NAP) 1-like 1 (NAP1L1) and NAP1-like 4 (NAP1L4) are identified as novel DGK? binding partners. NAP1Ls constitutively shuttle between the nucleus and the cytoplasm in transfected HEK293 cells. The molecular interaction of DGK? and NAP1Ls prohibits nuclear import of DGK? because binding of NAP1Ls to DGK? blocks import carrier proteins, Qip1 and NPI1, to interact with DGK?, leading to cytoplasmic tethering of DGK?. In addition, overexpression of NAP1Ls exerts a protective effect against doxorubicin-induced cytotoxicity. These findings suggest that NAP1Ls are involved in a novel molecular basis for the regulation of nucleocytoplasmic shuttling of DGK? and provide a clue to examine functional significance of its translocation under pathological conditions. PMID:21996351
Okada, Masashi; Hozumi, Yasukazu; Ichimura, Tohru; Tanaka, Toshiaki; Hasegawa, Hiroshi; Yamamoto, Masakazu; Takahashi, Nobuya; Iseki, Ken; Yagisawa, Hitoshi; Shinkawa, Takashi; Isobe, Toshiaki; Goto, Kaoru
HIV-1 capsid undergoes coupled binding and isomerization by the nuclear pore protein NUP358
Background Lentiviruses such as HIV-1 can be distinguished from other retroviruses by the cyclophilin A-binding loop in their capsid and their ability to infect non-dividing cells. Infection of non-dividing cells requires transport through the nuclear pore but how this is mediated is unknown. Results Here we present the crystal structure of the N-terminal capsid domain of HIV-1 in complex with the cyclophilin domain of nuclear pore protein NUP358. The structure reveals that HIV-1 is positioned to allow single-bond resonance stabilisation of exposed capsid residue P90. NMR exchange experiments demonstrate that NUP358 is an active isomerase, which efficiently catalyzes cis-trans isomerization of the HIV-1 capsid. In contrast, the distantly related feline lentivirus FIV can bind NUP358 but is neither isomerized by it nor requires it for infection. Conclusion Isomerization by NUP358 may be preserved by HIV-1 to target the nuclear pore and synchronize nuclear entry with capsid uncoating.
Efficient protein production using a Bombyx mori nuclear polyhedrosis virus lacking the cysteine proteinase gene.
Infection by a baculovirus (Bombyx mori nuclear polyhedrosis virus, BmNPV) in silkworm (Bombyx mori) larvae is highly efficient as an expression system for the production of useful proteins. However, the amount of the protein of interest expressed tends to decrease in the later stages of infection presumably due, in part, to a proteinase produced in the larval haemolymph. The N-terminal amino acid sequence of a proteinase purified from the haemolymph of BmNPV-infected larvae was identical to the internal amino acid sequence of the viral cysteine proteinase gene of BmNPV, suggesting that the cysteine proteinase in the haemolymph originated from the BmNPV gene. We constructed a mutant virus (CPd) which had a deletion in the cysteine proteinase gene. No proteinase activity corresponding to this proteinase was detected in the haemolymph of silkworm larvae infected with CPd. The firefly luciferase and the human growth hormone genes were separately introduced into CPd under control of the polyhedrin promoter. These constructs produced these proteins very efficiently, because of a greatly reduced degree of degradation of these proteins. A BmNPV vector system using CPd enhances the stability of foreign expressed proteins, especially for those that are cysteine proteinase-sensitive. PMID:9400955
Suzuki, T; Kanaya, T; Okazaki, H; Ogawa, K; Usami, A; Watanabe, H; Kadono-Okuda, K; Yamakawa, M; Sato, H; Mori, H; Takahashi, S; Oda, K
Nuclear transport of Ras-associated tumor suppressor proteins: different transport receptor binding specificities for arginine-rich nuclear targeting signals.
Ras proteins regulate a wide range of biological processes by interacting with a variety of effector proteins. In addition to the known role in tumorigensis, the activated form of Ras exhibits growth-inhibitory effects by unknown mechanisms. Several Ras effector proteins identified as mediators of apoptosis and cell-cycle arrest also exhibit properties normally associated with tumor suppressor proteins. Here, we show that Ras effector RASSF5/NORE-1 binds strongly to K-Ras but weakly to both N-Ras and H-Ras. RASSF5 was found to localize both in the nucleus and the nucleolus in contrast to other Ras effector proteins, RASSF1C and RASSF2, which are localized in the nucleus and excluded from nucleolus. A 50 amino acid residue transferable arginine-rich nucleolar localization signal (NoLS) identified in RASSF5 is capable of interacting with importin-beta and transporting the cargo into the nucleolus. Surprisingly, similar arginine-rich signals identified in RASSF1C and RASSF2 interact with importin-alpha and transport the heterologous cytoplasmic proteins to the nucleus. Interestingly, mutation of arginine residues within these nuclear targeting signals prevented interaction of Ras effector proteins with respective transport receptors and abolished their nuclear translocation. These results provide evidence for the first time that arginine-rich signals are able to recognize different nuclear import receptors and transport the RASSF proteins into distinct sub-cellular compartments. In addition, our data suggest that the nuclear localization of RASSF5 is critical for its cell growth control activity. Together, these data suggest that the transport of Ras effector superfamily proteins into the nucleus/nucleolus may play a vital role in modulating Ras-mediated cell proliferation during tumorigenesis. PMID:17320110
Kumari, Gita; Singhal, Prabhat K; Rao, M R K Subba; Mahalingam, S
Assembly in vitro of nuclei active in nuclear protein transport: ATP is required for nucleoplasmin accumulation.
DNA (from bacteriophage lambda or Xenopus) is assembled into nucleus-like structures when mixed with an extract from Xenopus eggs. Electron microscopy shows that these in vitro-reconstituted nuclei possess complete double membranes; some, but not all, nuclei have pore complexes. Extracts depleted of their endogenous ATP (by addition of ATPases) cannot assemble nuclear envelopes visible by phase-contrast microscopy. Once synthetic nuclei are assembled, however, they are stable when ATP is subsequently depleted, although their chromatin becomes condensed. About one-fourth of the nuclei assembled in vitro from lambda DNA accumulate nuclear proteins such as nucleoplasmin. ATP depletion blocks nucleoplasmin accumulation both in vitro, in pre-assembled synthetic nuclei, and in vivo, in the nucleus of microinjected oocytes. However, nucleoplasmin previously accumulated by reconstituted nuclei or by the germinal vesicle in microinjected oocytes is retained after ATP depletion. Images Fig. 1. Fig. 2. Fig. 3. Fig. 5. Fig. 6. Fig. 7. Fig. 9.
Newmeyer, D D; Lucocq, J M; Burglin, T R; De Robertis, E M
UL31 and UL34 Proteins of Herpes Simplex Virus Type 1 Form a Complex That Accumulates at the Nuclear Rim and Is Required for Envelopment of Nucleocapsids
The herpes simplex virus type 1 (HSV-1) UL34 protein is likely a type II membrane protein that localizes within the nuclear membrane and is required for efficient envelopment of progeny virions at the nuclear envelope, whereas the UL31 gene product of HSV-1 is a nuclear matrix-associated phosphoprotein previously shown to interact with UL34 protein in HSV-1-infected cell lysates. For these studies, polyclonal antisera directed against purified fusion proteins containing UL31 protein fused to glutathione-S-transferase (UL31-GST) and UL34 protein fused to GST (UL34-GST) were demonstrated to specifically recognize the UL31 and UL34 proteins of approximately 34,000 and 30,000 Da, respectively. The UL31 and UL34 gene products colocalized in a smooth pattern throughout the nuclear rim of infected cells by 10 h postinfection. UL34 protein also accumulated in pleiomorphic cytoplasmic structures at early times and associated with an altered nuclear envelope late in infection. Localization of UL31 protein at the nuclear rim required the presence of UL34 protein, inasmuch as cells infected with a UL34 null mutant virus contained UL31 protein primarily in central intranuclear domains separate from the nuclear rim, and to a lesser extent in the cytoplasm. Conversely, localization of UL34 protein exclusively at the nuclear rim required the presence of the UL31 gene product, inasmuch as UL34 protein was detectable at the nuclear rim, in replication compartments, and in the cytoplasm of cells infected with a UL31 null virus. When transiently expressed in the absence of other viral factors, UL31 protein localized diffusely in the nucleoplasm, whereas UL34 protein localized primarily in the cytoplasm and at the nuclear rim. In contrast, coexpression of the UL31 and UL34 proteins was sufficient to target both proteins exclusively to the nuclear rim. The proteins were also shown to directly interact in vitro in the absence of other viral proteins. In cells infected with a virus lacking the US3-encoded protein kinase, previously shown to phosphorylate the UL34 gene product, UL31 and UL34 proteins colocalized in small punctate areas that accumulated on the nuclear rim. Thus, US3 kinase is required for even distribution of UL31 and UL34 proteins throughout the nuclear rim. Taken together with the similar phenotypes of the UL31 and UL34 deletion mutants, these data strongly suggest that the UL31 and UL34 proteins form a complex that accumulates at the nuclear membrane and plays an important role in nucleocapsid envelopment at the inner nuclear membrane.
Reynolds, Ashley E.; Ryckman, Brent J.; Baines, Joel D.; Zhou, Yuping; Liang, Li; Roller, Richard J.
Effects of cycloheximide and actinomycin D on production of acidic nuclear proteins in human lymphocytes activated by phytohemagglutinin.
Previous studies have implicated a role for acidic nuclear proteins in the PHA stimulated activation of human lymphocytes. This study examines the mechanism of synthesis of these proteins. Cycloheximide inhibited their production, while Actinomycin D did not. It is suggested that synthesis proceeds by translation of dormant messenger RNA rather than of newly synthesized messenger RNA. PMID:15633971
Dekio, S
Zinc Fingers Function Cooperatively with KRAB Domain for Nuclear Localization of KRAB-Containing Zinc Finger Proteins
Multiple nuclear localization domains have been identified in nuclear proteins, and they finely control nuclear import and functions of those proteins. ZNF268 is a typical KRAB-containing zinc finger protein (KRAB-ZFP), and previous studies have shown that the KRAB domain reinforces nuclear localization of KRAB-ZFPs by interacting with KAP1. In this study, we find that some of 24 zinc fingers of ZNF268 also possess nuclear localization activity. Results of mutagenesis studies suggest that KRAB and zinc fingers are both necessary, and they function both independently and cooperatively for the nuclear localization of ZNF268. However, the subnuclear targeting activities of KRAB and zinc fingers are different. KRAB targets proteins in nucleoplasm, but not in the nucleolus, which is mediated by interaction with KAP1, while zinc fingers target proteins in the whole nucleus uniformly. The cooperative activities of KAP1-KRAB-zinc fingers result in the precise nucleoplasmic, but not nucleolar localization of KRAB-ZFPs. Our studies reveal a novel mechanism for the subcellular localization of KRAB-ZFPs and may help us to further explore their biological functions.
Wang, Wei; Cai, Jinyang; Lin, Yi; Liu, Zikou; Ren, Qihao; Hu, Li; Huang, Zan; Guo, Mingxiong; Li, Wenxin
Expression and nuclear localization of the TATA-box-binding protein during baculovirus infection.
The TATA-box-binding protein (TBP) plays a key role in initiating eukaryotic transcription and is used by many viruses for viral transcription. We previously reported increased TBP levels during infection with the baculovirus Autographa californica multicapsid nuclear polyhedrovirus (AcMNPV). The TBP antiserum used in that study, however, cross-reacted with a baculoviral protein. Here, we reported that increased amounts of nuclear TBP were detected upon infection of Spodoptera frugiperda and TN-368 cells with a TBP-specific antiserum. TBP levels increased until 72 h post-infection (p.i.), whilst tbp transcripts decreased by 16 h p.i., which suggested a virus-induced influence on the TBP protein levels. To address a potential modification of the TBP degradation pathway during infection, we investigated the possible role of viral ubiquitin. Infection studies with AcMNPV recombinants carrying a mutated viral ubiquitin gene revealed that the TBP increase during infection was not altered. In addition, pulse-chase experiments indicated a high TBP half-life of ~60 h in uninfected cells, suggesting that a virus-induced increase of TBP stability was unlikely. This increase in TBP correlated with a redistribution to nuclear domains resembling sites of viral DNA synthesis. Furthermore, we observed colocalization of TBP with host RNA polymerase (RNAP) II, but only until 8 h p.i., whilst TBP, but not RNAPII, was present in the enlarged replication domains late during infection. Thus, we suggested that AcMNPV adapted a mechanism to accumulate the highly stable cellular TBP at sites of viral DNA replication and transcription. PMID:24676420
Mainz, Daniela; Quadt, Ilja; Stranzenbach, Andrea K; Voss, Daniel; Guarino, Linda A; Knebel-M�rsdorf, Dagmar
Identification of Nuclear Dicing Bodies Containing Proteins for MicroRNA Biogenesis in Living Arabidopsis Plants
MicroRNAs (miRNAs) are important for regulating gene expression in muticellular organisms. MiRNA processing is a two step process, in animal cells the first step is nuclear and the second step cytoplasmic, whereas in plant cells both steps occur in the nucleus via the enzyme Dicer-like1 (DCL1) [1, 2] and other proteins including the zinc finger domain protein Serrate (SE) [3, 4] and a double-stranded RNA (DsRNA) binding domain protein Hyponastic Leaves1 (HYL1) [5�7]. Furthermore, plant miRNAs are methylated by Hua Enhancer (HEN1) at their 3� ends [8] and loaded onto Argonuate1 (AGO1) [9]. However, little is known about the cellular basis of miRNA biogenesis. Using live-cell imaging, we show here that DCL1 and HYL1 colocalize in discrete nuclear bodies in addition to being present in a low level diffuse nucleoplasmic distribution. These bodies, which we refer to as nuclear dicing bodies (D-bodies), differ from Cajal bodies [10, 11]. A mutated DCL1 with impaired function in miRNA processing fails to target to D-bodies, and an introduced pri-miRNA transcrpt is recruited to D-bodies. Furthermore, bi-molecular fluorescence complementation (BiFC) shows that DCL1, HYL1 and SE interact in D-bodies. Based upon these data we propose that D-bodies are crucial for orchestrating pri-miRNA processing and/or storage/assembly of miRNA processing complexes in the nuclei of plant cells.
Fang, Yuda; Spector, David L.
A second CRM1-dependent nuclear export signal in the influenza A virus NS2 protein contributes to the nuclear export of viral ribonucleoproteins.
Influenza A virus NS2 protein, also called nuclear export protein (NEP), is crucial for the nuclear export of viral ribonucleoproteins. However, the molecular mechanisms of NEP mediation in this process remain incompletely understood. A leucine-rich nuclear export signal (NES2) in NEP, located at the predicted N2 helix of the N-terminal domain, was identified in the present study. NES2 was demonstrated to be a transferable NES, with its nuclear export activity depending on the nuclear export receptor chromosome region maintenance 1 (CRM1)-mediated pathway. The interaction between NEP and CRM1 is coordinately regulated by both the previously reported NES (NES1) and now the new NES2. Deletion of the NES1 enhances the interaction between NEP and CRM1, and deletion of the NES1 and NES2 motifs completely abolishes this interaction. Moreover, NES2 interacts with CRM1 in the mammalian two-hybrid system. Mutant viruses containing NES2 alterations generated by reversed genetics exhibit reduced viral growth and delay in the nuclear export of viral ribonucleoproteins (vRNPs). The NES2 motif is highly conserved in the influenza A and B viruses. The results demonstrate that leucine-rich NES2 is involved in the nuclear export of vRNPs and contributes to the understanding of nucleocytoplasmic transport of influenza virus vRNPs. PMID:23115280
Huang, Shengping; Chen, Jingjing; Chen, Quanjiao; Wang, Huadong; Yao, Yanfeng; Chen, Jianjun; Chen, Ze
Transcriptional activation of NAD{sup +}-dependent protein deacetylase SIRT1 by nuclear receptor TLX
An orphan nuclear receptor TLX is a transcriptional repressor that promotes the proliferation and self-renewal of neural precursor cells (NPCs). SIRT1, an NAD{sup +}-dependent protein deacetylase, is highly expressed in the NPCs and participates in neurogenesis. Here, we found that TLX colocalized with SIRT1 and knockdown of TLX by small interfering RNAs decreased SIRT1 levels in NPCs. TLX increased the SIRT1 expression by binding to the newly identified TLX-activating element in the SIRT1 gene promoter in HEK293 cells. Thus, TLX is an inducer of SIRT1 and may contribute to neurogenesis both as a transactivator and as a repressor.
Iwahara, Naotoshi [Department of Pharmacology, Sapporo Medical University, Sapporo 060-8556 (Japan)] [Department of Pharmacology, Sapporo Medical University, Sapporo 060-8556 (Japan); Hisahara, Shin; Hayashi, Takashi [Department of Pharmacology, Sapporo Medical University, Sapporo 060-8556 (Japan) [Department of Pharmacology, Sapporo Medical University, Sapporo 060-8556 (Japan); Department of Neurology, Sapporo Medical University, Sapporo 060-8556 (Japan); Horio, Yoshiyuki, E-mail: email@example.com [Department of Pharmacology, Sapporo Medical University, Sapporo 060-8556 (Japan)] [Department of Pharmacology, Sapporo Medical University, Sapporo 060-8556 (Japan)
Colitis and Colitis-Associated Cancer Are Exacerbated in Mice Deficient for Tumor Protein 53-Induced Nuclear Protein 1?
Tumor protein 53-induced nuclear protein 1 (TP53INP1) is an antiproliferative and proapoptotic protein involved in cell stress response. To address its physiological roles in colorectal cancer and colitis, we generated and tested the susceptibility of Trp53inp1-deficient mice to the development of colorectal tumors induced by injection of the carcinogen azoxymethane followed by dextran sulfate sodium (DSS)-induced chronic colitis. Trp53inp1-deficient mice showed an increased incidence and multiplicity of tumors compared to those of wild-type (WT) mice. Furthermore, acute colitis induced by DSS treatment was more severe in Trp53inp1-deficient mice than in WT mice. Treatment with the antioxidant N-acetylcysteine prevented colitis and colitis-associated tumorigenesis more efficiently in WT mice than in Trp53inp1-deficient mice, suggesting a higher oxidative load in the latter. Consistently, we demonstrated by electron spin resonance and spin trapping that colons derived from deficient mice produced more free radicals than those of the WT during colitis and that the basal blood level of the antioxidant ascorbate was decreased in Trp53inp1-deficient mice. Collectively, these results indicate that the oxidative load is higher in Trp53inp1-deficient mice than in WT mice, generating a more-severe DSS-induced colitis, which favors development of colorectal tumors in Trp53inp1-deficient mice. Therefore, TP53INP1 is a potential target for the prevention of colorectal cancer in patients with inflammatory bowel disease.
Gommeaux, Julien; Cano, Carla; Garcia, Stephane; Gironella, Meritxell; Pietri, Sylvia; Culcasi, Marcel; Pebusque, Marie-Josephe; Malissen, Bernard; Dusetti, Nelson; Iovanna, Juan; Carrier, Alice
Theory of the time dependent transferred nuclear Overhauser effect: Applications to structural analysis of ligand-protein complexes in solution
The theory of the time dependent transferred nuclear Overhauser effect (TRNOE) for the generalized case of an exchanging system containing multiple spins as applied to a ligand-protein complex is presented and discussed. It is shown that cross-relaxation rates between pairs of bound ligand protons and between a bound ligand proton and a proton of the protein in the ligand-protein complex
Deuterium nuclear magnetic resonance study of amino acid dynamics in the membrane protein, bacteriorhodopsin
Deuterium (/sup 2/H) Fourier transform nuclear magnetic resonance (NMR) spectra for many polycrystalline, deuterium-labelled amino acids in the solid state have been obtained. Where possible, these have been biosynthetically incorporated into the membrane protein, bacteriorhodopsin in the photosynthetic purple membrane of Halobacterium halobium. Deuterio-methyl group spin lattice relaxation times have been obtained as a function of temperature. The results yield the Arrhenius activation energies for methyl rotation, and through the use of a suitable mathematical model, rotation correlation time. The results are analyzed using a mathematical model for two-fold flipping about the C/sub 2/ axis. Overall, the results demonstrate a similarity between the dynamics in amino acid crystals and in membrane proteins.
Smith, R.L.
Nuclear magnetic resonance analysis of the acetylation pattern of the neuronal tau protein.
Lysine acetylation of the neuronal Tau protein was described as a novel mechanism of posttranslational regulation of Tau functions with important outcomes in microtubule binding and aggregation processes related to Alzheimer's disease. Here, we unravel at a per-residue resolution the acetylation pattern of full-length Tau by the Creb-binding protein (CBP) acetyltransferase using high-resolution nuclear magnetic resonance spectroscopy. Our study gives a quantitative overview of CBP-mediated acetylation and examines the catalytic proficiency because the nonenzymatic reaction with acetyl-coenzyme A occurs in vitro. Furthermore, we have investigated with this characterized acetylated Tau the effect of acetylation on Tau fibrillization in a heparin-induced aggregation assay and on heparin binding. PMID:24708343
Kamah, Amina; Huvent, Isabelle; Cantrelle, Fran�ois-Xavier; Qi, Haoling; Lippens, Guy; Landrieu, Isabelle; Smet-Nocca, Caroline
Greatwall kinase: a nuclear protein required for proper chromosome condensation and mitotic progression in Drosophila.
Mutations in the Drosophila gene greatwall cause improper chromosome condensation and delay cell cycle progression in larval neuroblasts. Chromosomes are highly undercondensed, particularly in the euchromatin, but nevertheless contain phosphorylated histone H3, condensin, and topoisomerase II. Cells take much longer to transit the period of chromosome condensation from late G2 through nuclear envelope breakdown. Mutant cells are also subsequently delayed at metaphase, due to spindle checkpoint activity. These mutant phenotypes are not caused by spindle aberrations, by global defects in chromosome replication, or by activation of a caffeine-sensitive checkpoint. The Greatwall proteins in insects and vertebrates are located in the nucleus and belong to the AGC family of serine/threonine protein kinases; the kinase domain of Greatwall is interrupted by a long stretch of unrelated amino acids. PMID:14970188
Yu, Jiangtao; Fleming, Shawna L; Williams, Byron; Williams, Erika V; Li, ZeXiao; Somma, Patrizia; Rieder, Conly L; Goldberg, Michael L
Liver-type fatty acid binding protein interacts with hepatocyte nuclear factor 4?.
Hepatocyte nuclear factor 4? (HNF4?) regulates liver type fatty acid binding protein (L-FABP) gene expression. Conversely as shown herein, L-FABP structurally and functionally also interacts with HNF4?. Fluorescence resonance energy transfer (FRET) between Cy3-HNF4? (donor) and Cy5-L-FABP (acceptor) as well as FRET microscopy detected L-FABP in close proximity (~80 �) to HNF4?, binding with high affinity Kd ~250-300 nM. Circular dichroism (CD) determined that the HNF4?/L-FABP interaction altered protein secondary structure. Finally, L-FABP potentiated transactivation of HNF4? in COS7 cells. Taken together, these data suggest that L-FABP provides a signaling path to HNF4? activation in the nucleus. PMID:24140341
McIntosh, Avery L; Petrescu, Anca D; Hostetler, Heather A; Kier, Ann B; Schroeder, Friedhelm
Functional eukaryotic nuclear localization signals are widespread in terminal proteins of bacteriophages
A number of prokaryotic proteins have been shown to contain nuclear localization signals (NLSs), although its biological role remains sometimes unclear. Terminal proteins (TPs) of bacteriophages prime DNA replication and become covalently linked to the genome ends. We predicted NLSs within the TPs of bacteriophages from diverse families and hosts and, indeed, the TPs of ?29, Nf, PRD1, Bam35, and Cp-1, out of seven TPs tested, were found to localize to the nucleus when expressed in mammalian cells. Detailed analysis of ?29 TP led us to identify a bona fide NLS within residues 1�37. Importantly, gene delivery into the eukaryotic nucleus is enhanced by the presence of ?29 TP attached to the 5? DNA ends. These findings show a common feature of TPs from diverse bacteriophages targeting the eukaryotic nucleus and suggest a possible common function by facilitating the horizontal transfer of genes between prokaryotes and eukaryotes.
Redrejo-Rodriguez, Modesto; Munoz-Espin, Daniel; Holguera, Isabel; Mencia, Mario; Salas, Margarita
Iterative Development of an Application to Support Nuclear Magnetic Resonance Data Analysis of Proteins
The CONNecticut Joint University Research (CONNJUR) team is a group of biochemical and software engineering researchers at multiple institutions. The vision of the team is to develop a comprehensive application that integrates a variety of existing analysis tools with workflow and data management to support the process of protein structure determination using Nuclear Magnetic Resonance (NMR). The use of multiple disparate tools and lack of data management, currently the norm in NMR data processing, provides strong motivation for such an integrated environment. This manuscript briefly describes the domain of NMR as used for protein structure determination and explains the formation of the CONNJUR team and its operation in developing the CONNJUR application. The manuscript also describes the evolution of the CONNJUR application through four prototypes and describes the challenges faced while developing the CONNJUR application and how those challenges were met.
Ellis, Heidi J. C.; Nowling, Ronald J.; Vyas, Jay; Martyn, Timothy O.; Gryk, Michael R.
The nuclear protein Artemis promotes AMPK activation by stabilizing the LKB1-AMPK complex
Highlights: Black-Right-Pointing-Pointer The nuclear protein Artemis physically interacts with AMPK{alpha}2. Black-Right-Pointing-Pointer Artemis co-localizes with AMPK{alpha}2 in the nucleus. Black-Right-Pointing-Pointer Artemis promotes phosphorylation and activation of AMPK. Black-Right-Pointing-Pointer The interaction between AMPK{alpha}2 and LKB1 is stabilized by Artemis. -- Abstract: AMP-activated protein kinase (AMPK) is a hetero-trimeric Ser/Thr kinase composed of a catalytic {alpha} subunit and regulatory {beta} and {gamma} subunits; it functions as an energy sensor that controls cellular energy homeostasis. In response to an increased cellular AMP/ATP ratio, AMPK is activated by phosphorylation at Thr172 in the {alpha}-subunit by upstream AMPK kinases (AMPKKs), including tumor suppressor liver kinase B1 (LKB1). To elucidate more precise molecular mechanisms of AMPK activation, we performed yeast two-hybrid screening and isolated the complementary DNA (cDNA) encoding the nuclear protein Artemis/DNA cross-link repair 1C (DCLRE1C) as an AMPK{alpha}2-binding protein. Artemis was found to co-immunoprecipitate with AMPK{alpha}2, and the co-localization of Artemis with AMPK{alpha}2 in the nucleus was confirmed by immunofluorescence staining in U2OS cells. Moreover, over-expression of Artemis enhanced the phosphorylation of AMPK{alpha}2 and the AMPK substrate acetyl-CoA carboxylase (ACC). Conversely, RNAi-mediated knockdown of Artemis reduced AMPK and ACC phosphorylation. In addition, Artemis markedly increased the physical association between AMPK{alpha}2 and LKB1. Taken together, these results suggest that Artemis functions as a positive regulator of AMPK signaling by stabilizing the LKB1-AMPK complex.
Nakagawa, Koji, E-mail: email@example.com [Department of Pathophysiology and Therapeutics, Division of Pharmascience, Faculty of Pharmaceutical Sciences, Hokkaido University, N12 W6, Kita-ku, Sapporo, Hokkaido 060-0812 (Japan)] [Department of Pathophysiology and Therapeutics, Division of Pharmascience, Faculty of Pharmaceutical Sciences, Hokkaido University, N12 W6, Kita-ku, Sapporo, Hokkaido 060-0812 (Japan); Uehata, Yasuko; Natsuizaka, Mitsuteru; Kohara, Toshihisa; Darmanin, Stephanie [Department of Gastroenterology and Hematology, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 (Japan)] [Department of Gastroenterology and Hematology, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 (Japan); Asaka, Masahiro [Department of Gastroenterology and Hematology, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 (Japan) [Department of Gastroenterology and Hematology, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 (Japan); Department of Cancer Preventive Medicine, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 (Japan); Takeda, Hiroshi [Department of Pathophysiology and Therapeutics, Division of Pharmascience, Faculty of Pharmaceutical Sciences, Hokkaido University, N12 W6, Kita-ku, Sapporo, Hokkaido 060-0812 (Japan) [Department of Pathophysiology and Therapeutics, Division of Pharmascience, Faculty of Pharmaceutical Sciences, Hokkaido University, N12 W6, Kita-ku, Sapporo, Hokkaido 060-0812 (Japan); Department of Gastroenterology and Hematology, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 (Japan); Kobayashi, Masanobu [Department of Cancer Preventive Medicine, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 (Japan) [Department of Cancer Preventive Medicine, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 (Japan); School of Nursing and Social Services, Health Sciences University of Hokkaido, Ishikari-Toubetsu, Hokkaido 061-0293 (Japan)
A novel mechanism for regulating the activity of proliferating cell nuclear antigen by a small protein.
Proliferating cell nuclear antigen (PCNA) forms a trimeric ring that associates with and influences the activity of many proteins participating in DNA metabolic processes and cell cycle progression. Previously, an uncharacterized small protein, encoded by TK0808 in the archaeon Thermococcus kodakarensis, was shown to stably interact with PCNA in vivo. Here, we show that this protein, designated Thermococcales inhibitor of PCNA (TIP), binds to PCNA in vitro and inhibits PCNA-dependent activities likely by preventing PCNA trimerization. Using hydrogen/deuterium exchange mass spectrometry and site-directed mutagenesis, the interacting regions of PCNA and TIP were identified. Most proteins bind to PCNA via a PCNA-interacting peptide (PIP) motif that interacts with the inter domain connecting loop (IDCL) on PCNA. TIP, however, lacks any known PCNA-interacting motif, suggesting a new mechanism for PCNA binding and regulation of PCNA-dependent activities, which may support the development of a new subclass of therapeutic biomolecules for inhibiting PCNA. PMID:24728986
Li, Zhuo; Huang, Richard Y-C; Yopp, Daniel C; Hileman, Travis H; Santangelo, Thomas J; Hurwitz, Jerard; Hudgens, Jeffrey W; Kelman, Zvi
Leukocyte protease binding to nucleic acids promotes nuclear localization and cleavage of nucleic Acid binding proteins.
Killer lymphocyte granzyme (Gzm) serine proteases induce apoptosis of pathogen-infected cells and tumor cells. Many known Gzm substrates are nucleic acid binding proteins, and the Gzms accumulate in the target cell nucleus by an unknown mechanism. In this study, we show that human Gzms bind to DNA and RNA with nanomolar affinity. Gzms cleave their substrates most efficiently when both are bound to nucleic acids. RNase treatment of cell lysates reduces Gzm cleavage of RNA binding protein targets, whereas adding RNA to recombinant RNA binding protein substrates increases in vitro cleavage. Binding to nucleic acids also influences Gzm trafficking within target cells. Preincubation with competitor DNA and DNase treatment both reduce Gzm nuclear localization. The Gzms are closely related to neutrophil proteases, including neutrophil elastase (NE) and cathepsin G. During neutrophil activation, NE translocates to the nucleus to initiate DNA extrusion into neutrophil extracellular traps, which bind NE and cathepsin G. These myeloid cell proteases, but not digestive serine proteases, also bind DNA strongly and localize to nuclei and neutrophil extracellular traps in a DNA-dependent manner. Thus, high-affinity nucleic acid binding is a conserved and functionally important property specific to leukocyte serine proteases. Furthermore, nucleic acid binding provides an elegant and simple mechanism to confer specificity of these proteases for cleavage of nucleic acid binding protein substrates that play essential roles in cellular gene expression and cell proliferation. PMID:24771851
Thomas, Marshall P; Whangbo, Jennifer; McCrossan, Geoffrey; Deutsch, Aaron J; Martinod, Kimberly; Walch, Michael; Lieberman, Judy
Most nuclear systemic autoantigens are extremely disordered proteins: implications for the etiology of systemic autoimmunity
Patients with systemic autoimmune diseases usually produce high levels of antibodies to self-antigens (autoantigens). The repertoire of common autoantigens is remarkably limited, yet no readily understandable shared thread links these apparently diverse proteins. Using computer prediction algorithms, we have found that most nuclear systemic autoantigens are predicted to contain long regions of extreme structural disorder. Such disordered regions would generally make poor B cell epitopes and are predicted to be under-represented as potential T cell epitopes. Consideration of the potential role of protein disorder may give novel insights into the possible role of molecular mimicry in the pathogenesis of autoimmunity. The recognition of extreme autoantigen protein disorder has led us to an explicit model of epitope spreading that explains many of the paradoxical aspects of autoimmunity � in particular, the difficulty in identifying autoantigen-specific helper T cells that might collaborate with the B cells activated in systemic autoimmunity. The model also explains the experimentally observed breakdown of major histocompatibility complex (MHC) class specificity in peptides associated with the MHC II proteins of activated autoimmune B cells, and sheds light on the selection of particular T cell epitopes in autoimmunity. Finally, the model helps to rationalize the relative rarity of clinically significant autoimmunity despite the prevalence of low specificity/low avidity autoantibodies in normal individuals.
Carl, Philip L; Temple, Brenda RS; Cohen, Philip L
Kin17, a mouse nuclear zinc finger protein that binds preferentially to curved DNA.
Kin17 is a 45 kDa protein encoded by the KIN17 gene located on mouse chromosome 2, band A. The kin17 amino acid sequence predicts two domains, which were shown to be functional: (i) a bipartite nuclear localization signal (NLS) that can drive the protein to the cell nucleus, (ii) a bona fide zinc finger of the C2H2 type. The zinc finger is involved in kin17 binding to double-stranded DNA since a mutant deleted of the zinc finger, kin17 delta 1, showed reduced binding. Single-stranded DNA was bound poorly by kin17. Interestingly, we found that kin17 protein showed preferential binding to curved DNA from either pBR322 or synthetic oligonucleotides. Binding of kin17 to a non-curved DNA segment increased after we had inserted into it a short curved synthetic oligonucleotide. Kin17 delta 2, a mutant deleted of 110 amino acids at the C-terminal end, still exhibited preferential binding to curved DNA and so did kin17 delta 1, suggesting that a domain recognizing curved DNA is located in the protein core. Images
Mazin, A; Timchenko, T; Menissier-de Murcia, J; Schreiber, V; Angulo, J F; de Murcia, G; Devoret, R
Function of Piwi, a nuclear Piwi/Argonaute protein, is independent of its slicer activity
The Piwi protein subfamily is essential for Piwi-interacting RNA (piRNA) biogenesis, transposon silencing, and germ-line development, all of which have been proposed to require Piwi endonuclease activity, as validated for two cytoplasmic Piwi proteins in mice. However, recent evidence has led to questioning of the generality of this mechanism for the Piwi members that reside in the nucleus. Drosophila offers a distinct opportunity to study the function of nuclear Piwi proteins because, among three Drosophila Piwi proteins�called Piwi, Aubergine, and Argonaute 3�Piwi is the only member of this subfamily that is localized in the nucleus and expressed in both germ-line and somatic cells in the gonad, where it is responsible for piRNA biogenesis and regulatory functions essential for fertility. In this study, we demonstrate beyond doubt that the slicer activity of Piwi is not required for any known functions in vivo. We show that, in transgenic flies with the DDX catalytic triad of PIWI mutated, neither primary nor secondary piRNA biogenesis is detectably affected, transposons remain repressed, and fertility is normal. Our observations demonstrate that the mechanism of Piwi is independent of its in vitro endonuclease activity. Instead, it is consistent with the alternative mode of Piwi function as a molecule involved in the piRNA-directed guidance of epigenetic factors to chromatin.
Darricarrere, Nicole; Liu, Na; Watanabe, Toshiaki; Lin, Haifan
Transfection of influenza A virus nuclear export protein induces the expression of tumor necrosis factor alpha.
Influenza A virus genomic segments eight codes for non-structural 1 (NS1) protein that is involved in evasion of innate antiviral response, and nuclear export protein (NEP) that participates in the export of viral ribonucleoprotein (RNP) complexes, transcription and replication. Tumor necrosis factor alpha (TNF-?) is highly expressed during influenza virus infections and is considered an anti-infective cytokine. NS1 and NEP proteins were overexpressed and their role on TNF-? expression was evaluated. Both TNF-? mRNA and protein increased in cells transfected with NEP but not with NS1. We further investigate if NS1 or NEP regulates the activity of TNF-? promoter. In the presence of NEP the activity of TNF-? promoter increased significantly compared with the control (83.5�2.9 vs. 30.9�2.8, respectively; p=0.001). This effect decreased 15-fold when the TNF-? promoter distal region was deleted, suggesting the involvement of mitogen-activated protein kinases (MAPK) and NF-kB response elements. This was corroborated by testing the effect produced on TNF-? promoter by the treatment with Raf/MEK/ERK (U0126), NF-kB (Bay-11-7082) and PI3K (Ly294-002) cell signaling inhibitors. Treatment with U0126 and Bay-117082 reduced the activity of TNF-? promoter mediated by NEP (41.5�3.2, 70% inhibition; and 80.6�7.4, 35% inhibition, respectively) compared to mock-treated control. The results suggest a new role for NEP protein that participates in the transcriptional regulation of human TNF-? expression. PMID:24657783
Lara-Sampablo, Alejandra; Flores-Alonso, Juan Carlos; De Jes�s-Ortega, Nereyda; Santos-L�pez, Gerardo; Vallejo-Ruiz, Ver�nica; Rosas-Murrieta, Nora; Reyes-Carmona, Sandra; Herrera-Camacho, Irma; Reyes-Leyva, Julio
Calpain 5 is highly expressed in the central nervous system (CNS), carries dual nuclear localization signals, and is associated with nuclear promyelocytic leukemia protein bodies.
Calpain 5 (CAPN5) is a non-classical member of the calpain family. It lacks the EF hand motif characteristic of classical calpains but retains catalytic and Ca(2+) binding domains, and it contains a unique C-terminal domain. TRA-3, an ortholog of CAPN5, has been shown to be involved in necrotic cell death in Caenorhabditis elegans. CAPN5 is expressed throughout the CNS, but its expression relative to other calpains and subcellular distribution has not been investigated previously. Based on relative mRNA levels, Capn5 is the second most highly expressed calpain in the rat CNS, with Capn2 mRNA being the most abundant. Unlike classical calpains, CAPN5 is a non-cytosolic protein localized to the nucleus and extra-nuclear locations. CAPN5 possesses two nuclear localization signals (NLS): an N-terminal monopartite NLS and a unique bipartite NLS closer to the C terminus. The C-terminal NLS contains a SUMO-interacting motif that contributes to nuclear localization, and mutation or deletion of both NLS renders CAPN5 exclusively cytosolic. Dual NLS motifs are common among transcription factors. Interestingly, CAPN5 is found in punctate domains associated with promyelocytic leukemia (PML) protein within the nucleus. PML nuclear bodies are implicated in transcriptional regulation, cell differentiation, cellular response to stress, viral defense, apoptosis, and cell senescence as well as protein sequestration, modification, and degradation. The roles of nuclear CAPN5 remain to be determined. PMID:24838245
Singh, Ranjana; Brewer, M Kathryn; Mashburn, Charles B; Lou, Dingyuan; Bondada, Vimala; Graham, Brantley; Geddes, James W
A short Id2 protein fragment containing the nuclear export signal forms amyloid-like fibrils
The negative regulator of DNA-binding/cell-differentiation Id2 is a small protein containing a central helix-loop-helix (HLH) motif and a C-terminal nuclear export signal (NES). Whereas the former is essential for Id2 dimerization and nuclear localization, the latter is responsible for the transport of Id2 from the nucleus to the cytoplasm. Whereas the isolated Id2 HLH motif is highly helical, large C-terminal Id2 fragments including the NES sequence are either unordered or aggregation-prone. To study the conformational properties of the isolated NES region, we synthesized the Id2 segment 103-124. The latter was insoluble in water and only temporarily soluble in water/alcohol mixtures, where it formed quickly precipitating {beta}-sheets. Introduction of a positively charged N-terminal tail prevented aggressive precipitation and led to aggregates consisting of long fibrils that bound thioflavin T. These results show an interesting structural aspect of the Id2 NES region, which might be of significance for both protein folding and function.
Colombo, Noemi [Fakultaet fuer Chemie und Pharmazie, Universitaet Regensburg, Universitaetsstrasse 31, 93053 Regensburg (Germany); Schroeder, Josef [Institut fuer Pathologie, Zentrales EM-Labor, Fakultaet fuer Medizin, Universitaet Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg (Germany); Cabrele, Chiara [Fakultaet fuer Chemie und Pharmazie, Universitaet Regensburg, Universitaetsstrasse 31, 93053 Regensburg (Germany)]. E-mail: email@example.com
Interaction between the human nuclear cap-binding protein complex and hnRNP F.
hnRNP F was identified in a screen for proteins that interact with human CBP80 and CBP20, the components of the nuclear cap-binding complex (CBC). In vitro interaction studies showed that hnRNP F can bind to both CBP20 and CBP80 individually. hnRNP F and CBC bind independently to RNA, but hnRNP F binds preferentially to CBC-RNA complexes rather than to naked RNA. The hnRNP H protein, which is 78% identical to hnRNP F and also interacts with both CBP80 and CBP20 in vitro, does not discriminate between naked RNA and CBC-RNA complexes, showing that this effect is specific. Depletion of hnRNP F from HeLa cell nuclear extract decreases the efficiency of pre-mRNA splicing, a defect which can be partially compensated by addition of recombinant hnRNP F. Thus, hnRNP F is required for efficient pre-mRNA splicing in vitro and may participate in the effect of CBC on pre-mRNA splicing.
Gamberi, C; Izaurralde, E; Beisel, C; Mattaj, I W
Nuclear translocation of the cytoskeleton-associated protein, smALP, upon induction of skeletal muscle differentiation
Highlights: {yields} The cytoskeleton-associated protein, smALP, is expressed in differentiated skeletal muscle. {yields} smALP is translocated from the cytoplasm to the nucleus of C2C12 myoblasts upon induction of myogenesis. {yields} The differentiation-dependent nuclear translocation of smALP occurs in parallel with the nuclear accumulation of myogenin. {yields} The LIM domain of smALP is essential for the nuclear accumulation of the protein. {yields} smALP might act in the nucleus to control some critical aspect of the muscle differentiation process. -- Abstract: The skALP isoform has been shown to play a critical role in actin organization and anchorage within the Z-discs of skeletal muscles, but no data is available on the function of the smALP isoform in skeletal muscle cells. Here, we show that upon induction of differentiation a nuclear translocation of smALP from the cytoplasm to the nucleus of C2C12 myoblasts, concomitant to an up-regulation of the protein expression, occurs in parallel with the nuclear accumulation of myogenin. Moreover, we demonstrate that the LIM domain of smALP is essential for the nuclear translocation of the protein.
Cambier, Linda [CNRS UMR5237, Universite Montpellier 1, Universite Montpellier 2, Centre de Recherche en Biochimie Macromoleculaire, Montpellier (France)] [CNRS UMR5237, Universite Montpellier 1, Universite Montpellier 2, Centre de Recherche en Biochimie Macromoleculaire, Montpellier (France); Pomies, Pascal, E-mail: email@example.com [CNRS UMR5237, Universite Montpellier 1, Universite Montpellier 2, Centre de Recherche en Biochimie Macromoleculaire, Montpellier (France)] [CNRS UMR5237, Universite Montpellier 1, Universite Montpellier 2, Centre de Recherche en Biochimie Macromoleculaire, Montpellier (France)
KIFC1-Like Motor Protein Associates with the Cephalopod Manchette and Participates in Sperm Nuclear Morphogenesis in Octopus tankahkeei
Background Nuclear morphogenesis is one of the most fundamental cellular transformations taking place during spermatogenesis. In rodents, a microtubule-based perinuclear structure, the manchette, and a C-terminal kinesin motor KIFC1 are believed to play crucial roles in this process. Spermatogenesis in Octopus tankahkeei is a good model system to explore whether evolution has created a cephalopod prototype of mammalian manchette-based and KIFC1-dependent sperm nuclear shaping machinery. Methodology/Principal Findings We detected the presence of a KIFC1-like protein in the testis, muscle, and liver of O. tankahkeei by Western Blot. Then we tracked its dynamic localization in spermatic cells at various stages using Immunofluorescence and Immunogold Electron Microscopy. The KIFC1-like protein was not expressed at early stages of spermatogenesis when no significant morphological changes occur, began to be present in early spermatid, localized around and in the nucleus of intermediate and late spermatids where the nucleus was dramatically elongated and compressed, and concentrated at one end of final spermatid. Furthermore, distribution of the motor protein during nuclear elongation and condensation overlapped with that of the cephalopod counterpart of manchette at a significant level. Conclusions/Significance The results support the assumption that the protein is actively involved in sperm nuclear morphogenesis in O. tankahkeei possibly through bridging the manchette-like perinuclear microtubules to the nucleus and assisting in the nucleocytoplasmic trafficking of specific cargoes. This study represents the first description of the role of a motor protein in sperm nuclear shaping in cephalopod.
Tan, Fu-Qing; Yang, Wan-Xi
Characterization of a nuclear 20S complex containing the survival of motor neurons (SMN) protein and a specific subset of spliceosomal Sm proteins.
Spinal muscular atrophy (SMA) is a neurodegenerative disease of motor neurons caused by reduced levels of functional survival of motor neurons (SMN) protein. Cytoplasmic SMN directly interacts with spliceosomal Sm proteins and facilitates their assembly onto U snRNAs. Nuclear SMN, in contrast, mediates recycling of pre-mRNA splicing factors. In this study, we have addressed the function of SMN in the nucleus. We show that a monoclonal antibody directed against SMN inhibits pre-mRNA splicing. Interestingly, the mode of inhibition suggests a novel role for SMN in splicing that occurs prior to, or in addition to, its role in recycling. Using biochemical fractionation and anti-SMN immunoaffinity chromatography, we identified two distinct nuclear SMN complexes termed NSC1 and NSC2. The biochemical properties and protein composition of NSC1 were determined in detail. NSC1 migrates in sucrose gradients as a U snRNA-free 20S complex containing at least 10 proteins. In addition to SMN, these include the SMN-interacting protein 1 (SIP-1), the putative helicase dp103/Gemin3, the novel dp103/Gemin3-interacting protein GIP1/Gemin4 and three additional proteins with apparent masses of 43, 33 and 18 kDa, respectively. Most surprisingly, NSC1 also contains a specific subset of spliceosomal Sm proteins. This shows that the SMN-Sm protein interaction is not restricted to the cytoplasm. Our data imply that nuclear SMN affects splicing by modulating the Sm protein composition of U snRNPs. PMID:10942426
Meister, G; B�hler, D; Laggerbauer, B; Zobawa, M; Lottspeich, F; Fischer, U
The movement protein BC1 promotes redirection of the nuclear shuttle protein BV1 of Abutilon mosaic geminivirus to the plasma membrane in fission yeast
Summary.��In order to monitor their interaction and cellular localisation, the movement protein (MP; syn. BC1) and the nuclear shuttle\\u000a protein (NSP; syn. BV1) of the geminivirus Abutilon mosaic virus (AbMV) were ectopically expressed in Schizosaccharomyces pombe cells, either alone or together under the control of an inducible promoter. For highest resolution, electron microscopy using\\u000a freeze-fracture immunolabelling served to detect these
S. Frischmuth; C. Wege; D. H�lser; H. Jeske
Characterization of a nuclear 20S complex containing the survival of motor neurons (SMN) protein and a specific subset of spliceosomal Sm proteins
Spinal muscular atrophy (SMA) is a neuro- degenerative disease of motor neurons caused by reduced levels of functional survival of motor neurons (SMN) protein. Cytoplasmic SMN directly interacts with spliceosomal Sm proteins and facilit- ates their assembly onto U snRNAs. Nuclear SMN, in contrast, mediates recycling of pre-mRNA splicing factors. In this study, we have addressed the function of SMN
Gunter Meister; Dirk B�hler; Bernhard Laggerbauer; Monika Zobawa; Friedrich Lottspeich; Utz Fischer
The nuclear localization of low risk HPV11 E7 protein mediated by its zinc binding domain is independent of nuclear import receptors
We investigated the nuclear import of low risk HPV11 E7 protein using 1) transfection assays in HeLa cells with EGFP fusion plasmids containing 11E7 and its domains and 2) nuclear import assays in digitonin-permeabilized HeLa cells with GST fusion proteins containing 11E7 and its domains. The EGFP-11E7 and EGFP-11cE7{sub 39-98} localized mostly to the nucleus. The GST-11E7 and GST-11cE7{sub 39-98} were imported into the nuclei in the presence of either Ran-GDP or RanG19V-GTP mutant and in the absence of nuclear import receptors. This suggests that 11E7 enters the nucleus via a Ran-dependent pathway, independent of nuclear import receptors, mediated by a nuclear localization signal located in its C-terminal domain (cNLS). This cNLS contains the zinc binding domain consisting of two copies of Cys-X-X-Cys motif. Mutagenesis of Cys residues in these motifs changed the localization of the EGFP-11cE7/-11E7 mutants to cytoplasmic, suggesting that the zinc binding domain is essential for nuclear localization of 11E7.
Piccioli, Zachary; McKee, Courtney H.; Leszczynski, Anna; Onder, Zeynep; Hannah, Erin C.; Mamoor, Shahan; Crosby, Lauren; Moroianu, Junona, E-mail: firstname.lastname@example.com
Purification and partial sequencing of the nuclear autoantigen RA33 shows that it is indistinguishable from the A2 protein of the heterogeneous nuclear ribonucleoprotein complex.
RA33 is a nuclear autoantigen with an apparent molecular mass of 33 kD. Autoantibodies against RA33 are found in about 30% of sera from RA patients, but only occasionally in sera from patients with other connective tissue diseases. To characterize RA33, the antigen was purified from HeLa cell nuclear extracts to more than 90% homogeneity by affinity chromatography on heparin-Sepharose and by chromatofocusing. Sequence analysis of five tryptic peptides revealed that their sequences matched corresponding sequences of the A2 protein of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex. Furthermore, RA33 was shown to be present in the 40S hnRNP complex and to behave indistinguishably from A2 in binding to single stranded DNA. In summary, these data strongly indicate that RA33 and A2 are the same protein, and thus identify on a molecular level a new autoantigen. Images
Steiner, G; Hartmuth, K; Skriner, K; Maurer-Fogy, I; Sinski, A; Thalmann, E; Hassfeld, W; Barta, A; Smolen, J S
Yes-Associated Protein (YAP) Promotes the Nuclear Import of p73
NASA Astrophysics Data System (ADS)
p73 has been identified as a structural and functional homolog of the tumor suppressor p53. However, mechanisms that regulate the localization of p73 have not been fully clarified. The Yes-associated protein (YAP) is a transcriptional coactivator. As a transcriptional coactivator, YAP needs to bind transcription factors to stimulate gene expression. p73 is a reported YAP target transcription factors and YAP has been shown to positively regulate p73 in promoting apoptosis. Previous studies show that p73 interacts with YAP through its PPPY motif, and increases p73 transactivation of apoptotic genes. In this study, we focused on YAP's regulation of the localization of p73. After transient transfection into Rat pheochromocytoma (PC12) cells and Human embryonic kidney 293T cells with GFP-YAP and/or YFP-p73, and incubated for 24 hours expression. p73 was fused to YFP to allow the examination of its subcellular localization. When expressed alone, YFP-p73 was distributed throughout the cell. When coexpressed with YAP, nuclear accumulation of YFP-p73 became evident. We quantitated the effect of YAP on the redistribution of YFP-p73 by counting cells with nuclear-only YFP signal. We found that YAP can influence the subcellular distribution of p73. Altogether, coexpression with YAP affected the subcellular distribution of the p73 protein. Our studies attribute a central role to YAP in regulating p73 accumulation and YAP, at least in part, might promote the nuclear import of p73.
Zhang, Heng; Wu, Shengnan
Interactions and Nuclear Import of the N and P Proteins of Sonchus Yellow Net Virus, a Plant Nucleorhabdovirus
We have characterized the interaction and nuclear localization of the nucleocapsid (N) protein and phosphoprotein (P) of sonchus yellow net nucleorhabdovirus. Expression studies with plant and yeast cells revealed that both N and P are capable of independent nuclear import. Site-specific mutagenesis and deletion analyses demonstrated that N contains a carboxy-terminal bipartite nuclear localization signal (NLS) located between amino acids 465 and 481 and that P contains a karyophillic region between amino acids 40 and 124. The N NLS was fully capable of functioning outside of the context of the N protein and was able to direct the nuclear import of a synthetic protein fusion consisting of green fluorescent protein fused to glutathione S-transferase (GST). Expression and mapping studies suggested that the karyophillic domain in P is located within the N-binding domain. Coexpression of N and P drastically affected their localization patterns relative to those of individually expressed proteins and resulted in a shift of both proteins to a subnuclear region. Yeast two-hybrid and GST pulldown experiments verified the N-P and P-P interactions, and deletion analyses have identified the N and P interacting domains. N NLS mutants were not transported to the nucleus by import-competent P, presumably because N binding masks the P NLS. Taken together, our results support a model for independent entry of N and P into the nucleus followed by associations that mediate subnuclear localization.
Goodin, Michael M.; Austin, Jennifer; Tobias, Renee; Fujita, Miki; Morales, Christina; Jackson, Andrew O.
Arrest of Nuclear Division in Plasmodium through Blockage of Erythrocyte Surface Exposed Ribosomal Protein P2
Malaria parasites reside inside erythrocytes and the disease manifestations are linked to the growth inside infected erythrocytes (IE). The growth of the parasite is mostly confined to the trophozoite stage during which nuclear division occurs followed by the formation of cell bodies (schizogony). The mechanism and regulation of schizogony are poorly understood. Here we show a novel role for a Plasmodium falciparum 60S stalk ribosomal acidic protein P2 (PfP2) (PFC0400w), which gets exported to the IE surface for 6�8 hrs during early schizogony, starting around 26�28 hrs post-merozoite invasion. The surface exposure is demonstrated using multiple PfP2-specific monoclonal antibodies, and is confirmed through transfection using PfP2-GFP. The IE surface-exposed PfP2-protein occurs mainly as SDS-resistant P2-homo-tetramers. Treatment with anti-PfP2 monoclonals causes arrest of IEs at the first nuclear division. Upon removal of the antibodies, about 80�85% of synchronized parasites can be released even after 24 hrs of antibody treatment. It has been reported that a tubovesicular network (TVN) is set up in early trophozoites which is used for nutrient import. Anti-P2 monoclonal antibodies cause a complete fragmentation of TVN by 36 hrs, and impairs lipid import in IEs. These may be downstream causes for the cell-cycle arrest. Upon antibody removal, the TVN is reconstituted, and the cell division progresses. Each of the above properties is observed in the rodent malaria parasite species P. yoelii and P. berghei. The translocation of the P2 protein to the IE surface is therefore likely to be of fundamental importance in Plasmodium cell division.
Das, Sudipta; Basu, Himanish; Korde, Reshma; Tewari, Rita; Sharma, Shobhona
Nuclear localization of the actin regulatory protein Palladin in sertoli cells.
In the testis, F-actin structures are involved in spermatid nuclear remodeling and cytoplasm reduction, maintenance of the blood-testis barrier, support of the spermatogonial stem cell niche, and release of spermatids into the tubular lumen. To gain a better understanding of actin regulation in Sertoli-germ cell interactions, we investigated the expression of the Palladin (Palld) gene, which encodes a widely expressed phosphoprotein that localizes to actin-rich cytoplasmic structures, including focal adhesions, cell-cell junctions, podosomes, and stress fibers, and serves as a molecular scaffold to bundle actin fibers. In germ cells, PALLD was concentrated along the tubulin- and F-actin-containing cytoplasmic manchette that forms adjacent to the elongating spermatid nucleus during spermiogenesis. To our surprise, PALLD relocated from the cytoplasm to the nucleus of Sertoli cells in the juvenile testis, coincident with the onset of puberty, and this localization was maintained in the adult. We provide evidence that the 140 kDa isoform of PALLD predominates in Sertoli cells, and that it is apparently cleaved, with the C-terminus localizing to the nucleus while the N-terminus remains cytoplasmic. We investigated the nuclear localization of the C-terminus of PALLD and found that it is regulated by a putative nuclear export signal. These results provide the foundation for future work employing Sertoli cell- and spermatid-specific Palld-knockout mice to study diverse roles of PALLD as both a nuclear-actin regulatory protein and as a potential regulator of manchette formation during spermatogenesis. PMID:23559268
Niedenberger, Bryan A; Chappell, Vesna K; Kaye, Evelyn P; Renegar, Randall H; Geyer, Christopher B
Nuclear Fragile X Mental Retardation Protein Is localized to Cajal Bodies
Fragile X syndrome is caused by loss of function of a single gene encoding the Fragile X Mental Retardation Protein (FMRP). This RNA-binding protein, widely expressed in mammalian tissues, is particularly abundant in neurons and is a component of messenger ribonucleoprotein (mRNP) complexes present within the translational apparatus. The absence of FMRP in neurons is believed to cause translation dysregulation and defects in mRNA transport essential for local protein synthesis and for synaptic development and maturation. A prevalent model posits that FMRP is a nucleocytoplasmic shuttling protein that transports its mRNA targets from the nucleus to the translation machinery. However, it is not known which of the multiple FMRP isoforms, resulting from the numerous alternatively spliced FMR1 transcripts variants, would be involved in such a process. Using a new generation of anti-FMRP antibodies and recombinant expression, we show here that the most commonly expressed human FMRP isoforms (ISO1 and 7) do not localize to the nucleus. Instead, specific FMRP isoforms 6 and 12 (ISO6 and 12), containing a novel C-terminal domain, were the only isoforms that localized to the nuclei in cultured human cells. These isoforms localized to specific p80-coilin and SMN positive structures that were identified as Cajal bodies. The Cajal body localization signal was confined to a 17 amino acid stretch in the C-terminus of human ISO6 and is lacking in a mouse Iso6 variant. As FMRP is an RNA-binding protein, its presence in Cajal bodies suggests additional functions in nuclear post-transcriptional RNA metabolism. Supporting this hypothesis, a missense mutation (I304N), known to alter the KH2-mediated RNA binding properties of FMRP, abolishes the localization of human FMRP ISO6 to Cajal bodies. These findings open unexplored avenues in search for new insights into the pathophysiology of Fragile X Syndrome.
Tremblay, Sandra; Rose, Timothy M.; Cote, Jocelyn; De Koninck, Paul; Khandjian, Edouard W.
Evolution of metamorphosis: role of environment on expression of mutant nuclear receptors and other signal-transduction proteins.
Various lipophilic signals, including ecdysone, retinoic acid, estradiol, cortisol, testosterone, and progesterone, act through nuclear receptors, a large group of transcription factors that regulate differentiation and development, which are central to metamorphosis. Here, we focus on environmental factors (for example climate and chemicals) in the evolution of nuclear receptors and other signal-transduction proteins that interact with heat-shock protein 90 (Hsp90), a chaperone that promotes the proper folding and trafficking in cells of proteins. Hsp90 also promotes functional folding of some mutant signal proteins, which would be otherwise destabilized. Stress diverts Hsp90 from stabilizing mutant signal-transduction proteins and toward promoting proper folding of stress-damaged proteins and preventing the aggregation of denatured proteins. Reduced Hsp90 levels allow expression of cryptic mutations in signal-transduction proteins and new developmental patterns. Thus, environmental stress in the form of extreme climate can influence the evolution of metamorphosis. We discuss how extreme cooling called "Snowball Earth," which occurred in the late Proterozoic, diverted Hsp90 from chaperoning signal-transduction proteins. As a result, pre-existing mutant signal-transduction proteins were expressed in animals. Some mutations were selectively advantageous in animals that are seen in the Cambrian, when diverse pathways for metamorphosis in metazoans first appear in the fossil record. Other environmental factors, such as biological chemicals (for example the antibiotic geldanamycin) can reduce the levels of active Hsp90 providing another mechanism for the emergence of mutant signaling pathways. PMID:21672786
Baker, Michael E
NF-?B p65 regulates nuclear translocation of Ku70 via degradation of heat shock cognate protein 70 in pancreatic acinar AR42J cells
Ku proteins such as Ku70 and Ku80 play key roles in multiple nuclear processes. Nuclear translocation of Ku70 is independent of Ku80 translocation and mediated by nuclear localization signal (NLS) receptors including importin-?. In the present study using pancreatic acinar AR42J cells, heat shock cognate protein 70 (Hsc70) was identified as the protein associated with NLS of Ku70. Interaction of
Joo Weon Lim; Kyung Hwan Kim; Hyeyoung Kim
Atypical I?B proteins - nuclear modulators of NF-?B signaling
Nuclear factor ?B (NF-?B) controls a multitude of physiological processes such as cell differentiation, cytokine expression, survival and proliferation. Since NF-?B governs embryogenesis, tissue homeostasis and the functions of innate and adaptive immune cells it represents one of the most important and versatile signaling networks known. Its activity is regulated via the inhibitors of NF-?B signaling, the I?B proteins. Classical I?Bs, like the prototypical protein I?B?, sequester NF-?B transcription factors in the cytoplasm by masking of their nuclear localization signals (NLS). Thus, binding of NF-?B to the DNA is inhibited. The accessibility of the NLS is controlled via the degradation of I?B?. Phosphorylation of the conserved serine residues 32 and 36 leads to polyubiquitination and subsequent proteasomal degradation. This process marks the central event of canonical NF-?B activation. Once their NLS is accessible, NF-?B transcription factors translocate into the nucleus, bind to the DNA and regulate the transcription of their respective target genes. Several studies described a distinct group of atypical I?B proteins, referred to as the BCL-3 subfamily. Those atypical I?Bs show entirely different sub-cellular localizations, activation kinetics and an unexpected functional diversity. First of all, their interaction with NF-?B transcription factors takes place in the nucleus in contrast to classical I?Bs, whose binding to NF-?B predominantly occurs in the cytoplasm. Secondly, atypical I?Bs are strongly induced after NF-?B activation, for example by LPS and IL-1? stimulation or triggering of B cell and T cell antigen receptors, but are not degraded in the first place like their conventional relatives. Finally, the interaction of atypical I?Bs with DNA-associated NF-?B transcription factors can further enhance or diminish their transcriptional activity. Thus, they do not exclusively act as inhibitors of NF-?B activity. The capacity to modulate NF-?B transcription either positively or negatively, represents their most important and unique mechanistic difference to classical I?Bs. Several reports revealed the importance of atypical I?B proteins for immune homeostasis and the severe consequences following their loss of function. This review summarizes insights into the physiological processes regulated by this protein class and the relevance of atypical I?B functioning.
Engineering light-inducible nuclear localization signals for precise spatiotemporal control of protein dynamics in living cells
The function of many eukaryotic proteins is regulated by highly dynamic changes in their nucleocytoplasmic distribution. The ability to precisely and reversibly control nuclear translocation would, therefore, allow dissecting and engineering cellular networks. Here we develop a genetically encoded, light-inducible nuclear localization signal (LINuS) based on the LOV2 domain of Avena sativa phototropin 1. LINuS is a small, versatile tag, customizable for different proteins and cell types. LINuS-mediated nuclear import is fast and reversible, and can be tuned at different levels, for instance, by introducing mutations that alter AsLOV2 domain photo-caging properties or by selecting nuclear localization signals (NLSs) of various strengths. We demonstrate the utility of LINuS in mammalian cells by controlling gene expression and entry into mitosis with blue light.
Niopek, Dominik; Benzinger, Dirk; Roensch, Julia; Draebing, Thomas; Wehler, Pierre; Eils, Roland; Di Ventura, Barbara
Engineering light-inducible nuclear localization signals for precise spatiotemporal control of protein dynamics in living cells.
The function of many eukaryotic proteins is regulated by highly dynamic changes in their nucleocytoplasmic distribution. The ability to precisely and reversibly control nuclear translocation would, therefore, allow dissecting and engineering cellular networks. Here we develop a genetically encoded, light-inducible nuclear localization signal (LINuS) based on the LOV2 domain of Avena sativa phototropin 1. LINuS is a small, versatile tag, customizable for different proteins and cell types. LINuS-mediated nuclear import is fast and reversible, and can be tuned at different levels, for instance, by introducing mutations that alter AsLOV2 domain photo-caging properties or by selecting nuclear localization signals (NLSs) of various strengths. We demonstrate the utility of LINuS in mammalian cells by controlling gene expression and entry into mitosis with blue light. PMID:25019686
Niopek, Dominik; Benzinger, Dirk; Roensch, Julia; Draebing, Thomas; Wehler, Pierre; Eils, Roland; Di Ventura, Barbara
Homodimeric and heterodimeric leucine zipper proteins and nuclear factors from parsley recognize diverse promoter elements with ACGT cores.
Four short nucleotide sequences (boxes I to IV) contribute to the light responsiveness of the parsley chalcone synthase promoter. The sequence-related boxes II and III resemble several plant, viral, and bacterial promoter elements that share ACGT core sequences and are associated with diversely regulated genes. We have analyzed the binding characteristics and protein-protein interactions of factors from nuclear extracts and of three putative leucine zipper (bZIP) transcription factors potentially involved in the regulation of this promoter. These common plant regulatory factors (CPRFs) bind specifically to boxes II and III as well as other ACGT-containing promoter elements (hex1, Em1a, and as-1), though with markedly different affinities. Intact bZIP domains are crucial for CPRF binding to DNA. Distinct ensembles of nuclear factors bind to boxes II and III, despite their sequence similarity. The parsley CPRFs bind to DNA as dimers, selectively form heterodimeric DNA binding complexes, and interact with nuclear proteins.
Armstrong, G A; Weisshaar, B; Hahlbrock, K
Arabidopsis ALF4 encodes a nuclear-localized protein required for lateral root formation.
Lateral root formation, the primary way plants increase their root mass, displays developmental plasticity in response to environmental changes. The aberrant lateral root formation (alf)4-1 mutation blocks the initiation of lateral roots, thus greatly altering root system architecture. We have positionally cloned the ALF4 gene and have further characterized its phenotype. The encoded ALF4 protein is conserved among plants and has no similarities to proteins from other kingdoms. The gene is present in a single copy in Arabidopsis. Using translational reporters for ALF4 gene expression, we have determined that the ALF4 protein is nuclear localized and that the gene is expressed in most plant tissues; however, ALF4 expression and ALF4's subcellular location are not regulated by auxin. These findings taken together with further genetic and phenotypic characterization of the alf4-1 mutant suggest that ALF4 functions independent from auxin signaling and instead functions in maintaining the pericycle in the mitotically competent state needed for lateral root formation. Our results provide genetic evidence that the pericycle shares properties with meristems and that this tissue plays a central role in creating the developmental plasticity needed for root system development. PMID:14731255
DiDonato, Raymond J; Arbuckle, Erin; Buker, Shane; Sheets, Jill; Tobar, Jos�; Totong, Ronald; Grisafi, Paula; Fink, Gerald R; Celenza, John L
Targeting proliferating cell nuclear antigen and its protein interactions induces apoptosis in multiple myeloma cells.
Multiple myeloma is a hematological cancer that is considered incurable despite advances in treatment strategy during the last decade. Therapies targeting single pathways are unlikely to succeed due to the heterogeneous nature of the malignancy. Proliferating cell nuclear antigen (PCNA) is a multifunctional protein essential for DNA replication and repair that is often overexpressed in cancer cells. Many proteins involved in the cellular stress response interact with PCNA through the five amino acid sequence AlkB homologue 2 PCNA-interacting motif (APIM). Thus inhibiting PCNA's protein interactions may be a good strategy to target multiple pathways simultaneously. We initially found that overexpression of peptides containing the APIM sequence increases the sensitivity of cancer cells to contemporary therapeutics. Here we have designed a cell-penetrating APIM-containing peptide, ATX-101, that targets PCNA and show that it has anti-myeloma activity. We found that ATX-101 induced apoptosis in multiple myeloma cell lines and primary cancer cells, while bone marrow stromal cells and primary healthy lymphocytes were much less sensitive. ATX-101-induced apoptosis was caspase-dependent and cell cycle phase-independent. ATX-101 also increased multiple myeloma cells' sensitivity against melphalan, a DNA damaging agent commonly used for treatment of multiple myeloma. In a xenograft mouse model, ATX-101 was well tolerated and increased the anti-tumor activity of melphalan. Therefore, targeting PCNA by ATX-101 may be a novel strategy in multiple myeloma treatment. PMID:23936203
M�ller, Rebekka; Misund, Kristine; Holien, Toril; Bachke, Siri; Gilljam, Karin M; V�tsveen, Thea K; R�, Torstein B; Bellacchio, Emanuele; Sundan, Anders; Otterlei, Marit
Towards a robust description of intrinsic protein disorder using nuclear magnetic resonance spectroscopy.
In order to understand the conformational behaviour of Intrinsically Disordered Proteins (IDPs), it is essential to develop a molecular representation of the partially folded state. Due to the very large number of degrees of conformational freedom available to such a disordered system, this problem is highly underdetermined. Characterisation therefore requires extensive experimental data, and novel analytical tools are required to exploit the specific conformational sensitivity of different experimental parameters. In this review we concentrate on the use of nuclear magnetic resonance (NMR) spectroscopy for the study of conformational behaviour of IDPs at atomic resolution. Each experimental NMR parameter is sensitive to different aspects of the structural and dynamic behaviour of the disordered state and requires specific consideration of the relevant averaging properties of the physical interaction. In this review we present recent advances in the description of disordered proteins and the selection of representative ensembles on the basis of experimental data using statistical coil sampling from flexible-meccano and ensemble selection using ASTEROIDS. Using these tools we aim to develop a unified molecular representation of the disordered state, combining complementary data sets to extract a meaningful description of the conformational behaviour of the protein. PMID:21874206
Schneider, Robert; Huang, Jie-rong; Yao, Mingxi; Communie, Guillaume; Ozenne, Val�ry; Mollica, Luca; Salmon, Lo�c; Jensen, Malene Ringkj�bing; Blackledge, Martin
Modulation of Epstein�Barr Virus Nuclear Antigen 2-dependent transcription by protein arginine methyltransferase 5
Highlights: ? Catalytic active PRMT5 substantially binds to the EBNA2 RG domain. ? PRMT5 augments the EBNA2-dependent transcription. ? PRMT5 triggers the symmetric dimethylation of the EBNA2 RG domain. ? PRMT5 enhances the promoter occupancy of EBNA2 on its target promoters. -- Abstract: Epstein�Barr Virus Nuclear Antigen (EBNA) 2 features an Arginine�Glycine repeat (RG) domain at amino acid positions 335�360, which is a known target for protein arginine methyltransferaser 5 (PRMT5). In this study, we performed protein affinity pull-down assays to demonstrate that endogenous PRMT5 derived from lymphoblastoid cells specifically associated with the protein bait GST-E2 RG. Transfection of a plasmid expressing PRMT5 induced a 2.5- to 3-fold increase in EBNA2-dependent transcription of both the LMP1 promoter in AKATA cells, which contain the EBV genome endogenously, and a Cp-Luc reporter plasmid in BJAB cells, which are EBV negative. Furthermore, we showed that there was a 2-fold enrichment of EBNA2 occupancy in target promoters in the presence of exogenous PRMT5. Taken together, we show that PRMT5 triggers the symmetric dimethylation of EBNA2 RG domain to coordinate with EBNA2-mediated transcription. This modulation suggests that PRMT5 may play a role in latent EBV infection.
Liu, Cheng-Der; Cheng, Chi-Ping; Fang, Jia-Shih; Chen, Ling-Chih [Department of Life Sciences, Tzu-Chi University, 701 Chung-Yang Rd. Sec 3, Hualien 97004, Taiwan (China)] [Department of Life Sciences, Tzu-Chi University, 701 Chung-Yang Rd. Sec 3, Hualien 97004, Taiwan (China); Zhao, Bo; Kieff, Elliott [Department of Medicine and Microbiology and Molecular Genetics, Channing Laboratory, Brigham and Women�s Hospital and Harvard Medical School, 181 Longwood Ave., Boston 02115, MA (United States)] [Department of Medicine and Microbiology and Molecular Genetics, Channing Laboratory, Brigham and Women�s Hospital and Harvard Medical School, 181 Longwood Ave., Boston 02115, MA (United States); Peng, Chih-Wen, E-mail: firstname.lastname@example.com [Department of Life Sciences, Tzu-Chi University, 701 Chung-Yang Rd. Sec 3, Hualien 97004, Taiwan (China)] [Department of Life Sciences, Tzu-Chi University, 701 Chung-Yang Rd. Sec 3, Hualien 97004, Taiwan (China)
Nuclear resonance vibrational spectroscopy of a protein active-site mimic
NASA Astrophysics Data System (ADS)
For many years, M�ssbauer spectroscopy has been applied to measure recoilless absorption of x-ray photons by nuclei. Recently, synchrotron radiation sources have enabled the observation of weaker features separated from the recoilless resonance by the energy of vibrational quanta. This enables a form of vibrational spectroscopy with a unique sensitivity to the probe nucleus. Biological applications are particularly promising, because it is possible to selectively probe vibrations of a single atom at the active site of a complex biomolecule, while avoiding interference from the vibrations of thousands of other atoms. In contrast with traditional site-selective vibrational spectroscopies, nuclear resonance vibrational spectroscopy (NRVS) is not hampered by solvent interference and faces selection rule limitations only if the probe nucleus lies on a symmetry element. Here, we formulate a mathematical language appropriate for understanding NRVS measurements on molecular systems and apply it to analyse NRVS data recorded on ferrous nitrosyl tetraphenylporphyrin, Fe(TPP)(NO). This compound mimics the haem group found at the active site of many proteins involved in the biological usage of oxygen and nitric oxide. Measurements on such model compounds provide a baseline for evaluating the extent to which vibrations are localized at the active site of a protein, with the goal of elucidating the mechanisms of biological processes, such as intersite communication in allosteric proteins.
Sage, J. T.; Paxson, C.; Wyllie, G. R. A.; Sturhahn, W.; Durbin, S. M.; Champion, P. M.; Alp, E. E.; Scheidt, W. R.
Targeting Proliferating Cell Nuclear Antigen and Its Protein Interactions Induces Apoptosis in Multiple Myeloma Cells
Multiple myeloma is a hematological cancer that is considered incurable despite advances in treatment strategy during the last decade. Therapies targeting single pathways are unlikely to succeed due to the heterogeneous nature of the malignancy. Proliferating cell nuclear antigen (PCNA) is a multifunctional protein essential for DNA replication and repair that is often overexpressed in cancer cells. Many proteins involved in the cellular stress response interact with PCNA through the five amino acid sequence AlkB homologue 2 PCNA-interacting motif (APIM). Thus inhibiting PCNA�s protein interactions may be a good strategy to target multiple pathways simultaneously. We initially found that overexpression of peptides containing the APIM sequence increases the sensitivity of cancer cells to contemporary therapeutics. Here we have designed a cell-penetrating APIM-containing peptide, ATX-101, that targets PCNA and show that it has anti-myeloma activity. We found that ATX-101 induced apoptosis in multiple myeloma cell lines and primary cancer cells, while bone marrow stromal cells and primary healthy lymphocytes were much less sensitive. ATX-101-induced apoptosis was caspase-dependent and cell cycle phase-independent. ATX-101 also increased multiple myeloma cells� sensitivity against melphalan, a DNA damaging agent commonly used for treatment of multiple myeloma. In a xenograft mouse model, ATX-101 was well tolerated and increased the anti-tumor activity of melphalan. Therefore, targeting PCNA by ATX-101 may be a novel strategy in multiple myeloma treatment.
Muller, Rebekka; Bachke, Siri; Gilljam, Karin M.; Vatsveen, Thea K.; R?, Torstein B.; Bellacchio, Emanuele; Sundan, Anders; Otterlei, Marit
Modulation of Epstein-Barr Virus Nuclear Antigen 2-Dependent Transcription by Protein Arginine Methyltransferase 5
Epstein-Barr Virus Nuclear Antigen (EBNA) 2 features an Arg-Gly repeat (RG) domain at amino acid positions 335-360, which is a known target for protein arginine methyltransferaser 5 (PRMT5). In this study, we performed protein affinity pull-down assays to demonstrate that endogenous PRMT5 derived from lymphoblastoid cells specifically associated with the protein bait GST-E2 RG. Transfection of a plasmid expressing PRMT5 induced a 2.5- to 3-fold increase in EBNA2-dependent transcription of both the LMP1 promoter in AKATA cells, which contain the EBV genome endogenously, and a Cp-Luc reporter plasmid in BJAB cells, which are EBV negative. Furthermore, we showed that there was a 2-fold enrichment of EBNA2 occupancy in target promoters in the presence of exogenous PRMT5. Taken together, we show that PRMT5 triggers the symmetric dimethylation of EBNA2 RG domain to coordinate with EBNA2-mediated transcription. This modulation suggests that PRMT5 may play a role in latent EBV infection.
Liu, Cheng-Der; Cheng, Chi-Ping; Fang, Jia-Shih; Chen, Ling-Chih; Zhao, Bo; Kieff, Elliott; Peng, Chih-Wen
Oxidative stress-induced assembly of PML nuclear bodies controls sumoylation of partner proteins.
The promyelocytic leukemia (PML) protein organizes PML nuclear bodies (NBs), which are stress-responsive domains where many partner proteins accumulate. Here, we clarify the basis for NB formation and identify stress-induced partner sumoylation as the primary NB function. NB nucleation does not rely primarily on intermolecular interactions between the PML SUMO-interacting motif (SIM) and SUMO, but instead results from oxidation-mediated PML multimerization. Oxidized PML spherical meshes recruit UBC9, which enhances PML sumoylation, allow partner recruitment through SIM interactions, and ultimately enhance partner sumoylation. Intermolecular SUMO-SIM interactions then enforce partner sequestration within the NB inner core. Accordingly, oxidative stress enhances NB formation and global sumoylation in vivo. Some NB-associated sumoylated partners also become polyubiquitinated by RNF4, precipitating their proteasomal degradation. As several partners are protein-modifying enzymes, NBs could act as sensors that facilitate and confer oxidative stress sensitivity not only to sumoylation but also to other post-translational modifications, thereby explaining alterations of stress response upon PML or NB loss. PMID:24637324
Sahin, Umut; Ferhi, Omar; Jeanne, Marion; Benhenda, Shirine; Berthier, Caroline; Jollivet, Florence; Niwa-Kawakita, Michiko; Faklaris, Orestis; Setterblad, Niclas; de Th�, Hugues; Lallemand-Breitenbach, Val�rie
Flowering and genome integrity control by a nuclear matrix protein in Arabidopsis.
The matrix attachment regions (MARs) binding proteins could finely orchestrate temporal and spatial gene expression during development. In Arabidopsis, transposable elements (TEs) and TE-like repeat sequences are transcriptionally repressed or attenuated by the coordination of many key players including DNA methyltransferases, histone deacetylases, histone methyltransferases and the siRNA pathway, which help to protect genomic integrity and control multiple developmental processes such as flowering. We have recently reported that an AT-hook nuclear matrix binding protein, TRANSPOSABLE ELEMENT SILENCING VIA AT-HOOK (TEK), participates in a histone deacetylation (HDAC) complex to silence TEs and genes containing a TE-like sequence, including AtMu1, FWA and FLOWERING LOCUS C (FLC) in Ler background. We have shown that TEK knockdown causes increased histone acetylation, reduced H3K9me2 and moderate reduction of DNA methylation in the target loci, leading to the de-repression of FLC and FWA, as well as TE reactivation. Here we discuss the role of TEK as a putative MAR binding protein which functions in the maintenance of genome integrity and in flowering control by silencing TEs and repeat-containing genes. PMID:23836195
Xu, Yifeng; Gan, Eng-Seng; He, Yuehui; Ito, Toshiro
Kaposi's Sarcoma-Associated Herpesvirus LANA Protein Downregulates Nuclear Glycogen Synthase Kinase 3 Activity and Consequently Blocks Differentiation
The Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen (LANA) protein interacts with glycogen synthase kinase 3 (GSK-3) and relocalizes GSK-3 in a manner that leads to stabilization of -catenin and upregulation of -catenin-responsive cell genes. The LANA-GSK-3 interaction was further examined to determine whether there were additional downstream consequences. In the present study, the nuclear GSK-3 bound to LANA in transfected
Jianyong Liu; Heather Martin; Meir Shamay; Crystal Woodard; Qi-Qun Tang; S. Diane Hayward
Ubiquitin-Regulated Nuclear-Cytoplasmic Trafficking of the Nipah Virus Matrix Protein Is Important for Viral Budding
Paramyxoviruses are known to replicate in the cytoplasm and bud from the plasma membrane. Matrix is the major structural protein in paramyxoviruses that mediates viral assembly and budding. Curiously, the matrix proteins of a few paramyxoviruses have been found in the nucleus, although the biological function associated with this nuclear localization remains obscure. We report here that the nuclear-cytoplasmic trafficking of the Nipah virus matrix (NiV-M) protein and associated post-translational modification play a critical role in matrix-mediated virus budding. Nipah virus (NiV) is a highly pathogenic emerging paramyxovirus that causes fatal encephalitis in humans, and is classified as a Biosafety Level 4 (BSL4) pathogen. During live NiV infection, NiV-M was first detected in the nucleus at early stages of infection before subsequent localization to the cytoplasm and the plasma membrane. Mutations in the putative bipartite nuclear localization signal (NLS) and the leucine-rich nuclear export signal (NES) found in NiV-M impaired its nuclear-cytoplasmic trafficking and also abolished NiV-M budding. A highly conserved lysine residue in the NLS served dual functions: its positive charge was important for mediating nuclear import, and it was also a potential site for monoubiquitination which regulates nuclear export of the protein. Concordantly, overexpression of ubiquitin enhanced NiV-M budding whereas depletion of free ubiquitin in the cell (via proteasome inhibitors) resulted in nuclear retention of NiV-M and blocked viral budding. Live Nipah virus budding was exquisitely sensitive to proteasome inhibitors: bortezomib, an FDA-approved proteasome inhibitor for treating multiple myeloma, reduced viral titers with an IC50 of 2.7 nM, which is 100-fold less than the peak plasma concentration that can be achieved in humans. This opens up the possibility of using an �off-the-shelf� therapeutic against acute NiV infection.
Wang, Yao E.; Park, Arnold; Lake, Michael; Pentecost, Mickey; Torres, Betsabe; Yun, Tatyana E.; Wolf, Mike C.; Holbrook, Michael R.
Aleurone nuclear proteins bind to similar elements in the promoter regions of two gibberellin-regulated ?-amylase genes
Binding of nuclear proteins from wild oat aleurone protoplasts to the promoter regions of two gibberellin-regulated wheat a-amylase genes (a-Amyl\\/18 and a-Amy2\\/54) has been studied by gel retardation and DNase 1 footprinting. Gel retardation studies using 300�430 bp fragments of the promoters showed similar binding characteristics with nuclear extracts from both gibberellin A1-treated and untreated protoplasts. DNase 1 footprints localised
Paul J. Rushton; Richard Hooley; Colin M. Lazarus
Biophysical and functional analyses suggest that adenovirus E4-ORF3 protein requires higher-order multimerization to function against promyelocytic leukemia protein nuclear bodies.
The early region 4 open reading frame 3 protein (E4-ORF3; UniProt ID P04489) is the most highly conserved of all adenovirus-encoded gene products at the amino acid level. A conserved attribute of the E4-ORF3 proteins of different human adenoviruses is the ability to disrupt PML nuclear bodies from their normally punctate appearance into heterogeneous filamentous structures. This E4-ORF3 activity correlates with the inhibition of PML-mediated antiviral activity. The mechanism of E4-ORF3-mediated reorganization of PML nuclear bodies is unknown. Biophysical analysis of the purified WT E4-ORF3 protein revealed an ordered secondary/tertiary structure and the ability to form heterogeneous higher-order multimers in solution. Importantly, a nonfunctional E4-ORF3 mutant protein, L103A, forms a stable dimer with WT secondary structure content. Because the L103A mutant is incapable of PML reorganization, this result suggests that higher-order multimerization of E4-ORF3 may be required for the activity of the protein. In support of this hypothesis, we demonstrate that the E4-ORF3 L103A mutant protein acts as a dominant-negative effector when coexpressed with the WT E4-ORF3 in mammalian cells. It prevents WT E4-ORF3-mediated PML track formation presumably by binding to the WT protein and inhibiting the formation of higher-order multimers. In vitro protein binding studies support this conclusion as demonstrated by copurification of coexpressed WT and L103A proteins in Escherichia coli and coimmunoprecipitation of WT�L103A E4-ORF3 complexes in mammalian cells. These results provide new insight into the properties of the Ad E4-ORF3 protein and suggest that higher-order protein multimerization is essential for E4-ORF3 activity. PMID:22573317
Patsalo, Vadim; Yondola, Mark A; Luan, Bowu; Shoshani, Ilana; Kisker, Caroline; Green, David F; Raleigh, Daniel P; Hearing, Patrick
DNA damage-dependent interaction of the nuclear matrix protein C1D with Translin-associated factor X (TRAX).
The nuclear matrix protein C1D is an activator of the DNA-dependent protein kinase (DNA-PK), which is essential for the repair of DNA double-strand breaks (DSBs) and V(D)J recombination. C1D is phosphorylated very efficiently by DNA-PK, and its mRNA and protein levels are induced upon gamma-irradiation, suggesting that C1D may play a role in repair of DSBs in vivo. In an attempt to identify the biological function of C1D, we have employed the yeast two-hybrid system and found that C1D interacts specifically with Translin-associated factor X, TRAX. Although the biological function of TRAX remains unknown, its bipartite nuclear targeting sequences suggest a role for TRAX in the movement of associated proteins, including Translin, into the nucleus. We show that C1D and TRAX interact specifically in both yeast and mammalian cells. Interestingly, however, interaction of these two proteins in mammalian cells only occur following gamma-irradiation, raising the possibility of involvement of TRAX in DNA double-strand break repair and providing evidence for biological functions of the nuclear matrix protein C1D and TRAX. Moreover, we show, using fluorescently tagged proteins, that the relative expression levels of TRAX and Translin affect their subcellular localization. These results suggest that one role for C1D may be to regulate TRAX/Translin complex formation. PMID:11801738
Erdemir, Tuba; Bilican, Bilada; Oncel, Dilhan; Goding, Colin R; Yavuzer, Ugur
C++ OPPS, a new software for the interpretation of protein dynamics from nuclear magnetic resonance measurements
NASA Astrophysics Data System (ADS)
Nuclear magnetic resonance (NMR) is a powerful tool for elucidating protein dynamics because of the possibility to interpret nuclear spin relaxation properties in terms of microdynamic parameters. Magnetic relaxation times T1, T2, and NOE depend on dipolar and quadrupolar interactions, on chemical shift anisotropy and cross-correlation effects. Within the framework of given motional model, it is possible to express the NMR relaxation times as functions of spectral densities (Abragam, The Principles of Nuclear Magnetism; Oxford University Press: Clarendon, London, 1961), obtaining the connection between macroscopic observables and microscopic properties. In this context, recently Meirovitch et al. (Shapiro et al., Biochemistry 2002, 41, 6271, Meirovitch et al., J Phys Chem B 2006, 110, 20615, Meirovitch et al., J Phys Chem B 2007, 111, 12865) applied the dynamical model introduced by Polimeno and Freed (Polimeno and Freed, Adv Chem Phys 1993, 83, 89, Polimeno and Freed, J Phys Chem 1995, 99, 10995), known as the slowly relaxing local structure (SRLS) model, to the study of NMR data. The program C++OPPS (http://www.chimica.unipd.it/licc/), developed in our laboratory, implements the SRLS model in an user-friendly way with a graphical user interface (GUI), introduced to simplify the work to users who do not feel at ease with the complex mathematics of the model and the difficulties of command line based programs. The program is an evolution of the old FORTRAN 77 implementation COPPS (COupled Protein Probe Smoluchowski) and presents a number of new features: the presence of an easy to use GUI written in JAVA; high calculation performance thanks to features of C++ language, employment of BLAS (basic linear algebra subprograms) library (Blackford et al., Trans Math Soft 2002, 28, 135) in handling matrix-vector operations and parallelization of the code under the MPI (message passing interface) paradigm (Gropp et al., Parallel Comput 1996, 22, 789, Gropp and Lusk, User's Guide for mpich, a Portable Implementation of MPI Mathematics and Computer Science Division; Argonne National Laboratory, 1996); possibility to predict the diffusion tensor of the protein via a hydrodynamic approach (Barone et al., J Comp Chem, in press). A cluster version of C++OPPS was also developed, which can be easily accessed by users via the web.
Zerbetto, Mirco; Polimeno, Antonino; Meirovitch, Eva
Effect of ascorbic acid and curcumin on quercetin-induced nuclear DNA damage, lipid peroxidation and protein degradation.
The effects of ascorbic acid and curcumin on quercetin-induced DNA damage, lipid peroxidation protein degradation were investigated in a model system of isolated rat-liver nuclei under aerobic conditions and in the presence of equimolar concentrations of iron or copper. Neither ascorbic acid nor curcumin inhibited quercetin-induced nuclear DNA damage, lipid peroxidation, or protein degradation. In fact, both antioxidants stimulated the oxidative damage to nuclear macromolecules. Ascorbic acid significantly increased the quercetin-induced nuclear DNA damage in the presence of either iron or copper. The increases in quercetin-induced nuclear lipid peroxidation and protein degradation by ascorbic acid were statistically significant only in the presence of iron or copper, respectively. Similarly, stimulation of quercetin-induced DNA damage and lipid peroxidation by curcumin was statistically significant only in the presence of copper or iron, respectively. Curcumin had no significant effect on nuclear protein degradation. These results demonstrate the pro-oxidant properties of ascorbic acid and curcumin, compounds that also demonstrate antioxidant and anticarcinogenic properties. Ascorbic acid and curcumin may therefore each have a dual role in carcinogenesis. PMID:1576592
Sahu, S C; Washington, M C
Nuclear Import and Dimerization of Tomato ASR1, a Water Stress-Inducible Protein Exclusive to Plants
The ASR (for ABA/water stress/ripening) protein family, first described in tomato as nuclear and involved in adaptation to dry climates, is widespread in the plant kingdom, including crops of high agronomic relevance. We show both nuclear and cytosolic localization for ASR1 (the most studied member of the family) in histological plant samples by immunodetection, typically found in small proteins readily diffusing through nuclear pores. Indeed, a nuclear localization was expected based on sorting prediction software, which also highlight a monopartite nuclear localization signal (NLS) in the primary sequence. However, here we prove that such an �NLS� of ASR1 from tomato is dispensable and non-functional, being the transport of the protein to the nucleus due to simple diffusion across nuclear pores. We attribute such a targeting deficiency to the misplacing in that cryptic NLS of two conserved contiguous lysine residues. Based on previous in vitro experiments regarding quaternary structure, we also carried out live cell imaging assays through confocal microscopy to explore dimer formation in planta. We found homodimers in both the cytosol and the nucleus and demonstrated that assembly of both subunits together can occur in the cytosol, giving rise to translocation of preformed dimers. The presence of dimers was further corroborated by means of in vivo crosslinking of nuclei followed by SDS-PAGE.
Ricardi, Martiniano M.; Guaimas, Francisco F.; Gonzalez, Rodrigo M.; Burrieza, Hernan P.; Lopez-Fernandez, Maria P.; Estevez, Jose M.; Iusem, Norberto D.
Substrate Recognition in Nuclear Protein Quality Control Degradation Is Governed by Exposed Hydrophobicity That Correlates with Aggregation and Insolubility*
Misfolded proteins present an escalating deleterious challenge to cells over the course of their lifetime. One mechanism the cell possesses to prevent misfolded protein accumulation is their destruction by protein quality control (PQC) degradation systems. In eukaryotes, PQC degradation typically proceeds via multiple ubiquitin-protein ligases that act throughout the cell to ubiquitinate misfolded proteins for proteasome degradation. What the exact feature of misfolding that each PQC ubiquitin-protein ligase recognizes in their substrates remains an open question. Our previous studies of the budding yeast nuclear ubiquitin-protein ligase San1 indicated that it recognizes exposed hydrophobicity within its substrates, with the threshold of hydrophobicity equivalent to that of 5 contiguous hydrophobic residues. Here, we uncover an additional parameter: the nature of the exposed hydrophobicity that confers San1-mediated degradation correlates with significant protein insolubility. San1 particularly targets exposed hydrophobicity that leads to insolubility and aggregation above a certain threshold. Our studies presented here provide additional insight into the details of misfolded nuclear protein recognition and demonstrate that there is selectivity for the type of exposed hydrophobicity.
Fredrickson, Eric K.; Gallagher, Pamela S.; Clowes Candadai, Sarah V.; Gardner, Richard G.
A cytochemical study of the transcriptional and translational regulation of nuclear transition protein 1 (TP1), a major chromosomal protein of mammalian spermatids
Immunocytochemical localization and in situ hybridization techniques were used to investigate the presence of spermatid nuclear transition protein 1 (TP1) and its mRNA during the various stages of spermatogenesis in the rat. A specific antiserum to TP1 was raised in a rabbit and used to show that TP1 is immunologically crossreactive among many mammals including humans. During spermatogenesis the protein appears in spermatids as they progress from step 12 to step 13, a period in which nuclear condensation is underway. The protein is lost during step 15. An asymmetric RNA probe generated from a TP1 cDNA clone identified TP1 mRNA in late round spermatids beginning in step 7. The message could no longer be detected in spermatids of step 15 or beyond. Thus, TP1 mRNA first appears well after meiosis in haploid cells but is not translated effectively for the several days required for these cells to progress to the stage of chromatin condensation. Message and then protein disappear as the spermatids enter step 15. In agreement with a companion biochemical study (Heidaran, M.A., and W.S. Kistler. J. Biol. Chem. 1987. 262:13309-13315), these results establish that translational control is involved in synthesis of this major spermatid nuclear protein. In addition, they suggest that TP1 plays a role in the completion but not the initiation of chromatin condensation in elongated spermatids.
The negative regulator of Gli, Suppressor of fused (Sufu), interacts with SAP18, Galectin3 and other nuclear proteins.
Sufu (Suppressor of fused) is a negative regulator of the Hedgehog signal-transduction pathway, interacting directly with the Gli family of transcription factors. However, its function remains poorly understood. In the present study, we determined the expression, tissue distribution and biochemical properties of mSufu (mouse Sufu) protein. We identified several mSufu variants of which some were phosphorylated. A yeast two-hybrid screen with mSufu as bait allowed us to identify several nuclear proteins as potential partners of mSufu. Most of these partners, such as SAP18 (Sin3-associated polypeptide 18), pCIP (p300/CBP-cointegrator protein) and PIAS1 (protein inhibitor of activated signal transduction and activators of transcription 1), are involved in either repression or activation of transcription and two of them, Galectin3 and hnRNPA1 (heterogeneous nuclear ribonucleoprotein A1), have a nuclear function in pre-mRNA splicing. We confirmed the mSufu-SAP18 and mSufu-Galectin3 interactions by independent biochemical assays. Using a cell transfection assay, we also demonstrated that mSufu protein (484 amino acids) is predominantly cytoplasmic but becomes mostly nuclear when a putative nuclear export signal is mutated or after treatment of the cells with leptomycin B. Moreover, mSufu is translocated to the nucleus when co-expressed with SAP18, which is normally found in this compartment. In contrast, Galectin3 is translocated to the cytoplasm when it is co-expressed with mSufu. Our findings indicate that mSufu is a shuttle protein that appears to be extremely versatile in its ability to bind different proteins in both the cytoplasm and nucleus.
Paces-Fessy, Melanie; Boucher, Dominique; Petit, Emile; Paute-Briand, Sandrine; Blanchet-Tournier, Marie-Francoise
A new spectrin, beta IV, has a major truncated isoform that associates with promyelocytic leukemia protein nuclear bodies and the nuclear matrix.
We isolated cDNAs that encode a 77-kDa peptide similar to repeats 10-16 of beta-spectrins. Its gene localizes to human chromosome 19q13.13-q13.2 and mouse chromosome 7, at 7.5 centimorgans. A 289-kDa isoform, similar to full-length beta-spectrins, was partially assembled from sequences in the human genomic DNA data base and completely cloned and sequenced. RNA transcripts are seen predominantly in the brain, and Western analysis shows a major peptide that migrates as a 72-kDa band. This new gene, spectrin betaIV, thus encodes a full-length minor isoform (SpbetaIVSigma1) and a truncated major isoform (SpbetaIVSigma5). Immunostaining of cells shows a micropunctate pattern in the cytoplasm and nucleus. In mesenchymal stem cells, the staining concentrates at nuclear dots that stain positively for the promyelocytic leukemia protein (PML). Expression of SpbetaIVSigma5 fused to green fluorescence protein in cells produces nuclear dots that include all PML bodies, which double in number in transfected cells. Deletion analysis shows that partial repeats 10 and 16 of SpbetaIVSigma5 are necessary for nuclear dot formation. Immunostaining of whole-mount nuclear matrices reveals diffuse positivity with accentuation at PML bodies. Spectrin betaIV is the first beta-spectrin associated with a subnuclear structure and may be part of a nuclear scaffold to which gene regulatory machinery binds. PMID:11294830
Tse, W T; Tang, J; Jin, O; Korsgren, C; John, K M; Kung, A L; Gwynn, B; Peters, L L; Lux, S E
Incorporation of the nuclear pore basket protein Nup153 into nuclear pore structures is dependent upon lamina assembly: evidence from cell-free extracts of Xenopus eggs
In cell-free extracts of Xenopus eggs that support the assembly of replication-competent nuclei, we found that lamin B3 specifically associates with four polypeptides (termed SLAPs, soluble lamin associated proteins). Here, one SLAP is identified as the nuclear pore complex protein Nup153, one member of the F/GXFG motif-containing nucleoporins. In vitro translated Nup153 and lamin B3 co-immunoprecipitate, and lamin B3 interacts specifically with the C-terminal domain of Nup153. During nuclear envelope assembly, other F/GXFG-containing nucleoporins are incorporated into the nuclear envelope preceding lamina assembly. Incorporation of Nup153 occurs at the same time as lamina assembly. When lamina assembly is prevented using the dominant-negative mutant XlaminB?2+, Nup153 does not appear at the nuclear envelope, while other F/GXFG-containing nucleoporins and Nup93 are recruited normally. When the lamina of pre-assembled nuclei is disrupted using the same dominant-negative mutant, the distribution of other nucleoporins is unaffected. However, Nup153 recruitment at the nuclear envelope is lost. Our results indicate that both the recruitment and maintenance of Nup153 at the pore are dependent upon the integrity of the lamina.
Smythe, Carl; Jenkins, Hazel E.; Hutchison, Christopher J.
Permeabilized mammalian cells as an experimental system for nuclear import of geminiviral karyophilic proteins and of synthetic peptides derived from their nuclear localization signal regions.
The plant-infecting geminiviruses deliver their genome and viral proteins into the host cell nucleus. Members of the family Geminiviridae possess either a bipartite genome composed of two approximately 2.6 kb DNAs or a monopartite genome of approximately 3.0 kb DNA. The bipartite genome of Bean dwarf mosaic virus (BDMV) encodes several karyophilic proteins, among them the capsid protein (CP) and BV1 (nuclear shuttle protein). A CP is also encoded by the monopartite genome of Tomato yellow leaf curl virus (TYLCV). Here, an in vitro assay system was used for direct demonstration of nuclear import of BDMV BV1 and TYLCV CP, as well as synthetic peptides containing their putative nuclear localization signals (NLSs). Full-length recombinant BDMV BV1 and TYLCV CP mediated import of conjugated fluorescently labelled BSA molecules into nuclei of permeabilized mammalian cells. Fluorescently labelled and biotinylated BSA conjugates bearing the synthetic peptides containing aa 3-20 of TYLCV CP (CP-NLS) or aa 84-106 of BDMV BV1 (BV1-NLS) were also imported into the nuclei of permeabilized cells. This import was blocked by the addition of unlabelled BSA-NLS peptide conjugates or excess unlabelled free NLS peptides. The CP- and BV1-NLS peptides also mediated nuclear import of fluorescently labelled BSA molecules into the nuclei of microinjected mesophyll cells of Nicotiana benthamiana leaves, demonstrating their biological function in intact plant tissue. BV1-NLS and CP-NLS were shown to mediate specific binding to importin alpha, both in vitro and in vivo. These results are consistent with a common nuclear-import pathway for CP and BV1, probably via importin alpha. PMID:16894212
Kass, Gideon; Arad, Gabriel; Rosenbluh, Joseph; Gafni, Yedidya; Graessmann, Adolf; Rojas, Maria R; Gilbertson, Robert L; Loyter, Abraham
G protein-coupled receptor signalling in the cardiac nuclear membrane: evidence and possible roles in physiological and pathophysiological function
G protein-coupled receptors (GPCRs) play key physiological roles in numerous tissues, including the heart, and their dysfunction influences a wide range of cardiovascular diseases. Recently, the notion of nuclear localization and action of GPCRs has become more widely accepted. Nuclear-localized receptors may regulate distinct signalling pathways, suggesting that the biological responses mediated by GPCRs are not solely initiated at the cell surface but may result from the integration of extracellular and intracellular signalling pathways. Many of the observed nuclear effects are not prevented by classical inhibitors that exclusively target cell surface receptors, presumably because of their structures, lipophilic properties, or affinity for nuclear receptors. In this topical review, we discuss specifically how angiotensin-II, endothelin, ?-adrenergic and opioid receptors located on the nuclear envelope activate signalling pathways, which convert intracrine stimuli into acute responses such as generation of second messengers and direct genomic effects, and thereby participate in the development of cardiovascular disorders.
Tadevosyan, Artavazd; Vaniotis, George; Allen, Bruce G; Hebert, Terence E; Nattel, Stanley
G protein-coupled receptor signalling in the cardiac nuclear membrane: evidence and possible roles in physiological and pathophysiological function.
G protein-coupled receptors (GPCRs) play key physiological roles in numerous tissues, including the heart, and their dysfunction influences a wide range of cardiovascular diseases. Recently, the notion of nuclear localization and action of GPCRs has become more widely accepted. Nuclear-localized receptors may regulate distinct signalling pathways, suggesting that the biological responses mediated by GPCRs are not solely initiated at the cell surface but may result from the integration of extracellular and intracellular signalling pathways. Many of the observed nuclear effects are not prevented by classical inhibitors that exclusively target cell surface receptors, presumably because of their structures, lipophilic properties, or affinity for nuclear receptors. In this topical review, we discuss specifically how angiotensin-II, endothelin, ?-adrenergic and opioid receptors located on the nuclear envelope activate signalling pathways, which convert intracrine stimuli into acute responses such as generation of second messengers and direct genomic effects, and thereby participate in the development of cardiovascular disorders. PMID:22183719
Tadevosyan, Artavazd; Vaniotis, George; Allen, Bruce G; H�bert, Terence E; Nattel, Stanley
CCAAT displacement protein regulates nuclear factor-kappa beta-mediated chemokine transcription in melanoma cells
Members of the nuclear factor-kappa beta (NF-?B) family maintain cellular homeostasis by enhancing the transcription of genes involved in inflammation, immune response, cell proliferation, and apoptosis. Melanoma tumor cells often express inflammatory mediators through enhanced activation of NF-?B. The NF-?B activation appears to result from the enhancer formation including NF-?B and lysine acetyl transferases such as p300, CREB (cyclic AMP-responsive element binding protein)-binding protein (CBP), and/or p300/CBP associating factor (PCAF). We observed that proteins expressed by Hs294T metastatic melanoma cells are highly acetylated compared with normal melanocytes, and dominant-negative PCAF reduced the basal and tumor necrosis factor-?-stimulated transcriptional activity of NF-?B. The promoter activity of NF-?B-regulated chemokines was also reduced by the expression of dominant-negative PCAF. The promoters of these chemokines contain a CCAAT displacement protein (CDP)-binding site near the NF-?B element. compared with vector-transduced cells, in CDP-transduced Hs294T cells: (i) over-expressed CDP bound efficiently to PCAF, (ii) tumor necrosis factor-? stimulated chemokine expression and NF-?B-mediated transcription were reduced, and (iii) the binding of CBP to Rel A was reduced. These data suggest that CDP inhibits cytokine-induced NF-?B-regulated chemokine transcription. This study contributes to our understanding of the role of CDP in an enhanceosome of NF-?B-mediated chemokine transcription in human melanoma cells.
Ueda, Yukiko; Su, Yingjun; Richmond, Ann
CCAAT displacement protein regulates nuclear factor-kappa beta-mediated chemokine transcription in melanoma cells.
Members of the nuclear factor-kappa beta (NF-kappaB) family maintain cellular homeostasis by enhancing the transcription of genes involved in inflammation, immune response, cell proliferation, and apoptosis. Melanoma tumor cells often express inflammatory mediators through enhanced activation of NF-kappaB. The NF-kappaB activation appears to result from the enhancer formation including NF-kappaB and lysine acetyl transferases such as p300, CREB (cyclic AMP-responsive element binding protein)-binding protein (CBP), and/or p300/CBP associating factor (PCAF). We observed that proteins expressed by Hs294T metastatic melanoma cells are highly acetylated compared with normal melanocytes, and dominant-negative PCAF reduced the basal and tumor necrosis factor-alpha-stimulated transcriptional activity of NF-kappaB. The promoter activity of NF-kappaB-regulated chemokines was also reduced by the expression of dominant-negative PCAF. The promoters of these chemokines contain a CCAAT displacement protein (CDP)-binding site near the NF-kappaB element. compared with vector-transduced cells, in CDP-transduced Hs294T cells: (i) over-expressed CDP bound efficiently to PCAF, (ii) tumor necrosis factor-alpha-stimulated chemokine expression and NF-kappaB-mediated transcription were reduced, and (iii) the binding of CBP to Rel A was reduced. These data suggest that CDP inhibits cytokine-induced NF-kappaB-regulated chemokine transcription. This study contributes to our understanding of the role of CDP in an enhanceosome of NF-kappaB-mediated chemokine transcription in human melanoma cells. PMID:17496784
Ueda, Yukiko; Su, Yingjun; Richmond, Ann
Cell cycle-regulated phosphorylation of the pre-mRNA-binding (heterogeneous nuclear ribonucleoprotein) C proteins.
Heterogeneous nuclear ribonucleoprotein (hnRNP) complexes, the structures that contain heterogeneous nuclear RNA and its associated proteins, constitute one of the most abundant components of the eukaryotic nucleus. hnRNPs appear to play important roles in the processing, and possibly also in the transport, of mRNA. hnRNP C proteins (C1, M(r) of 41,000; C2, M(r) of 43,000 [by sodium dodecyl sulfate-polyacrylamide gel electrophoresis]) are among the most abundant pre-mRNA-binding proteins, and they bind tenaciously to sequences relevant to pre-mRNA processing, including the polypyrimidine stretch of introns (when it is uridine rich). C proteins are found in the nucleus during the interphase, but during mitosis they disperse throughout the cell. They have been shown previously to be phosphorylated in vivo, and they can be phosphorylated in vitro by a casein kinase type II. We have identified and partially purified at least two additional C protein kinases. One of these, termed Cs kinase, caused a distinct mobility shift of C proteins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These phosphorylated C proteins, the Cs proteins, were the prevalent forms of C proteins during mitosis, and Cs kinase activity was also increased in extracts prepared from mitotic cells. Thus, hnRNP C proteins undergo cell cycle-dependent phosphorylation by a cell cycle-regulated protein kinase. Cs kinase activity appears to be distinct from the well-characterized mitosis-specific histone H1 kinase activity. Several additional hnRNP proteins are also phosphorylated during mitosis and are thus also potential substrates for Cs kinase. These novel phosphorylations may be important in regulating the assembly and disassembly of hnRNP complexes and in the function or cellular localization of RNA-binding proteins. Images
Pinol-Roma, S; Dreyfuss, G
Dual functions of CDC6: a yeast protein required for DNA replication also inhibits nuclear division.
The Saccharomyces cerevisiae gene CDC6, whose protein product is required for DNA replication, is transcribed only in late G1 and S phases. We have discovered a critical reason why CDC6 expression is regulated in this fashion. Constitutive CDC6 transcription greatly delayed the initiation of M phase without effecting the G1-S transition or growth rate. This occurred in both fission and budding yeasts. The CDC6-induced M phase delay was dependent on the wee1/mik1 mitotic inhibitor kinases and was greatly accentuated in strains defective for the cdc25/MIH1 mitotic inducer phosphatases, indicating that CDC6 indirectly inhibits activation of the p34cdc2/CDC28 M phase kinase. Thus CDC6 appears to have an important and perhaps unique dual role in S phase, it is first required for the initiation of DNA replication and then actively participates in the suppression of nuclear division. Images
Bueno, A; Russell, P
Scotin, a novel p53-inducible proapoptotic protein located in the ER and the nuclear membrane
p53 is a transcription factor that induces growth arrest or apoptosis in response to cellular stress. To identify new p53-inducible proapoptotic genes, we compared, by differential display, the expression of genes in spleen or thymus of normal and p53 nullizygote mice after ?-irradiation of whole animals. We report the identification and characterization of human and mouse Scotin homologues, a novel gene directly transactivated by p53. The Scotin protein is localized to the ER and the nuclear membrane. Scotin can induce apoptosis in a caspase-dependent manner. Inhibition of endogenous Scotin expression increases resistance to p53-dependent apoptosis induced by DNA damage, suggesting that Scotin plays a role in p53-dependent apoptosis. The discovery of Scotin brings to light a role of the ER in p53-dependent apoptosis.
Bourdon, J.-C.; Renzing, J.; Robertson, P.L.; Fernandes, K.N.; Lane, D.P.
Cytochemical studies of nuclear basic proteins in control and vitamin B12 starved Euglena.
In avitaminosis B12, Euglena gracilis Z is blocked in the cell cycle in the S/G2 phase. In these blocked cells, transcription and traduction go on and the amount of DNA is less than doubled and remains constant during the blockage. Chromatin clumps observed in situ with classical electron microscopic methods are always condensed in control cells but are not visualized in B12 starved cells. Two cytochemical reactions, ethanolic phosphotungstic acid and ammoniacal silver reaction, specific for lysine- or arginine-rich residues, are performed to reveal basic nuclear proteins of chromatin. With these two methods, control chromatin in situ always shows a condensed aspect, whereas the starved chromatin appears dispersed. These cytochemical differences might be considered to result from a different supramolecular organization of the two kinds of chromatin. PMID:6411805
Br�, M H; Pouphile, M; Delpech, S; Lefort-Tran, M
Dual functions of CDC6: a yeast protein required for DNA replication also inhibits nuclear division.
The Saccharomyces cerevisiae gene CDC6, whose protein product is required for DNA replication, is transcribed only in late G1 and S phases. We have discovered a critical reason why CDC6 expression is regulated in this fashion. Constitutive CDC6 transcription greatly delayed the initiation of M phase without effecting the G1-S transition or growth rate. This occurred in both fission and budding yeasts. The CDC6-induced M phase delay was dependent on the wee1/mik1 mitotic inhibitor kinases and was greatly accentuated in strains defective for the cdc25/MIH1 mitotic inducer phosphatases, indicating that CDC6 indirectly inhibits activation of the p34cdc2/CDC28 M phase kinase. Thus CDC6 appears to have an important and perhaps unique dual role in S phase, it is first required for the initiation of DNA replication and then actively participates in the suppression of nuclear division. PMID:1600944
Bueno, A; Russell, P
Proliferation dependent expression and nuclear localization of carbohydrate-binding protein 35 in cultured fibroblasts
A highly specific polyclonal antibody against carbohydrate-binding protein 35 (CBP35), was used to analyze the subcellular distribution of the galactose specific lectin in mouse 3T3 fibroblasts. Cell surface specific labeling with anti-CBP35 and /sup 125/I revealed the presence of small amounts of CBP35 externally exposed at the cell surface. However, the majority of CBP35 was localized intracellularly as revealed by immunofluorescent studies of fixed and permeabilized 3T3 cells. The staining pattern showed the presence of CBP35 on the nucleus and in the cytoplasm. Subcellular fractionation studies also indicated that CBP35 can be detected by immunoblotting procedures in the nuclear pellet, the cytoplasm, and the plasma membrane.
Moutsatsos, I.K.
Activation of AMP-activated protein kinase stimulates the nuclear localization of glyceraldehyde 3-phosphate dehydrogenase in human diploid fibroblasts
In addition to its well-known glycolytic activity, GAPDH displays multiple functions, such as nuclear RNA export, DNA replication and repair, and apoptotic cell death. This functional diversity depends on its intracellular localization. In this study, we explored the signal transduction pathways involved in the nuclear translocation of GAPDH using confocal laser scanning microscopy of immunostained human diploid fibroblasts (HDFs). GAPDH was present mainly in the cytoplasm when cultured with 10% FBS. Serum depletion by culturing cells in a serum-free medium (SFM) led to a gradual accumulation of GAPDH in the nucleus, and this nuclear accumulation was reversed by the re-addition of serum or growth factors, such as PDGF and lysophosphatidic acid. The nuclear export induced by the re-addition of serum or growth factors was prevented by LY 294002 and SH-5, inhibitors of phosphoinositide 3-kinase (PI3K) and Akt/protein kinase B, respectively, suggesting an involvement of the PI3K signaling pathway in the nuclear export of GAPDH. In addition, 5-aminoimidazole-4-carboxamide-1-?-D-ribofuranoside (AICAR), an activator of AMP-activated protein kinase (AMPK), stimulated the nuclear translocation of GAPDH and prevented serum- and growth factor-induced GAPDH export. AMPK inhibition by compound C or AMPK depletion by siRNA treatment partially prevented SFM- and AICAR-induced nuclear translocation of GAPDH. Our data suggest that the nuclear translocation of GAPDH might be regulated by the PI3K signaling pathway acting mainly as a nuclear export signal and the AMPK signaling pathway acting as a nuclear import signal.
Kwon, Hyun Jin; Rhim, Ji Heon; Jang, Ik-Soon; Kim, Go-Eun
NSDL National Science Digital Library
What part does nuclear energy play in satisfying energy demands? This informational piece, part of a series about the future of energy, introduces students to the uranium atom as an energy source. Here students read about the history of nuclear energy, how energy is derived from uranium, and benefits of nuclear energy. Information is also provided about limitations, particularly disposal problems and radioactivity, and geographical considerations of nuclear power in the United States. Thought-provoking questions afford students chances to reflect on what they've read about the uses of nuclear power. Articles and information on new nuclear plant design and nuclear accidents are available from a sidebar. Five energy-related PBS NewsHour links are provided. A web link to the U.S. Nuclear Regulatory Commission is included. Copyright 2005 Eisenhower National Clearinghouse
Project, Iowa P.
Electron-nuclear interactions as probes of domain motion in proteins
Long range interactions between nuclear spins and paramagnetic ions can serve as a sensitive monitor of internal motion of various parts of proteins, including functional loops and separate domains. In the case of interdomain motion, the interactions between the ion and NMR-observable nuclei are modulated in direction and magnitude mainly by a combination of overall and interdomain motions. The effects on observable parameters such as paramagnetic relaxation enhancement (PRE) and pseudocontact shift (PCS) can, in principle, be used to characterize motion. These parameters are frequently used for the purpose of structural refinements. However, their use to probe actual domain motions is less common and is lacking a proper theoretical treatment from a motional perspective. In this work, a suitable spin Hamiltonian is incorporated in a two body diffusion model to produce the time correlation function for the nuclear spin�paramagnetic ion interactions. Simulated observables for nuclei in different positions with respect to the paramagnetic ion are produced. Based on these simulations, it demonstrated that both the PRE and the PCS can be very sensitive probes of domain motion. Results for different nuclei within the protein sense different aspects of the motions. Some are more sensitive to the amplitude of the internal motion, others are more sensitive to overall diffusion rates, allowing separation of these contributions. Experimentally, the interaction strength can also be tuned by substitution of different paramagnetic ions or by varying magnetic field strength (in the case of lanthanides) to allow the use of more detailed diffusion models without reducing the reliability of data fitting.
Shapira, Boaz; Prestegard, James H.
Identification of retinoic acid-regulated nuclear matrix-associated protein as a novel regulator of gastric cancer
Background: Retinoic acid-regulated nuclear matrix-associated protein (RAMP) is a WD40 repeat-containing protein that is involved in various biological functions, but little is known about its role in human cancer. This study aims to delineate the oncogenic role of RAMP in gastric carcinogenesis.Methods: RAMP expression was examined by real-time quantitative RT-PCR, immunohistochemistry and western blotting. Inhibition of RAMP expression was performed
J Li; E K O Ng; Y P Ng; C Y P Wong; J Yu; H Jin; V Y Y Cheng; M Y Y Go; P K F Cheung; M P A Ebert; J Tong; K F To; F K L Chan; J J Y Sung; N Y Ip; W K Leung
Nuclear Factor 90, a cellular dsRNA binding protein inhibits the HIV Rev-export function
BACKGROUND: The HIV Rev protein is known to facilitate export of incompletely spliced and unspliced viral transcripts to the cytoplasm, a necessary step in virus life cycle. The Rev-mediated nucleo-cytoplasmic transport of nascent viral transcripts, dependents on interaction of Rev with the RRE RNA structural element present in the target RNAs. The C-terminal variant of dsRNA-binding nuclear protein 90 (NF90ctv)
Silvio Urcuqui-Inchima; Maria Eugenia Casta�o; Dani�le Hernandez-Verdun; Georges St-Laurent; Ajit Kumar
Organization and expression of the nuclear gene coding for the plastid-specific S22 ribosomal protein from spinach
We report here on the genomic organization and expression of a nuclear gene coding for a plastid ribosomal protein. The gene encodes the plastid-specific ribosomal protein S22 (formerly named CS-S5). Southern blot analysis suggests that the gene is present in one copy in the spinach genome. The gene consists of 5 exons of sizes ranging from 108 to 273 bp
Cordelia Bisanz-Seyer; R�gis Mache
Human T-Cell Leukemia Virus Type 1 Tax Protein Binds to Assembled Nuclear Proteasomes and Enhances Their Proteolytic Activity
The human T-cell leukemia virus type 1 (HTLV-1) Tax protein activates the HTLV-1 long terminal repeat and key regulatory proteins involved in inflammation, activation, and proliferation and may induce cell transformation. Tax is also the immunodominant target antigen for cytotoxic T cells in HTLV-1 infection. We found that Tax bound to assembled nuclear proteasomes, but Tax could not be detected
Diverse regulation of protein function by O-GlcNAc: a nuclear and cytoplasmic carbohydrate post-translational modification
N-Acetylglucosamine O-linked to serines and threonines of cytosolic and nuclear proteins (O-GlcNAc) is an abundant reversible post-translational modification found in all higher eukaryotes. Evidence for functional regulation of proteins by this dynamic saccharide is rapidly accumulating. Deletion of the gene encoding the enzyme that attaches O-GlcNAc (OGT) is lethal at the single cell level, indicating the fundamental requirement for this
Keith Vosseller; Kaoru Sakabe; Lance Wells; Gerald W Hart
Nicotine mediates hypochlorous Acid-induced nuclear protein damage in Mammalian cells.
Activated neutrophils secrete hypochlorous acid (HOCl) into the extracellular space of inflamed tissues. Because of short diffusion distance in biological fluids, HOCl-damaging effect is restricted to the extracellular compartment. The current study aimed at investigating the ability of nicotine, a component of tobacco and electronic cigarettes, to mediate HOCl-induced intracellular damage. We report, for the first time, that HOCl reacts with nicotine to produce nicotine chloramine (Nic-Cl). Nic-Cl caused dose-dependent damage to proliferating cell nuclear antigen (PCNA), a nuclear protein, in cultured mammalian lung and kidney cells. Vitamin C, vitamin E analogue (Trolox), glutathione, and N-acetyl-L-cysteine inhibited the Nic-Cl-induced PCNA damage, implicating oxidation in PCNA damage. These findings point out the ability of nicotine to mediate HOCl-induced intracellular damage and suggest antioxidants as protective measures. The results also raise the possibility that Nic-Cl can be created in the inflamed tissues of tobacco and electronic cigarette smokers and may contribute to smoking-related diseases. PMID:24357417
Salama, Samir A; Arab, Hany H; Omar, Hany A; Maghrabi, Ibrahim A; Snapka, Robert M
Human Dna2 is a nuclear and mitochondrial DNA maintenance protein.
Dna2 is a highly conserved helicase/nuclease that in yeast participates in Okazaki fragment processing, DNA repair, and telomere maintenance. Here, we investigated the biological function of human Dna2 (hDna2). Immunofluorescence and biochemical fractionation studies demonstrated that hDna2 was present in both the nucleus and the mitochondria. Analysis of mitochondrial hDna2 revealed that it colocalized with a subfraction of DNA-containing mitochondrial nucleoids in unperturbed cells. Upon the expression of disease-associated mutant forms of the mitochondrial Twinkle helicase which induce DNA replication pausing/stalling, hDna2 accumulated within nucleoids. RNA interference-mediated depletion of hDna2 led to a modest decrease in mitochondrial DNA replication intermediates and inefficient repair of damaged mitochondrial DNA. Importantly, hDna2 depletion also resulted in the appearance of aneuploid cells and the formation of internuclear chromatin bridges, indicating that nuclear hDna2 plays a role in genomic DNA stability. Together, our data indicate that hDna2 is similar to its yeast counterpart and is a new addition to the growing list of proteins that participate in both nuclear and mitochondrial DNA maintenance. PMID:19487465
Duxin, Julien P; Dao, Benjamin; Martinsson, Peter; Rajala, Nina; Guittat, Lionel; Campbell, Judith L; Spelbrink, Johannes N; Stewart, Sheila A
Serine 249 phosphorylation by ATM protein kinase regulates hepatocyte nuclear factor-1? transactivation.
Hepatocyte nuclear factor-1 alpha (HNF1?) exerts important effects on gene expression in multiple tissues. Several studies have directly or indirectly supported the role of phosphorylation processes in the activity of HNF1?. However, the molecular mechanism of this phosphorylation remains largely unknown. Using microcapillary liquid chromatography MS/MS and biochemical assays, we identified a novel phosphorylation site in HNF1? at Ser249. We also found that the ATM protein kinase phosphorylated HNF1? at Ser249 in vitro in an ATM-dependent manner and that ATM inhibitor KU55933 treatment inhibited phosphorylation of HNF1? at Ser249 in vivo. Coimmunoprecipitation assays confirmed the association between HNF1? and ATM. Moreover, ATM enhanced HNF1? transcriptional activity in a dose-dependent manner, whereas the ATM kinase-inactive mutant did not. The use of KU55933 confirmed our observation. Compared with wild-type HNF1?, a mutation in Ser249 resulted in a pronounced decrease in HNF1? transactivation, whereas no dominant-negative effect was observed. The HNF1?Ser249 mutant also exhibited normal nuclear localization but decreased DNA-binding activity. Accordingly, the functional studies of HNF1?Ser249 mutant revealed a defect in glucose metabolism. Our results suggested that ATM regulates the activity of HNF1? by phosphorylation of serine 249, particularly in glucose metabolism, which provides valuable insights into the undiscovered mechanisms of ATM in the regulation of glucose homeostasis. PMID:24821553
Zhao, Long; Chen, Hui; Zhan, Yi-Qun; Li, Chang-Yan; Ge, Chang-Hui; Zhang, Jian-Hong; Wang, Xiao-Hui; Yu, Miao; Yang, Xiao-Ming
A Novel Nuclear Trafficking Module Regulates the Nucleocytoplasmic Localization of the Rabies Virus Interferon Antagonist, P Protein*
Regulated nucleocytoplasmic transport of proteins is central to cellular function and dysfunction during processes such as viral infection. Active protein trafficking into and out of the nucleus is dependent on the presence within cargo proteins of intrinsic specific modular signals for nuclear import (nuclear localization signals, NLSs) and export (nuclear export signals, NESs). Rabies virus (RabV) phospho (P) protein, which is largely responsible for antagonising the host anti-viral response, is expressed as five isoforms (P1�P5). The subcellular trafficking of these isoforms is thought to depend on a balance between the activities of a dominant N-terminal NES (N-NES) and a distinct C-terminal NLS (C-NLS). Specifically, the N-NES-containing isoforms P1 and P2 are cytoplasmic, whereas the shorter P3�P5 isoforms, which lack the N-NES, are believed to be nuclear through the activity of the C-NLS. Here, we show for the first time that RabV P contains an additional strong NLS in the N-terminal region (N-NLS), which, intriguingly, overlaps with the N-NES. This arrangement represents a novel nuclear trafficking module where the N-NLS is inactive in P1 but becomes activated in P3, concomitant with truncation of the N-NES, to become the principal targeting signal conferring nuclear accumulation. Understanding this unique switch arrangement of overlapping, co-regulated NES/NLS sequences is vital to delineating the critical role of RabV P protein in viral infection.
Oksayan, Sibil; Wiltzer, Linda; Rowe, Caitlin L.; Blondel, Danielle; Jans, David A.; Moseley, Gregory W.
Improper Tagging of the Non-Essential Small Capsid Protein VP26 Impairs Nuclear Capsid Egress of Herpes Simplex Virus
To analyze the subcellular trafficking of herpesvirus capsids, the small capsid protein has been labeled with different fluorescent proteins. Here, we analyzed the infectivity of several HSV1(17+) strains in which the N-terminal region of the non-essential small capsid protein VP26 had been tagged at different positions. While some variants replicated with similar kinetics as their parental wild type strain, others were not infectious at all. Improper tagging resulted in the aggregation of VP26 in the nucleus, prevented efficient nuclear egress of viral capsids, and thus virion formation. Correlative fluorescence and electron microscopy showed that these aggregates had sequestered several other viral proteins, but often did not contain viral capsids. The propensity for aggregate formation was influenced by the type of the fluorescent protein domain, the position of the inserted tag, the cell type, and the progression of infection. Among the tags that we have tested, mRFPVP26 had the lowest tendency to induce nuclear aggregates, and showed the least reduction in replication when compared to wild type. Our data suggest that bona fide monomeric fluorescent protein tags have less impact on proper assembly of HSV1 capsids and nuclear capsid egress than tags that tend to dimerize. Small chemical compounds capable of inducing aggregate formation of VP26 may lead to new antiviral drugs against HSV infections.
Binz, Anne; Bauerfeind, Rudolf; Sodeik, Beate
Nuclear localization and intensity of staining of nm23 protein is useful marker for breast cancer progression
Background Breast cancer is the most common cause of cancer death in the western world. The expression differences of many proteins are associated with breast cancer progression or suppression. The purpose of the study was to determine the expression of nm23 protein in the invasion status and metastatic potential of breast cancer by using tissue microarray and to determine its role in breast cancer based on the expression of nm23 gene product. Method nm23 protein expression was examined by immunohistochemistry (IHC) using commercially available tissue microarray containing malignant and normal breast tissues from 216 patients. Results a similar percentage of cases showed positive cytoplasmic/nuclear staining for nm23 in normal breast tissue (85.7%), primary breast carcinoma node negative (97.5%) and carcinoma with lymph node metastasis (92.1%). Nuclear localization of staining for nm23 protein was higher in infiltrating ductal carcinoma (IDC) node positive (24.3%) and in matched lymph mode metastasis (18.9%) compared to IDC node negative (4.9%). Strong intensity of cytoplasmic/nucleus staining was observed in IDC node negative (42.6%), in IDC node positive (57.1%), and Infiltrating lobular carcinoma (ILC) node negative (44%) compared to normal breast tissue (16.7%). Conclusion nm23 protein expression appears widely expressed in normal breast, early and advanced breast cancer stages. Interestingly our study found that strong staining intensity and nuclear localization of nm23 protein may prove to be a useful marker of breast cancer progression.
Ismail, Nawfal I; Kaur, Gurjeet; Hashim, Hasnah; Hassan, Mohammed S
A New Family of Nuclear Receptor Coregulators That Integrate Nuclear Receptor Signaling through CREB-Binding Protein
We describe the cloning and characterization of a new family of nuclear receptor coregulators (NRCs) which modulate the function of nuclear hormone receptors in a ligand-dependent manner. NRCs are expressed as alternatively spliced isoforms which may exhibit different intrinsic activities and receptor specificities. The NRCs are organized into several modular structures and contain a single functional LXXLL motif which associates
A Novel Bipartite Nuclear Localization Signal Guides BPM1 Protein to Nucleolus Suggesting Its Cullin3 Independent Function
BPM1 belongs to the MATH-BTB family of proteins, which act as substrate-binding adaptors for the Cullin3-based E3 ubiquitin ligase. MATH-BTB proteins associate with Cullin3 via the BTB domain and with the substrate protein via the MATH domain. Few BPM1-interacting proteins with different functions are recognized, however, specific roles of BPM1, depending on its cellular localization have not been studied so far. Here, we found a novel bipartite nuclear localization signal at the C-terminus of the BPM1 protein, responsible for its nuclear and nucleolar localization and sufficient to drive the green fluorescent protein and cytoplasmic BPM4 protein into the nucleus. Co-localization analysis in live Nicotiana tabacum BY2 cells indicates a Cullin3 independent function since BPM1 localization is predominantly nucleolar and thus devoid of Cullin3. Treatment of BY2 cells with the proteasome inhibitor MG132 blocks BPM1 and Cullin3 degradation, suggesting turnover of both proteins through the ubiquitin�proteasome pathway. Possible roles of BPM1 in relation to its in vivo localization are discussed.
Leljak Levanic, Dunja; Horvat, Tomislav; Martincic, Jelena; Bauer, Natasa
A novel bipartite nuclear localization signal guides BPM1 protein to nucleolus suggesting its Cullin3 independent function.
BPM1 belongs to the MATH-BTB family of proteins, which act as substrate-binding adaptors for the Cullin3-based E3 ubiquitin ligase. MATH-BTB proteins associate with Cullin3 via the BTB domain and with the substrate protein via the MATH domain. Few BPM1-interacting proteins with different functions are recognized, however, specific roles of BPM1, depending on its cellular localization have not been studied so far. Here, we found a novel bipartite nuclear localization signal at the C-terminus of the BPM1 protein, responsible for its nuclear and nucleolar localization and sufficient to drive the green fluorescent protein and cytoplasmic BPM4 protein into the nucleus. Co-localization analysis in live Nicotiana tabacum BY2 cells indicates a Cullin3 independent function since BPM1 localization is predominantly nucleolar and thus devoid of Cullin3. Treatment of BY2 cells with the proteasome inhibitor MG132 blocks BPM1 and Cullin3 degradation, suggesting turnover of both proteins through the ubiquitin-proteasome pathway. Possible roles of BPM1 in relation to its in vivo localization are discussed. PMID:23251450
Leljak Levani?, Dunja; Horvat, Tomislav; Martin?i?, Jelena; Bauer, Nata�a
The GIP gamma-tubulin complex-associated proteins are involved in nuclear architecture in Arabidopsis thaliana
During interphase, the microtubular cytoskeleton of cycling plant cells is organized in both cortical and perinuclear arrays. Perinuclear microtubules (MTs) are nucleated from ?-Tubulin Complexes (?-TuCs) located at the surface of the nucleus. The molecular mechanisms of ?-TuC association to the nuclear envelope (NE) are currently unknown. The ?-TuC Protein 3 (GCP3)-Interacting Protein 1 (GIP1) is the smallest ?-TuC component identified so far. AtGIP1 and its homologous protein AtGIP2 participate in the localization of active ?-TuCs at interphasic and mitotic MT nucleation sites. Arabidopsis gip1gip2 mutants are impaired in establishing a fully functional mitotic spindle and exhibit severe developmental defects. In this study, gip1gip2 knock down mutants were further characterized at the cellular level. In addition to defects in both the localization of ?-TuC core proteins and MT fiber robustness, gip1gip2 mutants exhibited a severe alteration of the nuclear shape associated with an abnormal distribution of the nuclear pore complexes. Simultaneously, they showed a misorganization of the inner nuclear membrane protein AtSUN1. Furthermore, AtGIP1 was identified as an interacting partner of AtTSA1 which was detected, like the AtGIP proteins, at the NE. These results provide the first evidence for the involvement of a ?-TuC component in both nuclear shaping and NE organization. Functional hypotheses are discussed in order to propose a model for a GIP-dependent nucleo-cytoplasmic continuum.
Batzenschlager, Morgane; Masoud, Kinda; Janski, Natacha; Houlne, Guy; Herzog, Etienne; Evrard, Jean-Luc; Baumberger, Nicolas; Erhardt, Mathieu; Nomine, Yves; Kieffer, Bruno; Schmit, Anne-Catherine; Chaboute, Marie-Edith
Nuclear quadrupole interaction of111Cd on type-1 Cu-sites in blue copper proteins
NASA Astrophysics Data System (ADS)
The nuclear quadrupole interaction (NQI) of111Cd substituted for Cu(II) on type-1 sites in blue copper proteins is characterized by high values of ?0 in the region of 300 Mrad/s, close to that for the catalytic zinc site in alcohol dehydrogenase. Type-1 Cu has usually two sulfur ligands and two nitrogen ligands and in some cases an oxygen ligand in either a distorted tetrahedral geometry or in a trigonal bipyramidal geometry. The near tetrahedral arrangement together with the ligand sphere containing the same number of sulfur ligands explains the value of ?0 in the blue copper proteins. The present work determined the partial NQI for methionine using the known structure of azurin. This value was then used in the angular overlap model to calculate the NQI for ascorbate oxidase the structure of which is also known and gave good agreement with experiment. NQI data for laccase and stellacyanin the structures of which are unknown, are also given.
Tr�ger, W.; Butz, T.; Danielsen, E.; Bauer, R.; Thoenes, U.; Messerschmidt, A.; Huber, R.; Canters, G. W.; den Blaauwen, T.
Fluctuation-based imaging of nuclear Rac1 activation by protein oligomerisation.
Here we describe a fluctuation-based method to quantify how protein oligomerisation modulates signalling activity of a multifunctional protein. By recording fluorescence lifetime imaging microscopy (FLIM) data of a FRET biosensor in a format that enables concomitant phasor and cross Number and Brightness (cN&B) analysis, we measure the nuclear dynamics of a Rac1 FRET biosensor and assess how Rac1 homo-oligomers (N&B) regulate Rac1 activity (hetero-oligomerisation with the biosensor affinity reagent, PBD, by FLIM-FRET) or interaction with an unknown binding partner (cN&B). The high spatiotemporal resolution of this method allowed us to discover that upon DNA damage monomeric and active Rac1 in the nucleus is segregated from dimeric and inactive Rac1 in the cytoplasm. This reorganisation requires Rac1 GTPase activity and is associated with an importin-?2 redistribution. Only with this multiplexed approach can we assess the oligomeric state a molecular complex must form in order to regulate a complex signalling network. PMID:24573109
Hinde, Elizabeth; Yokomori, Kyoko; Gaus, Katharina; Hahn, Klaus M; Gratton, Enrico
Fluctuation-based imaging of nuclear Rac1 activation by protein oligomerisation
Here we describe a fluctuation-based method to quantify how protein oligomerisation modulates signalling activity of a multifunctional protein. By recording fluorescence lifetime imaging microscopy (FLIM) data of a FRET biosensor in a format that enables concomitant phasor and cross Number and Brightness (cN&B) analysis, we measure the nuclear dynamics of a Rac1 FRET biosensor and assess how Rac1 homo-oligomers (N&B) regulate Rac1 activity (hetero-oligomerisation with the biosensor affinity reagent, PBD, by FLIM-FRET) or interaction with an unknown binding partner (cN&B). The high spatiotemporal resolution of this method allowed us to discover that upon DNA damage monomeric and active Rac1 in the nucleus is segregated from dimeric and inactive Rac1 in the cytoplasm. This reorganisation requires Rac1 GTPase activity and is associated with an importin-?2 redistribution. Only with this multiplexed approach can we assess the oligomeric state a molecular complex must form in order to regulate a complex signalling network.
Hinde, Elizabeth; Yokomori, Kyoko; Gaus, Katharina; Hahn, Klaus M.; Gratton, Enrico
Cytokinesis proteins Tum and Pav have a nuclear role in Wnt regulation.
Wg/Wnt signals specify cell fates in both invertebrate and vertebrate embryos and maintain stem-cell populations in many adult tissues. Deregulation of the Wnt pathway can transform cells to a proliferative fate, leading to cancer. We have discovered that two Drosophila proteins that are crucial for cytokinesis have a second, largely independent, role in restricting activity of the Wnt pathway. The fly homolog of RacGAP1, Tumbleweed (Tum)/RacGAP50C, and its binding partner, the kinesin-like protein Pavarotti (Pav), negatively regulate Wnt activity in fly embryos and in cultured mammalian cells. Unlike many known regulators of the Wnt pathway, these molecules do not affect stabilization of Arm/beta-catenin (betacat), the principal effector molecule in Wnt signal transduction. Rather, they appear to act downstream of betacat stabilization to control target-gene transcription. Both Tum and Pav accumulate in the nuclei of interphase cells, a location that is spatially distinct from their cleavage-furrow localization during cytokinesis. We show that this nuclear localization is essential for their role in Wnt regulation. Thus, we have identified two modulators of the Wnt pathway that have shared functions in cell division, which hints at a possible link between cytokinesis and Wnt activity during tumorigenesis. PMID:20516152
Jones, Whitney M; Chao, Anna T; Zavortink, Michael; Saint, Robert; Bejsovec, Amy
Cytokinesis proteins Tum and Pav have a nuclear role in Wnt regulation
Wg/Wnt signals specify cell fates in both invertebrate and vertebrate embryos and maintain stem-cell populations in many adult tissues. Deregulation of the Wnt pathway can transform cells to a proliferative fate, leading to cancer. We have discovered that two Drosophila proteins that are crucial for cytokinesis have a second, largely independent, role in restricting activity of the Wnt pathway. The fly homolog of RacGAP1, Tumbleweed (Tum)/RacGAP50C, and its binding partner, the kinesin-like protein Pavarotti (Pav), negatively regulate Wnt activity in fly embryos and in cultured mammalian cells. Unlike many known regulators of the Wnt pathway, these molecules do not affect stabilization of Arm/?-catenin (?cat), the principal effector molecule in Wnt signal transduction. Rather, they appear to act downstream of ?cat stabilization to control target-gene transcription. Both Tum and Pav accumulate in the nuclei of interphase cells, a location that is spatially distinct from their cleavage-furrow localization during cytokinesis. We show that this nuclear localization is essential for their role in Wnt regulation. Thus, we have identified two modulators of the Wnt pathway that have shared functions in cell division, which hints at a possible link between cytokinesis and Wnt activity during tumorigenesis.
Jones, Whitney M.; Chao, Anna T.; Zavortink, Michael; Saint, Robert; Bejsovec, Amy
Caspase-mediated cleavage of C53/LZAP protein causes abnormal microtubule bundling and rupture of the nuclear envelope
Apoptotic nucleus undergoes distinct morphological and biochemical changes including nuclear shrinkage, chromatin condensation and DNA fragmentation, which are attributed to caspase-mediated cleavage of several nuclear substrates such as lamins. As most of active caspases reside in the cytoplasm, disruption of the nuclear-cytoplasmic barrier is essential for caspases to reach their nuclear targets. The prevailing proposed mechanism is that the increase in the permeability of nuclear pores induced by caspases allows the caspases and other apoptotic factors to diffuse into the nucleus, thereby resulting in the nuclear destruction. Here, we report a novel observation that physical rupture of the nuclear envelope (NE) occurs in the early stage of apoptosis. We found that the NE rupture was caused by caspase-mediated cleavage of C53/LZAP, a protein that has been implicated in various signaling pathways, including NF-?B signaling and DNA damage response, as well as tumorigenesis and metastasis. We also demonstrated that C53/LZAP bound indirectly to the microtubule (MT), and expression of the C53/LZAP cleavage product caused abnormal MT bundling and NE rupture. Taken together, our findings suggest a novel role of C53/LZAP in the regulation of MT dynamics and NE structure during apoptotic cell death. Our study may provide an additional mechanism for disruption of the nuclear-cytoplasmic barrier during apoptosis.
Wu, Jianchun; Jiang, Hai; Luo, Shouqing; Zhang, Mingsheng; Zhang, Yinghua; Sun, Fei; Huang, Shuang; Li, Honglin
Nuclear Import of Cytoplasmic Poly(A) Binding Protein Restricts Gene Expression via Hyperadenylation and Nuclear Retention of mRNA ?
Poly(A) tail length is emerging as an important marker of mRNA fate, where deviations from the canonical length can signal degradation or nuclear retention of transcripts. Pathways regulating polyadenylation thus have the potential to broadly influence gene expression. Here we demonstrate that accumulation of cytoplasmic poly(A) binding protein (PABPC) in the nucleus, which can occur during viral infection or other forms of cellular stress, causes mRNA hyperadenylation and nuclear accumulation of poly(A) RNA. This inhibits gene expression but does not affect mRNA stability. Unexpectedly, PABPC-induced hyperadenylation can occur independently of mRNA 3?-end processing yet requires the canonical mRNA poly(A) polymerase II. We find that nuclear PABPC-induced hyperadenylation is triggered by multiple divergent viral factors, suggesting that altering the subcellular localization of PABPC may be a commonly used mechanism to regulate cellular gene expression in a polyadenylation-linked manner.
Kumar, G. Renuka; Glaunsinger, Britt A.
A protein interaction atlas for the nuclear receptors: properties and quality of a hub-based dimerisation network
Background The nuclear receptors are a large family of eukaryotic transcription factors that constitute major pharmacological targets. They exert their combinatorial control through homotypic heterodimerisation. Elucidation of this dimerisation network is vital in order to understand the complex dynamics and potential cross-talk involved. Results Phylogeny, protein-protein interactions, protein-DNA interactions and gene expression data have been integrated to provide a comprehensive and up-to-date description of the topology and properties of the nuclear receptor interaction network in humans. We discriminate between DNA-binding and non-DNA-binding dimers, and provide a comprehensive interaction map, that identifies potential cross-talk between the various pathways of nuclear receptors. Conclusion We infer that the topology of this network is hub-based, and much more connected than previously thought. The hub-based topology of the network and the wide tissue expression pattern of NRs create a highly competitive environment for the common heterodimerising partners. Furthermore, a significant number of negative feedback loops is present, with the hub protein SHP [NR0B2] playing a major role. We also compare the evolution, topology and properties of the nuclear receptor network with the hub-based dimerisation network of the bHLH transcription factors in order to identify both unique themes and ubiquitous properties in gene regulation. In terms of methodology, we conclude that such a comprehensive picture can only be assembled by semi-automated text-mining, manual curation and integration of data from various sources.
Amoutzias, Gregory D; Pichler, Elgar E; Mian, Nina; De Graaf, David; Imsiridou, Anastasia; Robinson-Rechavi, Marc; Bornberg-Bauer, Erich; Robertson, David L; Oliver, Stephen G
Evidence for a dual role for TC4 protein in regulating nuclear structure and cell cycle progression
TC4, a ras-like G protein, has been impli- cated in the feedback pathway linking the onset of mi- tosis to the completion of DNA replication. In this re- port we find distinct roles for TC4 in both nuclear assembly and cell cycle progression. Mutant and wild- type forms of \\
Sally Kornbluth; Mary Dasso; John Newport
A role for nucleosome assembly protein 1 in the nuclear transport of histones H2A and H2B
Import of core histones into the nucleus is a prerequisite for their deposition onto DNA and the assembly of chromatin. Here we demonstrate that nucleosome assembly protein 1 (Nap1p), a protein previously implicated in the deposition of histones H2A and H2B, is also involved in the transport of these two histones. We demonstrate that Nap1p can bind directly to Kap114p, the primary karyopherin/importin responsible for the nuclear import of H2A and H2B. Nap1p also serves as a bridge between Kap114p and the histone nuclear localization sequence (NLS). Nap1p acts cooperatively to increase the affinity of Kap114p for these NLSs. Nuclear accumulation of histone NLS�green fluorescent protein (GFP) reporters was decreased in ?nap1 cells. Furthermore, we demonstrate that Nap1p promotes the association of the H2A and H2B NLSs specifically with the karyopherin Kap114p. Localization studies demonstrate that Nap1p is a nucleocytoplasmic shuttling protein, and genetic experiments suggest that its shuttling is important for maintaining chromatin structure in vivo. We propose a model in which Nap1p links the nuclear transport of H2A and H2B to chromatin assembly.
Mosammaparast, Nima; Ewart, Courtney S.; Pemberton, Lucy F.
Nuclear targeting of a viral-cointernalized protein by a short signal sequence from human retinoic acid receptors.
The synthesis of a putative nuclear localization signal from the human retinoic acid receptor is described. Its ability to target a carrier protein to the nucleus is demonstrated following microinjection or a new technique of cell internalization in the presence of adenovirus 3. PMID:1665078
Hamy, F; Verwaerde, P; Helbecque, N; Formstecher, P; H�nichart, J P
A Polynomial-Time Algorithm for De Novo Protein Backbone Structure Determination from Nuclear Magnetic Resonance Data
We describe an efficient algorithm for protein backbone structure determination from solu- tion Nuclear Magnetic Resonance (NMR) data. A key feature of our algorithm is that it finds the conformation and orientation of secondary structure elements as well as the global fold in polynomial time. This is the first polynomial-time algorithm for de novo high-resolution biomacromolecular structure determination using experimentally
Lincong Wang; Ramgopal R. Mettu; Bruce Randall Donald
A Visual Screen of a GFP-fusion Library Identifies a New Type of Nuclear Envelope Membrane Protein
The nuclear envelope (NE) is a distinct sub- domain of the ER, but few membrane components have been described that are specific to it. We per- formed a visual screen in tissue culture cells to identify proteins targeted to the NE. This approach does not re- quire assumptions about the nature of the association with the NE or the physical
Melissa M. Rolls; Pascal A. Stein; Stephen S. Taylor; Edward Ha; Frank McKeon; Tom A. Rapoport
Transactivation activity of Maf nuclear oncoprotein is modulated by Jun, Fos and small Maf proteins.
The v-maf oncogene encodes a nuclear bZip protein which specifically recognizes relatively long palindromic sequences related to an AP-1 site. In this study, we investigated the relationship of transactivation and transformation activity of Maf. The amino-terminal two thirds of the molecule were dispensable for its DNA-binding activity but conferred its transactivation potential. Transactivation activities of a set of deletion mutants correlated well with their cell transforming abilities. However, a point mutant associated with enhanced oncogenic activity was not more effective in transactivation than the wild type, suggesting that some other function(s) of Maf is also important for its transforming ability. We also examined the effect of other bZip proteins on the transactivation activity of Maf. Three small Maf family proteins (MafK, MafF and MafG), which are missing the transactivation domain of v-Maf, competitively inhibited transactivation by Maf. Co-expression of Jun or Fos also affected the transactivation potential of Maf by forming Maf/Jun or Maf/Fos heterodimers of distinct DNA-binding specificities. In addition to these factors, we noticed the presence of a strong endogenous transactivating activity associated with a sequence related to an NF-E2 site rather than the typical AP-1 site in fibroblast cells. These results indicate that AP-1 site-like cis-regulatory elements of eukaryotic genes are regulated by multiple sets of bZip dimers with different DNA-binding and transactivation properties. PMID:8552399
Kataoka, K; Noda, M; Nishizawa, M
Sde2: A novel nuclear protein essential for telomeric silencing and genomic stability in Schizosaccharomyces pombe
Research highlights: {yields} Sde2 is essential for telomere silencing. {yields} Sde2 is involved in the maintenance of genomic stability. {yields} Sde2 promotes the recruitment of SHREC, a histone deacetylase complex, to telomeres. -- Abstract: Telomeres, specialized domains assembled at the ends of linear chromosomes, are essential for genomic stability in eukaryotes. The formation and maintenance of telomeres are governed by numerous factors such as telomeric repeats, telomere-binding proteins, heterochromatin proteins, and telomerase. Here, we report Sde2, a novel nuclear protein essential for telomeric silencing and genomic stability in the fission yeast Schizosaccharomyces pombe. A deficiency in sde2 results in the derepression of the ura4{sup +} gene inserted near telomeric repeats, and the noncoding transcripts from telomeric regions accumulate in sde2{Delta} cells. The loss of Sde2 function compromises transcriptional silencing at telomeres, and this silencing defect is accompanied by increased levels of acetylated histone H3K14 and RNA polymerase II occupancy at telomeres as well as reduced recruitment of the SNF2 ATPase/histone deacetylase-containing complex SHREC to telomeres. Deletion of sde2 also leads to a higher frequency of mitotic minichromosome loss, and sde2{Delta} cells often form asci that contain spores in abnormal numbers, shapes, or both. In addition, sde2{Delta} cells are highly sensitive to several stresses, including high/low temperatures, bleomycin, which induces DNA damage, and thiabendazole, a microtubule-destabilizing agent. Furthermore, Sde2 genetically interacts with the telomere regulators Taz1, Pof3, and Ccq1. These findings demonstrate that Sde2 cooperates with other telomere regulators to maintain functional telomeres, thereby preventing genomic instability.
Sugioka-Sugiyama, Rie [Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577 (Japan) [Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577 (Japan); Initiative for the Promotion of Young Scientists' Independent Research, University of Tsukuba, Tsukuba, Ibaraki 305-8577 (Japan); Sugiyama, Tomoyasu, E-mail: firstname.lastname@example.com [Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577 (Japan) [Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577 (Japan); Initiative for the Promotion of Young Scientists' Independent Research, University of Tsukuba, Tsukuba, Ibaraki 305-8577 (Japan); Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), Kawaguchi, Saitama 332-0012 (Japan)
In vivo characterization of chimeric PCV DNA clones containing heterogeneous capsid protein nuclear localization signals (NLS)
Background PCV ORF2 capsid protein was predicted to contribute to the control of replication via an interaction between the Cap and Rep proteins in the nucleoplasm. We previously showed that the nuclear localization signal (NLS) on the capsid protein plays an accessory role in the replication of PCV in vitro. To further evaluate the in vivo characteristics of NLS-chimeric PCV DNA clones, BALB/C mice were inoculated intranasally and intraperitoneally with the DNA clones. Results As expected, no gross lesions were detected during the study of the inoculated animals. The chimeric PCV12-, PCV1-NLS2- and PCV2-NLS1-inoculated animals had significantly fewer and less severe histopathological lesions in lymphoid tissues than the PCV2-inoculated animals (P?0.05). PCV12 induced a specific antibody response against PCV2 ORF2 comparable to that induced by wild-type PCV2 but demonstrated a shorter period of viremia and much lower level of virus loads in sera than those in PCV2-inoculated mice. Remarkably, the PCV2-NLS1 and PCV1-NLS2 chimeras replicated in inoculated mice and induced specific antibody responses but failed to produce viral antigens in the lymph nodes or a detectable viremia. Conclusions The chimeric PCV2-NLS1 and PCV1-NLS2 demonstrated a lower replication level as compared with wild type of PCV2 or PCV1 in vivo, suggesting that ORF2 NLSs played an accessory role in PCV replication. The chimeric PCV12 is a good candidate for vaccination against PCV2 infection.
Helicobacter pylori tumor necrosis factor-? inducing protein promotes cytokine expression via nuclear factor-?B
AIM: To study the effects of Helicobacter pylori (H. pylori) tumor necrosis factor-? (TNF) inducing protein (Tip-?) on cytokine expression and its mechanism. METHODS: We cloned Tip-? from the H. pylori strain 26695, transformed Escherichia coli with an expression plasmid, and then confirmed the expression product by Western blotting. Using different concentrations of Tip-? that affected SGC7901 and GES-1 cells at different times, we assessed cytokine levels using enzyme-linked immunosorbent assay. We blocked SGC7901 cells with pyrrolidine dithiocarbamate (PDTC), a specific inhibitor of nuclear factor ?B (NF-?B). We then detected interleukin (IL)-1? and TNF-? levels in SGC7901 cells. RESULTS: Western blot analysis using an anti-Tip-? antibody revealed a 23-kDa protein, which indicated that recombinant Tip-? protein was recombined successfully. The levels of IL-1?, IL-8 and TNF-? were significantly higher following Tip-? interference, whether GES-1 cells or SGC-7901 cells were used (P < 0.05). However, the levels of cytokines (including IL-1?, IL-8 and TNF-?) secreted by SGC-7901 cells were greater than those secreted by GES-1 cells following treatment with Tip-? at the same concentration and for the same duration (P < 0.05). After blocking NF-?B with PDTC, the cells (GES-1 cells and SGC-7901 cells) underwent interference with Tip-?. We found that IL-1? and TNF-? levels were significantly decreased compared to cells that only underwent Tip-? interference (P < 0.05). CONCLUSION: Tip-? plays an important role in cytokine expression through NF-?B.
Tang, Chun-Li; Hao, Bo; Zhang, Guo-Xin; Shi, Rui-Hua; Cheng, Wen-Fang

subset: commoncrawl