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Yeast prions and human prion-like proteins: sequence features and prediction methods.
Prions are self-propagating infectious protein isoforms. A growing number of prions have been identified in yeast, each resulting from the conversion of soluble proteins into an insoluble amyloid form. These yeast prions have served as a powerful model system for studying the causes and consequences of prion aggregation. Remarkably, a number of human proteins containing prion-like domains, defined as domains with compositional similarity to yeast prion domains, have recently been linked to various human degenerative diseases, including amyotrophic lateral sclerosis. This suggests that the lessons learned from yeast prions may help in understanding these human diseases. In this review, we examine what has been learned about the amino acid sequence basis for prion aggregation in yeast, and how this information has been used to develop methods to predict aggregation propensity. We then discuss how this information is being applied to understand human disease, and the challenges involved in applying yeast prediction methods to higher organisms. PMID:24390581
Cascarina, Sean M; Ross, Eric D
PrionHome: A Database of Prions and Other Sequences Relevant to Prion Phenomena
Prions are units of propagation of an altered state of a protein or proteins; prions can propagate from organism to organism, through cooption of other protein copies. Prions contain no necessary nucleic acids, and are important both as both pathogenic agents, and as a potential force in epigenetic phenomena. The original prions were derived from a misfolded form of the mammalian Prion Protein PrP. Infection by these prions causes neurodegenerative diseases. Other prions cause non-Mendelian inheritance in budding yeast, and sometimes act as diseases of yeast. We report the bioinformatic construction of the PrionHome, a database of >2000 prion-related sequences. The data was collated from various public and private resources and filtered for redundancy. The data was then processed according to a transparent classification system of prionogenic sequences (i.e., sequences that can make prions), prionoids (i.e., proteins that propagate like prions between individual cells), and other prion-related phenomena. There are eight PrionHome classifications for sequences. The first four classifications are derived from experimental observations: prionogenic sequences, prionoids, other prion-related phenomena, and prion interactors. The second four classifications are derived from sequence analysis: orthologs, paralogs, pseudogenes, and candidate-prionogenic sequences. Database entries list: supporting information for PrionHome classifications, prion-determinant areas (where relevant), and disordered and compositionally-biased regions. Also included are literature references for the PrionHome classifications, transcripts and genomic coordinates, and structural data (including comparative models made for the PrionHome from manually curated alignments). We provide database usage examples for both vertebrate and fungal prion contexts. Using the database data, we have performed a detailed analysis of the compositional biases in known budding-yeast prionogenic sequences, showing that the only abundant bias pattern is for asparagine bias with subsidiary serine bias. We anticipate that this database will be a useful experimental aid and reference resource. It is freely available at: http://libaio.biol.mcgill.ca/prion.
Harbi, Djamel; Parthiban, Marimuthu; Gendoo, Deena M. A.; Ehsani, Sepehr; Kumar, Manish; Schmitt-Ulms, Gerold; Sowdhamini, Ramanathan; Harrison, Paul M.
Sequence variations of the bovine prion protein gene (PRNP) in native Korean Hanwoo cattle
Bovine spongiform encephalopathy (BSE) is one of the fatal neurodegenerative diseases known as transmissible spongiform encephalopathies (TSEs) caused by infectious prion proteins. Genetic variations correlated with susceptibility or resistance to TSE in humans and sheep have not been reported for bovine strains including those from Holstein, Jersey, and Japanese Black cattle. Here, we investigated bovine prion protein gene (PRNP) variations in Hanwoo cattle [Bos (B.) taurus coreanae], a native breed in Korea. We identified mutations and polymorphisms in the coding region of PRNP, determined their frequency, and evaluated their significance. We identified four synonymous polymorphisms and two non-synonymous mutations in PRNP, but found no novel polymorphisms. The sequence and number of octapeptide repeats were completely conserved, and the haplotype frequency of the coding region was similar to that of other B. taurus strains. When we examined the 23-bp and 12-bp insertion/deletion (indel) polymorphisms in the non-coding region of PRNP, Hanwoo cattle had a lower deletion allele and 23-bp del/12-bp del haplotype frequency than healthy and BSE-affected animals of other strains. Thus, Hanwoo are seemingly less susceptible to BSE than other strains due to the 23-bp and 12-bp indel polymorphisms.
Choi, Sangho
Prion protein in ESC regulation
A large number of studies have analyzed the putative functions of the prion protein (PrPC) in mammals. Although its sequence conservation over a wide range of different animals may indicate that this protein could have a key role in prion diseases, an absolutely accepted involvement has not been found so far. We have recently reported that PrPC regulates Nanog mRNA expression, the first non-redundant function of PrPC in embryonic stem cells (ESC), which translates into control of pluripotency and early differentiation. Contrary to what is believed, the other two members of the prion protein family, Doppel and Shadoo, cannot replace the absence of PrPC, causing the appearance of a new embryoid body (EB) population in our in vitro culture. The similarities between EB and an early post-implantation embryo suggest that this might also occur in vivo, enhancing the importance of this finding. On the other hand, our data may support the hypothesis of a relationship between the loss of PrPC function and neuronal degeneration in prion diseases. A reduction in brain stem cells pluripotency after PrPC is misfolded into the pathological conformation (PrPSc) could lead to a delay or a disappearance of the normal brain damage recovery.
Pericuesta, Eva
Recombinant Human Prion Protein Inhibits Prion Propagation in vitro
Prion diseases are associated with the conformational conversion of the cellular prion protein (PrPC) into the pathological scrapie isoform (PrPSc) in the brain. Both the in vivo and in vitro conversion of PrPC into PrPSc is significantly inhibited by differences in amino acid sequence between the two molecules. Using protein misfolding cyclic amplification (PMCA), we now report that the recombinant full-length human PrP (rHuPrP23-231) (that is unglycosylated and lacks the glycophosphatidylinositol anchor) is a strong inhibitor of human prion propagation. Furthermore, rHuPrP23-231 also inhibits mouse prion propagation in a scrapie-infected mouse cell line. Notably, it binds to PrPSc, but not PrPC, suggesting that the inhibitory effect of recombinant PrP results from blocking the interaction of brain PrPC with PrPSc. Our findings suggest a new avenue for treating prion diseases, in which a patient's own unglycosylated and anchorless PrP is used to inhibit PrPSc propagation without inducing immune response side effects.
Yuan, Jue; Zhan, Yi-An; Abskharon, Romany; Xiao, Xiangzhu; Martinez, Manuel Camacho; Zhou, Xiaochen; Kneale, Geoff; Mikol, Jacqueline; Lehmann, Sylvain; Surewicz, Witold K.; Castilla, Joaquin; Steyaert, Jan; Zhang, Shulin; Kong, Qingzhong; Petersen, Robert B.; Wohlkonig, Alexandre; Zou, Wen-Quan
Fishing for Prion Protein Function
The prion protein is infamous for its role in devastating neurological diseases, but its normal, physiological function has remained mysterious. A new study uses the experimentally tractable zebrafish model to obtain fresh clues to this puzzle.
Roberto Chiesa; David A. Harris
Scrapie prion proteins are synthesized in neurons.
Scrapie is a slow degenerative encephalopathy of animals caused by unusual infectious particles termed prions. A cDNA encoding the only apparent component of the prion, a protein designated PrP 27-30, has recently been cloned and sequenced. By measuring mRNA levels using in situ hybridization with the PrP cDNA, the authors found that prion proteins are synthesized almost exclusively within neurons. The levels of PrP mRNA varied among different types of neurons, but did not change during scrapie infection. A cDNA encoding glial fibrillary acidic protein (GFAP) was a positive control; GFAP mRNA was confined to astrocytes. Our finding of PrP mRNA in neurons may explain the degeneration and vacuolation that occurs in these cells during scrapie infection. Images Figure 1 Figure 2 Figure 3
Kretzschmar, H. A.; Prusiner, S. B.; Stowring, L. E.; DeArmond, S. J.
Prion protein in Caenorhabditis elegans
The infectious agent of prion diseases is believed to be nucleic acid-free particles composed of misfolded conformational isomers of a host protein known as prion protein (PrP). Although this �protein-only� concept is generally accepted, decades of extensive research have not been able to elucidate the mechanisms by which PrP misfolding leads to neurodegeneration and infectivity. The challenges in studying prion diseases relate in part to the limitations of mammalian prion models, which include the long incubation period post-infection until symptoms develop, the high expense of maintaining mammals for extended periods, as well as safety issues. In order to develop prion models incorporating a genetically tractable simple system with a well-defined neuronal system, we generated transgenic C. elegans expressing the mouse PrP behind the pan-neuronal ric-19 promoter (Pric-19). We show here that high expression of Pric-19::PrP in C. elegans can result in altered morphology, defective mobility and shortened lifespan. Low expression of Pric-19::PrP, however, does not cause any detectable harm. Using the dopamine neuron specific promoter Pdat-1, we also show that expression of the murine BAX, a pro-apoptotic member of the Bcl-2 family, causes dopamine neuron destruction in the nematode. However, co-expression of PrP inhibits BAX-mediated dopamine neuron degeneration, demonstrating for the first time that PrP has anti-BAX activity in living animals. Thus, these distinct PrP-transgenic C. elegans lines recapitulate a number of functional and neuropathological features of mammalian prion models and provide an opportunity for facile identification of genetic and environmental contributors to prion-associated pathology.
Park, Kyung-Won
A bipolar personality of yeast prion proteins
Prions are infectious, self-propagating protein conformations. [PSI+], [RNQ+] and [URE3] are well characterized prions in Saccharomyces cerevisiae and represent the aggregated states of the translation termination factor Sup35, a functionally unknown protein Rnq1 and a regulator of nitrogen metabolism Ure2, respectively. Overproduction of Sup35 induces the de novo appearance of the [PSI+] prion in [RNQ+] or [URE3] strain, but not in non-prion strain. However, [RNQ+] and [URE3] prions themselves, as well as overexpression of a mutant Rnq1 protein, Rnq1?100 and Lsm4, hamper the maintenance of [PSI+]. These findings point to a bipolar activity of [RNQ+], [URE3], Rnq1?100 and Lsm4, and probably other yeast prion proteins as well, for the fate of [PSI+] prion. Possible mechanisms underlying the apparent bipolar activity of yeast prions will be discussed.
Kurahashi, Hiroshi; Oishi, Keita
Prion Diseases: From Protein to Cell Pathology
Prion diseases or transmissible spongiform encephalopathies are fatal neurodegenerative conditions in humans and animals that originate spontaneously, genetically or by infection. Conformational change of the normal (cellular) form of prion protein (PrPc) to a pathological, disease-associated form (PrPTSE) is considered central to pathogenesis and formation of the infectious agent or prion. Neuronal damage is central to clinical manifestation of prion diseases but poorly understood. In this review, we analyze the major pathogenetic pathways that lead to tissue pathology in different forms of disease. Neuropathogenesis of prion diseases evolves in complex ways on several front lines, most but not all of which exist also in other neurodegenerative as well as infectious diseases. Whereas intracellular accumulation of PrP forms might significantly impair cell function and lead to cytopathology, mere extracellular deposition of PrPTSE is questionable as a direct cytotoxic factor. Tissue damage may result from several parallel, interacting, or subsequent pathways. Future studies should clarify the trigger(s) and sequence of these processes and whether, and which, one is dominating or decisive.
Kovacs, Gabor G.; Budka, Herbert
The Prion Protein Knockout Mouse
The key pathogenic event in prion disease involves misfolding and aggregation of the cellular prion protein (PrP). Beyond this fundamental observation, the mechanism by which PrP misfolding in neurons leads to injury and death remains enigmatic. Prion toxicity may come about by perverting the normal function of PrP. If so, understanding the normal function of PrP may help to elucidate the molecular mechansim of prion disease. Ablation of the Prnp gene, which encodes PrP, was instrumental for determining that the continuous production of PrP is essential for replicating prion infectivity. Since the structure of PrP has not provided any hints to its possible function, and there is no obvious phenotype in PrP KO mice, studies of PrP function have often relied on intuition and serendipity. Here, we enumerate the multitude of phenotypes described in PrP deficient mice, many of which manifest themselves only upon physiological challenge. We discuss the pleiotropic phenotypes of PrP deficient mice in relation to the possible normal function of PrP. The critical question remains open: which of these phenotypes are primary effects of PrP deletion and what do they tell us about the function of PrP?
Lindquist, Susan; Aguzzi, Adriano
Prion protein self-interaction in prion disease therapy approaches.
Transmissible spongiform encephalopathies (TSEs) or prion diseases are unique disorders that are not caused by infectious micro-organisms (bacteria or fungi), viruses or parasites, but rather seem to be the result of an infectious protein. TSEs are comprised of fatal neurodegenerative disorders affecting both human and animals. Prion diseases cause sponge-like degeneration of neuronal tissue and include (among others) Creutzfeldt-Jacob disease in humans, bovine spongiform encephalopathy (BSE) in cattle and scrapie in sheep. TSEs are characterized by the formation and accumulation of transmissible (infectious) disease-associated protease-resistant prion protein (PrP(Sc)), mainly in tissues of the central nervous system. The exact molecular processes behind the conversion of PrP(C) into PrP(Sc) are not clearly understood. Correlations between prion protein polymorphisms and disease have been found, however in what way these polymorphisms influence the conversion processes remains an enigma; is stabilization or destabilization of the prion protein the basis for a higher conversion propensity? Apart from the disease-associated polymorphisms of the prion protein, the molecular processes underlying conversion are not understood. There are some notions as to which regions of the prion protein are involved in refolding of PrP(C) into PrP(Sc) and where the most drastic structural changes take place. Direct interactions between PrP(C) molecules and/or PrP(Sc) are likely at the basis of conversion, however which specific amino acid domains are involved and to what extent these domains contribute to conversion resistance/sensitivity of the prion protein or the species barrier is still unknown. PMID:22029882
Rigter, Alan; Priem, Jan; Langeveld, Jan P M; Bossers, Alex
Signal Transduction Through Prion Protein
The cellular prion protein PrPc is a glycosylphosphatidylinositol-anchored cell-surface protein whose biological function is unclear. We used the murine 1C11 neuronal differentiation model to search for PrPc-dependent signal transduction through antibody-mediated cross-linking. A caveolin-1-dependent coupling of PrPc to the tyrosine kinase Fyn was observed. Clathrin might also contribute to this coupling. The ability of the 1C11 cell line to trigger
S. Mouillet-Richard; M. Ermonval; C. Chebassier; J. L. Laplanche; S. Lehmann; J. M. Launay; O. Kellermann
Manganese Enhances Prion Protein Survival in Model Soils and Increases Prion Infectivity to Cells
Prion diseases are considered to be transmissible. The existence of sporadic forms of prion diseases such as scrapie implies an environmental source for the infectious agent. This would suggest that under certain conditions the prion protein, the accepted agent of transmission, can survive in the environment. We have developed a novel technique to extract the prion protein from soil matrices.
Paul Davies; David R. Brown; Tsuneya Ikezu
Copper Binding in the Prion Protein�
A conformational change of the prion protein is responsible for a class of neurodegenerative diseases called the transmissible spongiform encephalopathies that include mad cow disease and the human afflictions kuru and Creutzfeldt�Jakob disease. Despite the attention given to these diseases, the normal function of the prion protein in healthy tissue is unknown. Research over the past few years, however, demonstrates that the prion protein is a copper binding protein with high selectivity for Cu2+. The structural features of the Cu2+ binding sites have now been characterized and are providing important clues about the normal function of the prion protein and perhaps how metals or loss of protein function play a role in disease. The link between prion protein and copper may provide insight into the general, and recently appreciated, role of metals in neurodegenerative disease.
Recombinant prion protein induces a new transmissible prion disease in wild-type animals
Prion disease is a neurodegenerative malady, which is believed to be transmitted via a prion protein in its abnormal conformation\\u000a (PrPSc). Previous studies have failed to demonstrate that prion disease could be induced in wild-type animals using recombinant\\u000a prion protein (rPrP) produced in Escherichia coli. Here, we report that prion infectivity was generated in Syrian hamsters after inoculating full-length rPrP
Natallia Makarava; Gabor G. Kovacs; Olga Bocharova; Regina Savtchenko; Irina Alexeeva; Herbert Budka; Robert G. Rohwer; Ilia V. Baskakov
Cyclic Amplification of Prion Protein Misfolding
Protein Misfolfing Cyclic amplification (PMCA) is a technique that take advantage of the nucleation-dependent prion replication process to accelerate the conversion of PrPC into PrPSc in the test tube. PMCA uses ultrasound waves to fragment the PrPSc polymers, increasing the amount of seeds present in the infected sample without affecting their ability to act as conversion nucleus. Over the past 5 years PMCA has became an invaluable technique to study diverse aspects of prions. The PMCA technology has been used by several groups to understand the molecular mechanism of prion replication, the cellular factors involved in prion propagation, the intriguing phenomena of prion strains and species barriers, to detect PrPSc in tissues and biological fluids and to screen for inhibitors against prion replication. In this article we describe a detailed protocol of the PMCA technique, highlighting some of the important technical aspects to obtain a successful and reproducible application of the technology.
Barria, Marcelo A; Gonzalez-Romero, Dennisse; Soto, Claudio
Protein misfolding cyclic amplification of infectious prions
Prions are proteinaceous infectious agents responsible for the transmission of prion diseases. The lack of a procedure for cultivating prions in the laboratory has been a major limitation to the study of the unorthodox nature of this infectious agent and the molecular mechanism by which the normal prion protein (PrPC) is converted into the abnormal isoform (PrPSc). Protein misfolding cyclic amplification (PMCA ), described in detail in this protocol, is a simple, fast and efficient methodology to mimic prion replication in the test tube. PMCA involves incubating materials containing minute amounts of infectious prions with an excess of PrPC and boosting the conversion by cycles of sonication to fragment the converting units, thereby leading to accelerated prion replication. PMCA is able to detect the equivalent of a single molecule of infectious PrPSc and propagate prions that maintain high infectivity, strain properties and species specificity. A single PMCA assay takes little more than 3 d to replicate a large amount of prions, which could take years in an in vivo situation. Since its invention 10 years ago, PMCA has helped to answer fundamental questions about this intriguing infectious agent and has been broadly applied in research areas that include the food industry, blood bank safety and human and veterinary disease diagnosis.
Morales, Rodrigo; Duran-Aniotz, Claudia; Diaz-Espinoza, Rodrigo; Camacho, Manuel V; Soto, Claudio
Epithelial and endothelial expression of the green fluorescent protein reporter gene under the control of bovine prion protein (PrP) gene regulatory sequences in transgenic mice
NASA Astrophysics Data System (ADS)
The expression of the cellular form of the prion protein (PrPc) gene is required for prion replication and neuroinvasion in transmissible spongiform encephalopathies. The identification of the cell types expressing PrPc is necessary to understanding how the agent replicates and spreads from peripheral sites to the central nervous system. To determine the nature of the cell types expressing PrPc, a green fluorescent protein reporter gene was expressed in transgenic mice under the control of 6.9 kb of the bovine PrP gene regulatory sequences. It was shown that the bovine PrP gene is expressed as two populations of mRNA differing by alternative splicing of one 115-bp 5' untranslated exon in 17 different bovine tissues. The analysis of transgenic mice showed reporter gene expression in some cells that have been identified as expressing PrP, such as cerebellar Purkinje cells, lymphocytes, and keratinocytes. In addition, expression of green fluorescent protein was observed in the plexus of the enteric nervous system and in a restricted subset of cells not yet clearly identified as expressing PrP: the epithelial cells of the thymic medullary and the endothelial cells of both the mucosal capillaries of the intestine and the renal capillaries. These data provide valuable information on the distribution of PrPc at the cellular level and argue for roles of the epithelial and endothelial cells in the spread of infection from the periphery to the brain. Moreover, the transgenic mice described in this paper provide a model that will allow for the study of the transcriptional activity of the PrP gene promoter in response to scrapie infection.
Lemaire-Vieille, Catherine; Schulze, Tobias; Podevin-Dimster, Val�rie; Follet, J�rome; Bailly, Yannick; Blanquet-Grossard, Fran�oise; Decavel, Jean-Pierre; Heinen, Ernst; Cesbron, Jean-Yves
Self Prion Protein Peptides are Immunogenic in Lewis Rats
Prion diseases are caused by abnormal folding of the prion protein. The paradigm is that the prion protein is not immunogenic because the immune system must be tolerant to such a self protein. In an attempt to identify immunogenic prion peptides, we immunized Lewis rats with peptides that fitted the MHC class II RT1.B1motif. Both humoral and cellular immunity to
Lina Souan; Raanan Margalit; Ori Brenner; Irun R. Cohen; Felix Mor
Did the prion protein become vulnerable to misfolding after an evolutionary divide and conquer event?
Despite high sequence identity among mammalian prion proteins (PrPs), mammals have varying rates of susceptibility to prion disease resulting in a so-called species barrier. The species barrier follows no clear pattern, with closely related species or similar sequences being no more likely to infect each other, and remains an unresolved enigma. Variation of the conformationally flexible regions may alter the thermodynamics of the conformational change, commonly referred to as the conformational conversion, which occurs in the pathogenic process of the mammalian prion protein. A conformational ensemble scenario is supported by the species barrier in prion disease and evidence that there are strains of pathogenic prion with different conformations within species. To study how conformational flexibility has evolved in the prion protein, an investigation was undertaken on the evolutionary dynamics of structurally disordered regions in the mammalian prion protein, non-mammalian prion protein that is not vulnerable to prion disease, and remote homologs Doppel and Shadoo. Structural disorder prediction analyzed in an evolutionary context revealed that the occurrence of increased or altered conformational flexibility in mammalian PrPs coincides with key events among PrP, Doppel, and Shadoo. Comparatively rapid evolutionary dynamics of conformational flexibility in the prion protein suggest that the species barrier is not a static phenomenon. A small number of amino acid substitutions can repopulate the conformational ensemble and have a disproportionately large effect on pathogenesis. PMID:23859022
Richmond, Kacy; Masterson, Patrick; Ortiz, Juan Felipe; Siltberg-Liberles, Jessica
Yeast prion architecture explains how proteins can be genes
NASA Astrophysics Data System (ADS)
Prions (infectious proteins) transmit information without an accompanying DNA or RNA. Most yeast prions are self-propagating amyloids that inactivate a normally functional protein. A single protein can become any of several prion variants, with different manifestations due to different amyloid structures. We showed that the yeast prion amyloids of Ure2p, Sup35p and Rnq1p are folded in-register parallel beta sheets using solid state NMR dipolar recoupling experiments, mass-per-filament-length measurements, and filament diameter measurements. The extent of beta sheet structure, measured by chemical shifts in solid-state NMR and acquired protease-resistance on amyloid formation, combined with the measured filament diameters, imply that the beta sheets must be folded along the long axis of the filament. We speculate that prion variants of a single protein sequence differ in the location of these folds. Favorable interactions between identical side chains must hold these structures in-register. The same interactions must guide an unstructured monomer joining the end of a filament to assume the same conformation as molecules already in the filament, with the turns at the same locations. In this way, a protein can template its own conformation, in analogy to the ability of a DNA molecule to template its sequence by specific base-pairing.
Wickner, Reed
Cytosolic Prion Protein Toxicity Is Independent of Cellular Prion Protein Expression and Prion Propagation?
Prion diseases are transmissible neurodegenerative diseases caused by a conformational isoform of the prion protein (PrP), a host-encoded cell surface sialoglycoprotein. Recent evidence suggests a cytosolic fraction of PrP (cyPrP) functions either as an initiating factor or toxic element of prion disease. When expressed in cultured cells, cyPrP acquires properties of the infectious conformation of PrP (PrPSc), including insolubility, protease resistance, aggregation, and toxicity. Transgenic mice (2D1 and 1D4 lines) that coexpress cyPrP and PrPC exhibit focal cerebellar atrophy, scratching behavior, and gait abnormalities suggestive of prion disease, although they lack protease-resistant PrP. To determine if the coexpression of PrPC is necessary or inhibitory to the phenotype of these mice, we crossed Tg1D4(Prnp+/+) mice with PrP-ablated mice (TgPrnpo/o) to generate Tg1D4(Prnpo/o) mice and followed the development of disease and pathological phenotype. We found no difference in the onset of symptoms or the clinical or pathological phenotype of disease between Tg1D4(Prnp+/+) and Tg1D4(Prnpo/o) mice, suggesting that cyPrP and PrPC function independently in the disease state. Additionally, Tg1D4(Prnpo/o) mice were resistant to challenge with mouse-adapted scrapie (RML), suggesting cyPrP is inaccessible to PrPSc. We conclude that disease phenotype and cellular toxicity associated with the expression of cyPrP are independent of PrPC and the generation of typical prion disease.
Norstrom, Eric M.; Ciaccio, Mark F.; Rassbach, Benjamin; Wollmann, Robert; Mastrianni, James A.
Mutant prion protein-mediated aggregation of normal prion protein in the endoplasmic reticulum: implications for prion propagation and neurotoxicity.
Familial prion disorders are believed to result from spontaneous conversion of mutant prion protein (PrPM) to the pathogenic isoform (PrPSc). While most familial cases are heterozygous and thus express the normal (PrPC) and mutant alleles of PrP, the role of PrPC in the pathogenic process is unclear. Plaques from affected cases reveal a heterogeneous picture; in some cases only PrPM is detected, whereas in others both PrPC and PrPM are transformed to PrPSc. To understand if the coaggregation of PrPC is governed by PrP mutations or is a consequence of the cellular compartment of PrPM aggregation, we coexpressed PrPM and PrPC in neuroblastoma cells, the latter tagged with green fluorescent protein (PrPC-GFP) for differentiation. Two PrPM forms (PrP231T, PrP217R/231T) that aggregate spontaneously in the endoplasmic reticulum (ER) were generated for this analysis. We report that PrPC-GFP aggregates when coexpressed with PrP231T or PrP217R/231T, regardless of sequence homology between the interacting forms. Furthermore, intracellular aggregates of PrP231T induce the accumulation of a C-terminal fragment of PrP, most likely derived from a potentially neurotoxic transmembrane form of PrP (CtmPrP) in the ER. These findings have implications for prion pathogenesis in familial prion disorders, especially in cases where transport of PrPM from the ER is blocked by the cellular quality control. PMID:12485397
Gu, Yaping; Verghese, Susamma; Mishra, Ravi Shankar; Xu, Xeumin; Shi, Yongchang; Singh, Neena
Regional Mapping of Prion Proteins in Brain
Scrapie is characterized by the accumulation of a protease-resistant isoform of the prion protein PrPSc. Limited proteolysis and chaotropes were used to map the distribution of PrPSc in cryostat sections of scrapie-infected brain blotted onto nitrocellulose membranes, designated histoblots. Proteolysis was omitted in order to map the cellular isoform of the prion protein (PrP^C) in uninfected brains. Compared with immunohistochemistry,
Albert Taraboulos; Klaus Jendroska; Dan Serban; Shu-Lian Yang; Stephen J. Dearmond
The role of the prion protein membrane anchor in prion infection
Normal cellular and abnormal disease-associated forms of prion protein (PrP) contain a C-terminal glycophosphatidyl-inositol (GPI) membrane anchor. The importance of the GPI membrane anchor in prion diseases is unclear but there are data to suggest that it both is and is not required for abnormal prion protein formation and prion infection. Utilizing an in vitro model of prion infection we have recently demonstrated that, while the GPI anchor is not essential for the formation of abnormal prion protein in a cell, it is necessary for the establishment of persistent prion infection. In combination with previously published data, our results suggest that GPI anchored PrP is important in the amplification and spread of prion infectivity from cell to cell.
McNally, Kristin L
Role of microglia and host prion protein in neurotoxicity of a prion protein fragment
THE prion protein PrPc is a glycoprotein of unknown function1 normally found in neurons2 and glia3. It is involved in diseases such as bovine spongiform encephalopathy (BSE), scrapie and Creutzfeldt-Jakob disease4. PrPSc, an altered isoform of PrPc that is associated with disease, shows greater protease resistance and is part of the infectious agent, the prion5,6. Prion diseases are characterized by
David R. Brown; Bernhard Schmidt; Hans A. Kretzschmar
Selective Oxidation of Methionine Residues in Prion Proteins
Prion proteins are central to the pathogenesis of several neurodegenerative diseases through the postulated conversion of the endogenous cellular isoform (PrPc) into a pathogenic isoform (PrPSc). Although the cellular function of normal prion protein remains unresolved a number of studies have shown that prion proteins may be involved in the cellular response to oxidative stress. Here, using purified recombinant sources
Boon-Seng Wong; Hui Wang; David R. Brown; Ian M. Jones
Conformational variations in an infectious protein determine prion strain differences
A remarkable feature of prion biology is the strain phenomenon wherein prion particles apparently composed of the same protein lead to phenotypically distinct transmissible states. To reconcile the existence of strains with the `protein-only' hypothesis of prion transmission, it has been proposed that a single protein can misfold into multiple distinct infectious forms, one for each different strain. Several studies
Motomasa Tanaka; Peter Chien; Nariman Naber; Roger Cooke; Jonathan S. Weissman
Interaction of metals with prion protein: Possible role of divalent cations in the pathogenesis of prion diseases
Prion diseases are fatal neurodegenerative disorders that affect both humans and animals. The rapid clinical progression, change in protein conformation, cross-species transmission and massive neuronal degeneration are some key features of this devastating degenerative condition. Although the etiology is unknown, aberrant processing of cellular prion proteins is well established in the pathogenesis of prion diseases. Normal cellular prion protein (PrPc)
Christopher J. Choi; Arthi Kanthasamy; Vellareddy Anantharam; Anumantha G. Kanthasamy
Prions and the potential transmissibility of protein misfolding diseases.
Prions, or infectious proteins, represent a major frontier in the study of infectious agents. The prions responsible for mammalian transmissible spongiform encephalopathies (TSEs) are due primarily to infectious self-propagation of misfolded prion proteins. TSE prion structures remain ill-defined, other than being highly structured, self-propagating, and often fibrillar protein multimers with the capacity to seed, or template, the conversion of their normal monomeric precursors into a pathogenic form. Purified TSE prions usually take the form of amyloid fibrils, which are self-seeding ultrastructures common to many serious protein misfolding diseases such as Alzheimer's, Parkinson's, Huntington's and Lou Gehrig's (amytrophic lateral sclerosis). Indeed, recent reports have now provided evidence of prion-like propagation of several misfolded proteins from cell to cell, if not from tissue to tissue or individual to individual. These findings raise concerns that various protein misfolding diseases might have spreading, prion-like etiologies that contribute to pathogenesis or prevalence. PMID:23808331
Kraus, Allison; Groveman, Bradley R; Caughey, Byron
Prion Strain Mutation Determined by Prion Protein Conformational Compatibility and Primary Structure
Prions are infectious proteins composed of the abnormal disease-causing isoform PrPSc, which induces conformational conversion of the host-encoded normal cellular prion protein PrPC to additional PrPSc. The mechanism underlying prion strain mutation in the absence of nucleic acids remains unresolved. Additionally, the frequency of strains causing chronic wasting disease (CWD), a burgeoning prion epidemic of cervids, is unknown. Using susceptible transgenic mice, we identified two prevalent CWD strains with divergent biological properties but composed of PrPSc with indistinguishable biochemical characteristics. Although CWD transmissions indicated stable, independent strain propagation by elk PrPC, strain coexistence in the brains of deer and transgenic mice demonstrated unstable strain propagation by deer PrPC. The primary structures of deer and elk prion proteins differ at residue 226, which, in concert with PrPSc conformational compatibility, determines prion strain mutation in these cervids.
Angers, Rachel C.; Kang, Hae-Eun; Napier, Dana; Browning, Shawn; Seward, Tanya; Mathiason, Candace; Balachandran, Aru; McKenzie, Debbie; Castilla, Joaquin; Soto, Claudio; Jewell, Jean; Graham, Catherine; Hoover, Edward A.; Telling, Glenn C.
Interaction Networks of Prion, Prionogenic and Prion-Like Proteins in Budding Yeast, and Their Role in Gene Regulation
Prions are transmissible, propagating alternative states of proteins. Prions in budding yeast propagate heritable phenotypes and can function in large-scale gene regulation, or in some cases occur as diseases of yeast. Other �prionogenic� proteins are likely prions that have been determined experimentally to form amyloid in vivo, and to have prion-like domains that are able to propagate heritable states. Furthermore, there are over 300 additional �prion-like� yeast proteins that have similar amino-acid composition to prions (primarily a bias for asparagines and glutamines). Here, we examine the protein functional and interaction networks that involve prion, prionogenic and prion-like proteins. Set against a marked overall preference for N/Q-rich prion-like proteins not to interact with each other, we observe a significant tendency of prion/prionogenic proteins to interact with other, N/Q-rich prion-like proteins. This tendency is mostly due to a small number of networks involving the proteins NUP100p, LSM4p and PUB1p. In general, different data analyses of functional and interaction networks converge to indicate a strong linkage of prionogenic and prion-like proteins, to stress-granule assembly and related biological processes. These results further elucidate how prions may impact gene regulation, and reveal a broader horizon for the functional relevance of N/Q-rich prion-like domains.
Harbi, Djamel; Harrison, Paul M.
Role of Prion Protein Aggregation in Neurotoxicity
In several neurodegenerative diseases, such as Parkinson, Alzheimer�s, Huntington, and prion diseases, the deposition of aggregated misfolded proteins is believed to be responsible for the neurotoxicity that characterizes these diseases. Prion protein (PrP), the protein responsible of prion diseases, has been deeply studied for the peculiar feature of its misfolded oligomers that are able to propagate within affected brains, inducing the conversion of the natively folded PrP into the pathological conformation. In this review, we summarize the available experimental evidence concerning the relationship between aggregation status of misfolded PrP and neuronal death in the course of prion diseases. In particular, we describe the main findings resulting from the use of different synthetic (mainly PrP106-126) and recombinant PrP-derived peptides, as far as mechanisms of aggregation and amyloid formation, and how these different spatial conformations can affect neuronal death. In particular, most data support the involvement of non-fibrillar oligomers rather than actual amyloid fibers as the determinant of neuronal death.
Corsaro, Alessandro; Thellung, Stefano; Villa, Valentina; Nizzari, Mario; Florio, Tullio
Copper Binding to the N-Terminal Tandem Repeat Regions of Mammalian and Avian Prion Protein
Mammalian prion protein (PrP) is a normal cellular protein (PrPc) which through post-translational modification produces the infectious prion protein (PrPsc). We have shown, using mass spectrometry, that synthetic peptides containing three or four copies of an octapeptide repeat sequence (PHGGGWGQ), found in a highly conserved N-terminal domain of PrP, preferentially bind copper over other metals. Peptides from the analogous region
M. P. Hornshaw; J. R. Mcdermott; J. M. Candy
Prion biology problem space: Mad cows, itchy sheep and protein structure
NSDL National Science Digital Library
Prions are infectious protein particles associated with various neurodegenerative diseases in humans and animals. This problem space introduces basic skills in protein structure and sequence exploration that can be used to analyze some unusual properties of prions, and develop testable hypotheses that can be explored through these tools. Students will be challenged to use a systems-biology approach to link structure, evolution and function of proteins.
Sebrenka Robic (Agnes Scott College;); Maryam Qureshi (Boston University;)
Prion biology problem space: Mad cows, itchy sheep and protein structure
NSDL National Science Digital Library
Prions are infectious protein particles associated with various neurodegenerative diseases in humans and animals. This problem space introduces basic skills in protein structure and sequence exploration that can be used to analyze some unusual properties of prions, and develop testable hypotheses that can be explored through these tools. Students will be challenged to use a systems-biology approach to link structure, evolution and function of proteins.
Sebrenka Robic (Agnes Scott College;Biology); Maryam Qureshi (Boston University;)
By biological and medical criteria, prions are infectious agents; however, many of their properties differ profoundly from those of conventional microbes. Prions are "encoded" by alterations in protein conformation rather than in nucleic acid or amino acid sequence. New epidemic prion diseases (bovine spongiform encephalopathy and new variant Creutzfeldt-Jakob disease) have recently emerged under the active surveillance of the modern world. The risk of contracting prion disease from blood products or other biologicals is now a focus of worldwide concern. Much has been discovered about prions and prion diseases, but much remains to be done.
Cashman, N R
A prion protein epitope selective for the pathologically misfolded conformation
Conformational conversion of proteins in disease is likely to be accompanied by molecular surface exposure of previously sequestered amino-acid side chains. We found that induction of ?-sheet structures in recombinant prion proteins is associated with increased solvent accessibility of tyrosine. Antibodies directed against the prion protein repeat motif, tyrosine-tyrosine-arginine, recognize the pathological isoform of the prion protein but not the
Eustache Paramithiotis; Marc Pinard; Trebor Lawton; Sylvie LaBoissiere; Valerie L Leathers; Wen-Quan Zou; Lisa A Estey; Julie Lamontagne; Marty T Lehto; Leslie H Kondejewski; Gregory P Francoeur; Maria Papadopoulos; Ashkan Haghighat; Stephen J Spatz; Mark Head; Robert Will; James Ironside; Katherine O'Rourke; Quentin Tonelli; Harry C Ledebur; Avi Chakrabartty; Neil R Cashman
Prion protein interaction with soil humic substances: environmental implications.
Transmissible spongiform encephalopathies (TSE) are fatal neurodegenerative disorders caused by prions. Animal TSE include scrapie in sheep and goats, and chronic wasting disease (CWD) in cervids. Effective management of scrapie in many parts of the world, and of CWD in North American deer population is complicated by the persistence of prions in the environment. After shedding from diseased animals, prions persist in soil, withstanding biotic and abiotic degradation. As soil is a complex, multi-component system of both mineral and organic components, it is important to understand which soil compounds may interact with prions and thus contribute to disease transmission. Several studies have investigated the role of different soil minerals in prion adsorption and infectivity; we focused our attention on the interaction of soil organic components, the humic substances (HS), with recombinant prion protein (recPrP) material. We evaluated the kinetics of recPrP adsorption, providing a structural and biochemical characterization of chemical adducts using different experimental approaches. Here we show that HS act as potent anti-prion agents in prion infected neuronal cells and in the amyloid seeding assays: HS adsorb both recPrP and prions, thus sequestering them from the prion replication process. We interpreted our findings as highly relevant from an environmental point of view, as the adsorption of prions in HS may affect their availability and consequently hinder the environmental transmission of prion diseases in ruminants. PMID:24937266
Giachin, Gabriele; Narkiewicz, Joanna; Scaini, Denis; Ngoc, Ai Tran; Margon, Alja; Sequi, Paolo; Leita, Liviana; Legname, Giuseppe
Prion Protein Interaction with Soil Humic Substances: Environmental Implications
Transmissible spongiform encephalopathies (TSE) are fatal neurodegenerative disorders caused by prions. Animal TSE include scrapie in sheep and goats, and chronic wasting disease (CWD) in cervids. Effective management of scrapie in many parts of the world, and of CWD in North American deer population is complicated by the persistence of prions in the environment. After shedding from diseased animals, prions persist in soil, withstanding biotic and abiotic degradation. As soil is a complex, multi-component system of both mineral and organic components, it is important to understand which soil compounds may interact with prions and thus contribute to disease transmission. Several studies have investigated the role of different soil minerals in prion adsorption and infectivity; we focused our attention on the interaction of soil organic components, the humic substances (HS), with recombinant prion protein (recPrP) material. We evaluated the kinetics of recPrP adsorption, providing a structural and biochemical characterization of chemical adducts using different experimental approaches. Here we show that HS act as potent anti-prion agents in prion infected neuronal cells and in the amyloid seeding assays: HS adsorb both recPrP and prions, thus sequestering them from the prion replication process. We interpreted our findings as highly relevant from an environmental point of view, as the adsorption of prions in HS may affect their availability and consequently hinder the environmental transmission of prion diseases in ruminants.
Giachin, Gabriele; Narkiewicz, Joanna; Scaini, Denis; Ngoc, Ai Tran; Margon, Alja; Sequi, Paolo; Leita, Liviana; Legname, Giuseppe
Ion channels induced by the prion protein
Prion diseases comprise a group of rapidly progressive and invariably fatal neurodegenerative disorders for which there are no effective treatments. While conversion of the cellular prion protein (PrPC) to a ?-sheet rich isoform (PrPSc) is known to be a critical event in propagation of infectious prions, the identity of the neurotoxic form of PrP and its mechanism of action remain unclear. Insights into this mechanism have been provided by studying PrP molecules harboring deletions and point mutations in the conserved central region, encompassing residues 105�125. When expressed in transgenic mice, PrP deleted for these residues (?105�125) causes a spontaneous neurodegenerative illness that is reversed by co-expression of wild-type PrP. In cultured cells, ?105�125 PrP confers hypersensitivity to certain cationic antibiotics and induces spontaneous ion channel activity that can be recorded by electrophysiological techniques. We have utilized these drug-hypersensitization and current-inducing activities to identify which PrP domains and subcellular locations are required for toxicity. We present an ion channel model for the toxicity of ?105�125 PrP and related mutants and speculate how a similar mechanism could mediate PrPSc-associated toxicity. Therapeutic regimens designed to inhibit prion-induced toxicity, as well as formation of PrPSc, may prove to be the most clinically beneficial.
Solomon, Isaac H; Biasini, Emiliano
Unique quadruplex structure and interaction of an RNA aptamer against bovine prion protein
RNA aptamers against bovine prion protein (bPrP) were obtained, most of the obtained aptamers being found to contain the r(GGAGGAGGAGGA) (R12) sequence. Then, it was revealed that R12 binds to both bPrP and its b-isoform with high affinity. Here, we present the structure of R12. This is the first report on the structure of an RNA aptamer against prion protein.
Tsukasa Mashima; Akimasa Matsugami; Fumiko Nishikawa; Satoshi Nishikawa; Masato Katahira
Neurotoxicity of a prion protein fragment
THE cellular prion protein (PrPc) is a sialoglycoprotein of Mr 33-35K that is expressed predominantly in neurons1-3. In transmissible and genetic neurodegenerative disorders such as scrapie of sheep, spongiform encephalopathy of cattle and Creutzfeldt-Jakob or Gerstmann-Str�ussler-Scheinker diseases of humans4,5, PrPc is converted into an altered form (termed PrPSc) which is distinguishable from its normal homologue by its relative resistance to
Gianluigi Forloni; Nadia Angeretti; Roberto Chiesa; Enrico Monzani; Mario Salmona; Orso Bugiani; Fabrizio Tagliavini
Evolutionary Implications of Metal Binding Features in Different Species' Prion Protein: An Inorganic Point of View
Prion disorders are a group of fatal neurodegenerative conditions of mammals. The key molecular event in the pathogenesis of such diseases is the conformational conversion of prion protein, PrPC, into a misfolded form rich in ?-sheet structure, PrPSc, but the detailed mechanistic aspects of prion protein conversion remain enigmatic. There is uncertainty on the precise physiological function of PrPC in healthy individuals. Several evidences support the notion of its role in copper homeostasis. PrPC binds Cu2+ mainly through a domain composed by four to five repeats of eight amino acids. In addition to mammals, PrP homologues have also been identified in birds, reptiles, amphibians and fish. The globular domain of protein is retained in the different species, suggesting that the protein carries out an essential common function. However, the comparison of amino acid sequences indicates that prion protein has evolved differently in each vertebrate class. The primary sequences are strongly conserved in each group, but these exhibit a low similarity with those of mammals. The N-terminal domain of different prions shows tandem amino acid repeats with an increasing amount of histidine residues going from amphibians to mammals. The difference in the sequence affects the number of copper binding sites, the affinity and the coordination environment of metal ions, suggesting that the involvement of prion in metal homeostasis may be a specific characteristic of mammalian prion protein. In this review, we describe the similarities and the differences in the metal binding of different species� prion protein, as revealed by studies carried out on the entire protein and related peptide fragments.
La Mendola, Diego; Rizzarelli, Enrico
Recombinant prion protein induces a new transmissible prion disease in wild-type animals.
Prion disease is a neurodegenerative malady, which is believed to be transmitted via a prion protein in its abnormal conformation (PrP(Sc)). Previous studies have failed to demonstrate that prion disease could be induced in wild-type animals using recombinant prion protein (rPrP) produced in Escherichia coli. Here, we report that prion infectivity was generated in Syrian hamsters after inoculating full-length rPrP that had been converted into the cross-beta-sheet amyloid form and subjected to annealing. Serial transmission gave rise to a disease phenotype with highly unique clinical and neuropathological features. Among them were the deposition of large PrP(Sc) plaques in subpial and subependymal areas in brain and spinal cord, very minor lesioning of the hippocampus and cerebellum, and a very slow progression of disease after onset of clinical signs despite the accumulation of large amounts of PrP(Sc) in the brain. The length of the clinical duration is more typical of human and large animal prion diseases, than those of rodents. Our studies establish that transmissible prion disease can be induced in wild-type animals by inoculation of rPrP and introduce a valuable new model of prion diseases. PMID:20052481
Makarava, Natallia; Kovacs, Gabor G; Bocharova, Olga; Savtchenko, Regina; Alexeeva, Irina; Budka, Herbert; Rohwer, Robert G; Baskakov, Ilia V
Cellular prion protein and Alzheimer disease
Soluble oligomeric amyloid-? (A?) has been suggested to impair synaptic and neuronal function, leading to neurodegeneration that is clinically observed as the memory and cognitive dysfunction characteristic of Alzheimer disease, while the precise mechanism(s) whereby oligomeric A? causes neurotoxicity remains unknown. Recently, the cellular prion protein (PrPC) was reported to be an essential co-factor in mediating the neurotoxic effect of oligomeric A?. Our recent study showed that Prnp?/? mice are resistant to the neurotoxic effect of oligomeric A? in vivo and in vitro. Furthermore, application of an anti-PrPC antibody or PrPC peptide was able to block oligomeric A?-induced neurotoxicity. These findings demonstrate that PrPC may be involved in neuropathologic conditions other than conventional prion diseases, i.e., Creutzfeldt-Jakob disease.
Kudo, Wataru; Petersen, Robert B.
Scrapie prion protein accumulation by scrapie-infected neuroblastoma cells abrogated by exposure to a prion protein antibody
Exposure of susceptible neuroblastoma N2a cells to mouse scrapie prions leads to infection, as evidenced by the continued presence of the scrapie form of the prion protein (PrPSc) and infectivity after 300 or more cell doublings. We find that exposure to phosphatidylinositol-specific phospholipase C (PIPLC) or to the monoclonal anti-prion protein (PrP) antibody 6H4 not only prevents infection of susceptible
Masato Enari; Eckhard Flechsig; Charles Weissmann
Ultraviolet-ozone treatment reduces levels of disease-associated prion protein and prion infectivity
Background. Transmissible spongiform encephalopathies (TSEs) are a group of fatal neurodegenerative diseases caused by novel infectious agents referred to as prions. Prions appear to be composed primarily, if not exclusively, of a misfolded isoform of the cellular prion protein. TSE infectivity is remarkably stable and can resist many aggressive decontamination procedures, increasing human, livestock and wildlife exposure to TSEs. Findings. We tested the hypothesis that UV-ozone treatment reduces levels of the pathogenic prion protein and inactivates the infectious agent. We found that UV-ozone treatment decreased the carbon and prion protein content in infected brain homogenate to levels undetectable by dry-ashing carbon analysis or immunoblotting, respectively. After 8 weeks of ashing, UV-ozone treatment reduced the infectious titer of treated material by a factor of at least 105. A small amount of infectivity, however, persisted despite UV-ozone treatment. When bound to either montmorillonite clay or quartz surfaces, PrPTSE was still susceptible to degradation by UV-ozone. Conclusion. Our findings strongly suggest that UV-ozone treatment can degrade pathogenic prion protein and inactivate prions, even when the agent is associated with surfaces. Using larger UV-ozone doses or combining UV-ozone treatment with other decontaminant methods may allow the sterilization of TSE-contaminated materials. ?? 2009 Aiken et al; licensee BioMed Central Ltd.
Johnson, C. J.; Gilbert, P.; McKenzie, D.; Pedersen, J. A.; Aiken, J. M.
Mechanisms of prion protein assembly into amyloid.
The conversion of the alpha-helical, cellular isoform of the prion protein (PrP(C)) to the insoluble, beta-sheet-rich, infectious, disease-causing isoform (PrP(Sc)) is the key event in prion diseases. In an earlier study, several forms of PrP were converted into a fibrillar state by using an in vitro conversion system consisting of low concentrations of SDS and 250 mM NaCl. Here, we characterize the structure of the fibril precursor state, that is, the soluble state under fibrillization conditions. CD spectroscopy, analytical ultracentrifugation, and chemical cross-linking indicate that the precursor state exists in a monomer-dimer equilibrium of partially denatured, alpha-helical PrP, with a well defined contact site of the subunits in the dimer. Using fluorescence with thioflavin T, we monitored and quantitatively described the kinetics of seeded fibril formation, including dependence of the reaction on substrate and seed concentrations. Exponential, seed-enhanced growth can be achieved in homogeneous solution, which can be enhanced by sonication. From these data, we propose a mechanistic model of fibrillization, including the presence of several intermediate structures. These studies also provide a simplified amplification system for prions. PMID:18268326
St�hr, Jan; Weinmann, Nicole; Wille, Holger; Kaimann, Tina; Nagel-Steger, Luitgard; Birkmann, Eva; Panza, Giannantonio; Prusiner, Stanley B; Eigen, Manfred; Riesner, Detlev
Mechanisms of prion protein assembly into amyloid
The conversion of the ?-helical, cellular isoform of the prion protein (PrPC) to the insoluble, ?-sheet-rich, infectious, disease-causing isoform (PrPSc) is the key event in prion diseases. In an earlier study, several forms of PrP were converted into a fibrillar state by using an in vitro conversion system consisting of low concentrations of SDS and 250 mM NaCl. Here, we characterize the structure of the fibril precursor state, that is, the soluble state under fibrillization conditions. CD spectroscopy, analytical ultracentrifugation, and chemical cross-linking indicate that the precursor state exists in a monomer-dimer equilibrium of partially denatured, ?-helical PrP, with a well defined contact site of the subunits in the dimer. Using fluorescence with thioflavin T, we monitored and quantitatively described the kinetics of seeded fibril formation, including dependence of the reaction on substrate and seed concentrations. Exponential, seed-enhanced growth can be achieved in homogeneous solution, which can be enhanced by sonication. From these data, we propose a mechanistic model of fibrillization, including the presence of several intermediate structures. These studies also provide a simplified amplification system for prions.
Stohr, Jan; Weinmann, Nicole; Wille, Holger; Kaimann, Tina; Nagel-Steger, Luitgard; Birkmann, Eva; Panza, Giannantonio; Prusiner, Stanley B.; Eigen, Manfred; Riesner, Detlev
Spontaneous Neurodegeneration in Transgenic Mice with Mutant Prion Protein
Transgenic mice were created to assess genetic linkage between Gerstmann-Straussler-Scheinker syndrome and a leucine substitution at codon 102 of the human prion protein gene. Spontaneous neurologic disease with spongiform degeneration and gliosis similar to that in mouse scrapie developed at a mean age of 166 days in 35 mice expressing mouse prion protein with the leucine substitution. Thus, many of
Karen K. Hsiao; Michael Scott; Dallas Foster; Darlene F. Groth; Stephen J. Dearmond
The complete CDS of the prion protein (PRNP) gene of African lion (Panthera leo).
We provide the complete PRNP CDS sequence for the African lion, which is different from the previously published sequence and more similar to other carnivore sequences. The newly obtained prion protein sequence differs from the domestic cat sequence at three amino acid positions and contains only four octapeptide repeats. We recommend that this sequence be used as the reference sequence for future studies of the PRNP gene for this species. PMID:18256917
Maj, Andrzej; Spellman, Garth M; Sarver, Shane K
Protease-Resistant Prions Selectively Decrease Shadoo Protein
The central event in prion diseases is the conformational conversion of the cellular prion protein (PrPC) into PrPSc, a partially protease-resistant and infectious conformer. However, the mechanism by which PrPSc causes neuronal dysfunction remains poorly understood. Levels of Shadoo (Sho), a protein that resembles the flexibly disordered N-terminal domain of PrPC, were found to be reduced in the brains of mice infected with the RML strain of prions [1], implying that Sho levels may reflect the presence of PrPSc in the brain. To test this hypothesis, we examined levels of Sho during prion infection using a variety of experimental systems. Sho protein levels were decreased in the brains of mice, hamsters, voles, and sheep infected with different natural and experimental prion strains. Furthermore, Sho levels were decreased in the brains of prion-infected, transgenic mice overexpressing Sho and in infected neuroblastoma cells. Time-course experiments revealed that Sho levels were inversely proportional to levels of protease-resistant PrPSc. Membrane anchoring and the N-terminal domain of PrP both influenced the inverse relationship between Sho and PrPSc. Although increased Sho levels had no discernible effect on prion replication in mice, we conclude that Sho is the first non-PrP marker specific for prion disease. Additional studies using this paradigm may provide insight into the cellular pathways and systems subverted by PrPSc during prion disease.
Watts, Joel C.; Stohr, Jan; Bhardwaj, Sumita; Wille, Holger; Oehler, Abby; DeArmond, Stephen J.; Giles, Kurt; Prusiner, Stanley B.
Attachment of pathogenic prion protein to model oxide surfaces.
Prions are the infectious agents in the class of fatal neurodegenerative diseases known as transmissible spongiform encephalopathies, which affect humans, deer, sheep, and cattle. Prion diseases of deer and sheep can be transmitted via environmental routes, and soil is has been implicated in the transmission of these diseases. Interaction with soil particles is expected to govern the transport, bioavailability and persistence of prions in soil environments. A mechanistic understanding of prion interaction with soil components is critical for understanding the behavior of these proteins in the environment. Here, we report results of a study to investigate the interactions of prions with model oxide surfaces (Al2O3, SiO2) using quartz crystal microbalance with dissipation monitoring and optical waveguide light mode spectroscopy. The efficiency of prion attachment to Al2O3 and SiO2 depended strongly on pH and ionic strength in a manner consistent with electrostatic forces dominating interaction with these oxides. The presence of the N-terminal portion of the protein appeared to promote attachment to Al2O3 under globally electrostatically repulsive conditions. We evaluated the utility of recombinant prion protein as a surrogate for prions in attachment experiments and found that its behavior differed markedly from that of the infectious agent. Our findings suggest that prions would tend to associate with positively charged mineral surfaces in soils (e.g., iron and aluminum oxides). PMID:23611152
Jacobson, Kurt H; Kuech, Thomas R; Pedersen, Joel A
Prion protein induced signaling cascades in monocytes
Prion proteins play a central role in transmission and pathogenesis of transmissible spongiform encephalopathies. The cellular prion protein (PrP{sup C}), whose physiological function remains elusive, is anchored to the surface of a variety of cell types including neurons and cells of the lymphoreticular system. In this study, we investigated the response of a mouse monocyte/macrophage cell line to exposure with PrP{sup C} fusion proteins synthesized with a human Fc-tag. PrP{sup C} fusion proteins showed an attachment to the surface of monocyte/macrophages in nanomolar concentrations. This was accompanied by an increase of cellular tyrosine phosphorylation as a result of activated signaling pathways. Detailed investigations exhibited activation of downstream pathways through a stimulation with PrP fusion proteins, which include phosphorylation of ERK{sub 1,2} and Akt kinase. Macrophages opsonize and present antigenic structures, contact lymphocytes, and deliver cytokines. The findings reported here may become the basis of understanding the molecular function of PrP{sup C} in monocytes and macrophages.
Krebs, Bjarne [Center for Neuropathology and Prion Research, Ludwig-Maximilians-University Munich, Muenchen (Germany); Dorner-Ciossek, Cornelia [CNS Research III, Boehringer Ingelheim Pharma GmbH and Co KG, Biberach/Riss (Germany); Schmalzbauer, Ruediger [Center for Neuropathology and Prion Research, Ludwig-Maximilians-University Munich, Munich (Germany); Vassallo, Neville [Department of Physiology and Biochemistry, University of Malta, Msida (Malta); Herms, Jochen [Center for Neuropathology and Prion Research, Ludwig-Maximilians-University Munich, Munich (Germany); Kretzschmar, Hans A. [Center for Neuropathology and Prion Research, Ludwig-Maximilians-University Munich, Munich (Germany)]. E-mail: email@example.com
Prion protein induced signaling cascades in monocytes.
Prion proteins play a central role in transmission and pathogenesis of transmissible spongiform encephalopathies. The cellular prion protein (PrP(C)), whose physiological function remains elusive, is anchored to the surface of a variety of cell types including neurons and cells of the lymphoreticular system. In this study, we investigated the response of a mouse monocyte/macrophage cell line to exposure with PrP(C) fusion proteins synthesized with a human Fc-tag. PrP(C) fusion proteins showed an attachment to the surface of monocyte/macrophages in nanomolar concentrations. This was accompanied by an increase of cellular tyrosine phosphorylation as a result of activated signaling pathways. Detailed investigations exhibited activation of downstream pathways through a stimulation with PrP fusion proteins, which include phosphorylation of ERK(1,2) and Akt kinase. Macrophages opsonize and present antigenic structures, contact lymphocytes, and deliver cytokines. The findings reported here may become the basis of understanding the molecular function of PrP(C) in monocytes and macrophages. PMID:16343423
Krebs, Bjarne; Dorner-Ciossek, Cornelia; Schmalzbauer, R�diger; Vassallo, Neville; Herms, Jochen; Kretzschmar, Hans A
Prion neuropathology follows the accumulation of alternate prion protein isoforms after infective titre has peaked
Prions are lethal infectious agents thought to consist of multi-chain forms (PrPSc) of misfolded cellular prion protein (PrPC). Prion propagation proceeds in two distinct mechanistic phases: an exponential phase 1, which rapidly reaches a fixed level of infectivity irrespective of PrPC expression level, and a plateau (phase 2), which continues until clinical onset with duration inversely proportional to PrPC expression level. We hypothesized that neurotoxicity relates to distinct neurotoxic species produced following a pathway switch when prion levels saturate. Here we show a linear increase of proteinase K-sensitive PrP isoforms distinct from classical PrPSc at a rate proportional to PrPC concentration, commencing at the phase transition and rising until clinical onset. The unaltered level of total PrP during phase 1, when prion infectivity increases a million-fold, indicates that prions comprise a small minority of total PrP. This is consistent with PrPC concentration not being rate limiting to exponential prion propagation and neurotoxicity relating to critical concentrations of alternate PrP isoforms whose production is PrPC concentration dependent.
Sandberg, Malin K.; Al-Doujaily, Huda; Sharps, Bernadette; De Oliveira, Michael Wiggins; Schmidt, Christian; Richard-Londt, Angela; Lyall, Sarah; Linehan, Jacqueline M.; Brandner, Sebastian; Wadsworth, Jonathan D. F.; Clarke, Anthony R.; Collinge, John
Live-cell FRET imaging reveals clustering of the prion protein at the cell surface induced by infectious prions.
Prion diseases are associated to the conversion of the prion protein into a misfolded pathological isoform. The mechanism of propagation of protein misfolding by protein templating remains largely unknown. Neuroblastoma cells were transfected with constructs of the prion protein fused to both CFP-GPI-anchored and to YFP-GPI-anchored and directed to its cell membrane location. Live-cell FRET imaging between the prion protein fused to CFP or YFP was measured giving consistent values of 10�2%. This result was confirmed by fluorescence lifetime imaging microscopy and indicates intermolecular interactions between neighbor prion proteins. In particular, considering that a maximum FRET efficiency of 17�2% was determined from a positive control consisting of a fusion CFP-YFP-GPI-anchored. A stable cell clone expressing the two fusions containing the prion protein was also selected to minimize cell-to-cell variability. In both, stable and transiently transfected cells, the FRET efficiency consistently increased in the presence of infectious prions - from 4�1% to 7�1% in the stable clone and from 10�2% to 16�1% in transiently transfected cells. These results clearly reflect an increased clustering of the prion protein on the membrane in the presence of infectious prions, which was not observed in negative control using constructs without the prion protein and upon addition of non-infected brain. Our data corroborates the recent view that the primary site for prion conversion is the cell membrane. Since our fluorescent cell clone is not susceptible to propagate infectivity, we hypothesize that the initial event of prion infectivity might be the clustering of the GPI-anchored prion protein. PMID:24530636
Tavares, Evandro; Macedo, Joana A; Paulo, Pedro M R; Tavares, Catarina; Lopes, Carlos; Melo, Eduardo P
Shadoo, a new protein highly conserved from fish to mammals and with similarity to prion protein
We report evidence from cDNA isolation and expression analysis as well as analyses of genome, expressed sequence tag (EST), cDNA and expression databases for a new gene named SPRN (shadow of prion protein). SPRN comprises two exons, with the open reading frame (ORF) contained within exon 2, and codes for a protein of 130�150 amino acids named Shadoo (Japanese shadow),
Marko Premzl; Lorenzo Sangiorgio; Bice Strumbo; Jennifer A Marshall Graves; Tatjana Simonic; Jill E Gready
SSCP analysis and sequencing of the human prion protein gene (PRNP) detects two different 24 bp deletions in an atypical Alzheimer`s disease family
Alzheimer`s disease (AD) is a progressive, degenerative neurological disorder of the central nervous system. AD is the fourth leading cause of death in elderly persons 65 years or older in Western industrialized societies. The etiology of AD is unknown, but clinical, pathological, epidemiological, and molecular investigations suggest it is etiologically heterogeneous. Mutations in the amyloid protein are rare and segregate with the disease in a few early-onset familial AD (FAD) families. Similarities between AD and the unconventional viral (UCV) diseases, and between the amyloid and prion proteins, implicate the human prion protein gene (PRNP) as another candidate gene. Single strand conformation polymorphism (SSCP) analysis was used to screen for mutations at this locus in 82 AD patients from 54 families (30 FAD), vs. 39 age-matched controls. A 24-bp deletion around codon 68 that codes for one of five Gly-Pro rich octarepeats was identified in two affected sibs and one offspring of one late-onset FAD family. Two other affected sibs, three unaffected sibs, and three offspring from this family, in addition to one sporadic AD patient and three age-matched controls, were heterozygous for another octarepeat deletion located around codon 82. Two of the four affected sibs had features of PD, including one who was autopsy-verified AD and PD. Although these deletions were found infrequently in other AD patients and controls, they appear to be a rare polymorphism that is segregating in this FAD family. It does not appear that mutations at the PRNP locus are frequently associated with AD in this population. 54 refs., 4 figs.
Perry, R.T.; Go, R.C.P.; Harrell, L.E.; Acton, R.T. [Univ. of Alabama, Birmingham, AL (United States)
Prions consist mainly, if not entirely, of PrPSc, an aggregated conformer of the host protein PrPC. Prions come in different strains, all based on the same PrPC sequence, but differing in their conformations. The efficiency of prion transmission between species is usually low, but increases after serial transmission in the new host, suggesting a process involving mutation and selection. Even within the same species, the transfer of prions between cell types entails a selection of favoured 'substrains', and propagation of prions in the presence of an inhibitory drug can result in the appearance of drug-resistant prion populations. We propose that prion populations are comprised of a variety of conformers, constituting 'quasi-species', from which the one replicating most efficiently in a particular environment is selected.
Weissmann, Charles; Li, Jiali; Mahal, Sukhvir P.; Browning, Shawn
Prions consist mainly, if not entirely, of PrP(Sc), an aggregated conformer of the host protein PrP(C). Prions come in different strains, all based on the same PrP(C) sequence, but differing in their conformations. The efficiency of prion transmission between species is usually low, but increases after serial transmission in the new host, suggesting a process involving mutation and selection. Even within the same species, the transfer of prions between cell types entails a selection of favoured 'substrains', and propagation of prions in the presence of an inhibitory drug can result in the appearance of drug-resistant prion populations. We propose that prion populations are comprised of a variety of conformers, constituting 'quasi-species', from which the one replicating most efficiently in a particular environment is selected. PMID:21997298
Weissmann, Charles; Li, Jiali; Mahal, Sukhvir P; Browning, Shawn
Evidence for Protein X Binding to a Discontinuous Epitope on the Cellular Prion Protein during Scrapie Prion Propagation
Studies on the transmission of human (Hu) prions to transgenic (Tg) mice suggested that another molecule provisionally designated protein X participates in the formation of nascent scrapie isoform of prion protein (PrPSc). We report the identification of the site at which protein X binds to the cellular isoform of PrP (PrPC) using scrapie-infected mouse (Mo) neuroblastoma cells transfected with chimeric
Kiyotoshi Kaneko; Laurence Zulianello; Michael Scott; Carol M. Cooper; Andrew C. Wallace; Thomas L. James; Fred E. Cohen
Normal host prion protein necessary for scrapie-induced neurotoxicity
ACCUMULATION of the prion protein PrPSc, a pathological and protease-resistant isoform of the normal host protein PrPc, is a feature of prion disease such as scrapie1,2. It is still unknown whether scrapie pathology comes about by neurotoxicity of PrPSc, acute depletion of PrPc, or some other mechanism. Here we investigate this question by grafting neural tissue overexpressing PrPc into the
Sebastian Brandner; Stefan Isenmann; Alex Raeber; Marek Fischer; Andreas Sailer; Yasushi Kobayashi; Silvia Marino; Charles Weissmann; Adriano Aguzzi
Prevalence of lymphoreticular prion protein accumulation in UK tissue samples
This study aims to provide an estimate of the number of individuals in the UK who may be incubating variant Creutzfeldt-Jakob disease and at risk of causing iatrogenic spread of the disease. Lymphoreticular accumulation of prion protein is a consistent feature of variant Creutzfeldt-Jakob at autopsy and has also been demonstrated in the pre-clinical phase. Immunohistochemical accumulation of prion protein
David A Hilton; Azra C Ghani; Lisa Conyers; Philip Edwards; Linda McCardle; Diane Ritchie; Mark Penney; Doha Hegazy; James W Ironside
Structural studies of the scrapie prion protein by electron crystallography
Because the insolubility of the scrapie prion protein (PrPSc) has frustrated structural studies by x-ray crystallography or NMR spectroscopy, we used electron crystallography to characterize the structure of two infectious variants of the prion protein. Isomorphous two-dimensional crystals of the N-terminally truncated PrPSc (PrP 27-30) and a miniprion (PrPSc106) were identified by negative stain electron microscopy. Image processing allowed the
Holger Wille; Melissa D. Michelitsch; Vincent Gu�nebaut; Surachai Supattapone; Ana Serban; Fred E. Cohen; David A. Agard
The cellular prion protein mediates neurotoxic signalling of ?-sheet-rich conformers independent of prion replication
Formation of aberrant protein conformers is a common pathological denominator of different neurodegenerative disorders, such as Alzheimer's disease or prion diseases. Moreover, increasing evidence indicates that soluble oligomers are associated with early pathological alterations and that oligomeric assemblies of different disease-associated proteins may share common structural features. Previous studies revealed that toxic effects of the scrapie prion protein (PrPSc), a ?-sheet-rich isoform of the cellular PrP (PrPC), are dependent on neuronal expression of PrPC. In this study, we demonstrate that PrPC has a more general effect in mediating neurotoxic signalling by sensitizing cells to toxic effects of various ?-sheet-rich (?) conformers of completely different origins, formed by (i) heterologous PrP, (ii) amyloid ?-peptide, (iii) yeast prion proteins or (iv) designed ?-peptides. Toxic signalling via PrPC requires the intrinsically disordered N-terminal domain (N-PrP) and the GPI anchor of PrP. We found that the N-terminal domain is important for mediating the interaction of PrPC with ?-conformers. Interestingly, a secreted version of N-PrP associated with ?-conformers and antagonized their toxic signalling via PrPC. Moreover, PrPC-mediated toxic signalling could be blocked by an NMDA receptor antagonist or an oligomer-specific antibody. Our study indicates that PrPC can mediate toxic signalling of various ?-sheet-rich conformers independent of infectious prion propagation, suggesting a pathophysiological role of the prion protein beyond of prion diseases.
Resenberger, Ulrike K; Harmeier, Anja; Woerner, Andreas C; Goodman, Jessica L; Muller, Veronika; Krishnan, Rajaraman; Vabulas, R Martin; Kretzschmar, Hans A; Lindquist, Susan; Hartl, F Ulrich; Multhaup, Gerd; Winklhofer, Konstanze F; Tatzelt, Jorg
Persistence of pathogenic prion protein during simulated wastewater treatment processes
Transmissible spongiform encephalopathies (TSEs, prion diseases) are a class of fatal neurodegenerative diseases affecting a variety of mammalian species including humans. A misfolded form of the prion protein (PrP TSE) is the major, if not sole, component of the infectious agent. Prions are highly resistant to degradation and to many disinfection procedures suggesting that, if prions enter wastewater treatment systems through sewers and/or septic systems (e.g., from slaughterhouses, necropsy laboratories, rural meat processors, private game dressing) or through leachate from landfills that have received TSE-contaminated material, prions could survive conventional wastewater treatment Here, we report the results of experiments examining the partitioning and persistence of PrPTSE during simulated wastewater treatment processes including activated and mesophilic anaerobic sludge digestion. Incubation with activated sludge did not result in significant PrPTSE degradation. PrPTSE and prion infectivity partitioned strongly to activated sludge solids and are expected to enter biosolids treatment processes. A large fraction of PrPTSE survived simulated mesophilic anaerobic sludge digestion. The small reduction in recoverable PrPTSE after 20-d anaerobic sludge digestion appeared attributable to a combination of declining extractability with time and microbial degradation. Our results suggest that if prions were to enter municipal wastewater treatment systems, most would partition to activated sludge solids, survive mesophilic anaerobic digestion, and be present in treated biosolids. ?? 2008 American Chemical Society.
Hinckley, G. T.; Johnson, C. J.; Jacobson, K. H.; Bartholomay, C.; Mcmahon, K. D.; Mckenzie, D.; Aiken, J. M.; Pedersen, J. A.
Persistence of pathogenic prion protein during simulated wastewater treatment processes.
Transmissible spongiform encephalopathies (TSEs, prion diseases) are a class of fatal neurodegenerative diseases affecting a variety of mammalian species including humans. A misfolded form of the prion protein (PrP(TSE)) is the major, if not sole, component of the infectious agent. Prions are highly resistant to degradation and to many disinfection procedures suggesting that, if prions enter wastewater treatment systems through sewers and/or septic systems (e.g., from slaughterhouses, necropsy laboratories, rural meat processors, private game dressing) or through leachate from landfills that have received TSE-contaminated material, prions could survive conventional wastewater treatment. Here, we report the results of experiments examining the partitioning and persistence of PrPTSE during simulated wastewater treatment processes including activated and mesophilic anaerobic sludge digestion. Incubation with activated sludge did not result in significant PrPTSE degradation. PrPTSE and prion infectivity partitioned strongly to activated sludge solids and are expected to enter biosolids treatment processes. A large fraction of PrPTSE survived simulated mesophilic anaerobic sludge digestion. The small reduction in recoverable PrPTSE after 20-d anaerobic sludge digestion appeared attributable to a combination of declining extractability with time and microbial degradation. Our results suggest that if prions were to enter municipal wastewater treatment systems, most would partition to activated sludge solids, survive mesophilic anaerobic digestion, and be present in treated biosolids. PMID:18754377
Hinckley, Glen T; Johnson, Christopher J; Jacobson, Kurt H; Bartholomay, Christian; McMahon, Katherine D; McKenzie, Debbie; Aiken, Judd M; Pedersen, Joel A
Influence of the maillard reaction to prion protein aggregation.
Prion diseases are fatal neurodegenerative diseases that occur either spontaneously or genetically or are caused by infection. Spontaneously occurring prion diseases are age related. The infectious agents, called prions, are proteinaceous infectious particles, composed mainly of the host-encoded prion protein (PrP) in a misfolded, insoluble, and aggregated isoform. Advanced glycation end products (AGEs) are well known to contribute to protein misfolding, insolubility, and aggregation. Thus, we studied if AGE-modification could influence PrP aggregation. We analyzed PrP preparations immunochemically to determine if they contain AGE-modified PrP. We also studied the influence of AGE modifications on the PrP aggregation process in vitro. PMID:20370497
Panza, Giannantonio; Dumpitak, Christian; Birkmann, Eva
Developmental expression of the prion protein gene in glial cells
Replication of prions is dependent on the presence of the host protein PrPc. During the course of disease, PrPc is converted into an abnormal isoform, PrPsc, which accumulates in the brain. Attempts to identify the cell type(s) in which prion replication and PrP conversion occur have reached conflicting results. Although PrP mRNA is present in high amounts in neurons throughout
Markus Moser; Raymond J Colello; Uwe Pott; Bruno Oesch
Spontaneous and BSE-prion-seeded amyloid formation of full length recombinant bovine prion protein.
The conversion of the cellular isoform of the prion protein into the pathogenic isoform PrP(Sc) is the key event in prion diseases. The disease can occur spontaneously genetically or by infection. In earlier studies we presented an in vitro conversion system which simulates the structural transition in recPrP by varying low concentrations of SDS at constant NaCl. In the present study we adopted the conversion system from experimental Scrapie in hamster to bovine recPrP and generated amyloid fibrils. The intermediate state which is optimal for fibril formation is a soluble, beta-rich state. The system was extended using BSE-prions as seeds and led to an acceleration of fibril formation by orders of magnitude. This seeded amyloid formation assay avoids any PK-treatment, is therefore able to detect even PK-sensitive PrP(Sc) and does not require cellular components. PMID:18585368
Panza, Giannantonio; St�hr, Jan; Dumpitak, Christian; Papathanassiou, Dimitrios; Weiss, J�rgen; Riesner, Detlev; Willbold, Dieter; Birkmann, Eva
Low copper and high manganese levels in prion protein plaques
Accumulation of aggregates rich in an abnormally folded form of the prion protein characterize the neurodegeneration caused by transmissible spongiform encephalopathies (TSEs). The molecular triggers of plaque formation and neurodegeneration remain unknown, but analyses of TSE-infected brain homogenates and preparations enriched for abnormal prion protein suggest that reduced levels of copper and increased levels of manganese are associated with disease. The objectives of this study were to: (1) assess copper and manganese levels in healthy and TSE-infected Syrian hamster brain homogenates; (2) determine if the distribution of these metals can be mapped in TSE-infected brain tissue using X-ray photoelectron emission microscopy (X-PEEM) with synchrotron radiation; and (3) use X-PEEM to assess the relative amounts of copper and manganese in prion plaques in situ. In agreement with studies of other TSEs and species, we found reduced brain levels of copper and increased levels of manganese associated with disease in our hamster model. We also found that the in situ levels of these metals in brainstem were sufficient to image by X-PEEM. Using immunolabeled prion plaques in directly adjacent tissue sections to identify regions to image by X-PEEM, we found a statistically significant relationship of copper-manganese dysregulation in prion plaques: copper was depleted whereas manganese was enriched. These data provide evidence for prion plaques altering local transition metal distribution in the TSE-infected central nervous system.
Johnson, Christopher J.; Gilbert, P. U. P. A.; Abrecth, Mike; Baldwin, Katherine L.; Russell, Robin E.; Pedersen, Joel A.; McKenzie, Debbie
Cellular prion protein transduces neuroprotective signals
To test for a role for the cellular prion protein (PrPc) in cell death, we used a PrPc-binding peptide. Retinal explants from neonatal rats or mice were kept in vitro for 24�h, and anisomycin (ANI) was used to induce apoptosis. The peptide activated both cAMP/protein kinase�A (PKA) and Erk pathways, and partially prevented cell death induced by ANI in explants from wild-type rodents, but not from PrPc-null mice. Neuroprotection was abolished by treatment with phosphatidylinositol-specific phospholipase�C, with human peptide 106�126, with certain antibodies to PrPc or with a PKA inhibitor, but not with a MEK/Erk inhibitor. In contrast, antibodies to PrPc that increased cAMP also induced neuroprotection. Thus, engagement of PrPc transduces neuroprotective signals through a cAMP/PKA-dependent pathway. PrPc may function as a trophic receptor, the activation of which leads to a neuroprotective state.
Chiarini, Luciana B.; Freitas, Adriana R.O.; Zanata, Silvio M.; Brentani, Ricardo R.; Martins, Vilma R.; Linden, Rafael
Endogenous Prion Protein Attenuates Experimentally Induced Colitis
Although the cellular prion protein (PrPC) is expressed in the enteric nervous system and lamina propria, its function(s) in the gut is unknown. Because PrPC may exert a cytoprotective effect in response to various physiologic stressors, we hypothesized that PrPC expression levels might modulate the severity of experimental colitis. We evaluated the course of dextran sodium sulfate (DSS)�induced colitis in hemizygous Tga20 transgenic mice (approximately sevenfold overexpression of PrPC), Prnp?/? mice, and wild-type mice. On day 7, colon length, disease severity, and histologic activity indices were determined. Unlike DSS-treated wild-type and Prnp?/? animals, PrPC overexpressing mice were resistant to colitis induction, exhibited much milder histopathologic features, and did not exhibit weight loss or colonic shortening. In keeping with these results, pro-survival molecule expression and/or phosphorylation levels were elevated in DSS-treated Tga20 mice, whereas pro-inflammatory cytokine production and pSTAT3 levels were reduced. In contrast, DSS-treated Prnp?/? mice exhibited increased BAD protein expression and a cytokine expression profile predicted to favor inflammation and differentiation. PrPC expression from both the endogenous Prnp locus or the Tga20 transgene was increased in the colons of DSS-treated mice. Considered together, these findings demonstrate that PrPC has a previously unrecognized cytoprotective and/or anti-inflammatory function within the murine colon.
Martin, Gary R.; Keenan, Catherine M.; Sharkey, Keith A.; Jirik, Frank R.
Expression and characterisation of fully posttranslationally modified cellular prion protein in Pichia pastoris.
Prion diseases are fatal neurodegenerative diseases which occur as sporadic, genetic, and transmissible disorders. A molecular hallmark of prion diseases is the conformational conversion of the host-encoded cellular form of the prion protein (PrPC) into its misfolded pathogenic isoform (PrPSc). PrPSc is the main component of the pathological and infectious prion agent. The study of the conversion mechanism from PrPC to PrPSc is a major field in prion research. PrPC is glycosylated and attached to the plasma membrane via its glycosyl phosphatidyl inositol (GPI)-anchor. In this study we established and characterised the expression of fully posttranslationally modified mammalian Syrian golden hamster PrPC in the yeast Pichia pastoris using native PrPC-specific N- and C-terminal signal sequences. In vivo as well as in vitro-studies demonstrated that the signal sequences controlled posttranslational processing and trafficking of native PrPC, resulting in PrPC localised in the plasma membrane of P. pastoris. In addition, the glycosylation pattern of native PrPC could be confirmed. PMID:23893688
Marbach, Jendrik; Zentis, Peter; Ellinger, Philipp; M�ller, Henrik; Birkmann, Eva
Prion Peptide Uptake in Microglial Cells - The Effect of Naturally Occurring Autoantibodies against Prion Protein
In prion disease, a profound microglial activation that precedes neurodegeneration has been observed in the CNS. It is still not fully elucidated whether microglial activation has beneficial effects in terms of prion clearance or whether microglial cells have a mainly detrimental function through the release of pro-inflammatory cytokines. To date, no disease-modifying therapy exists. Several immunization attempts have been performed as one therapeutic approach. Recently, naturally occurring autoantibodies against the prion protein (nAbs-PrP) have been detected. These autoantibodies are able to break down fibrils of the most commonly used mutant prion variant PrP106-126 A117V and prevent PrP106-126 A117V-induced toxicity in primary neurons. In this study, we examined the phagocytosis of the prion peptide PrP106-126 A117V by primary microglial cells and the effect of nAbs-PrP on microglia. nAbs-PrP considerably enhanced the uptake of PrP106-126 A117V without inducing an inflammatory response in microglial cells. PrP106-126 A117V uptake was at least partially mediated through scavenger receptors. Phagocytosis of PrP106-126 A117V with nAbs-PrP was inhibited by wortmannin, a potent phosphatidylinositol 3-kinase inhibitor, indicating a separate uptake mechanism for nAbs-PrP mediated phagocytosis. These data suggest the possible mechanisms of action of nAbs-PrP in prion disease.
Roettger, Yvonne; Zerr, Inga; Dodel, Richard; Bach, Jan-Philipp
Cytoplasmic Expression of Mouse Prion Protein Causes Severe Toxicity in C. elegans
To test if Caenorhabditis elegans could be established as a model organism for prion study, we created transgenic C. elegans expressing the cytosolic form of the mouse prion .protein, MoPrP(23-231), which lacks the N-terminal signal sequence and the C-terminal glycosylphosphatidylinisotol (GPI) anchor site. We report here that transgenic worms expressing MoPrP(23-231)�CFP exhibited a wide range of distinct phenotypes: from normal growth and development, reduced mobility and development delay, complete paralysis and development arrest, to embryonic lethality. Similar levels of MoPrP (23-231)-CFP were produced in animals exhibiting these distinct phenotypes, suggesting that MoPrP (23-231)-CFP might have misfolded into distinct toxic species. In combining with the observation that mutations in PrP that affect prion pathogenesis also affect the toxic phenotypes in C. elegans, we conclude that the prion protein folding mechanism is similar in mammals and C. elegans. Thus, C. elegans can be a useful model organism for prion research.
Park, Kyung-Won; Li, Liming
Small RNA deep sequencing reveals a distinct miRNA signature released in exosomes from prion-infected neuronal cells
Prion diseases are transmissible neurodegenerative disorders affecting both humans and animals. The cellular prion protein, PrPC, and the abnormal infectious form, PrPSc, are found associated with exosomes, which are small 50�130 nm vesicles released from cells. Exosomes also contain microRNAs (miRNAs), a class of non-coding RNA, and have been utilized to identify miRNA signatures for diagnosis of disease. While some miRNAs are deregulated in prion-infected brain tissue, the role of miRNA in circulating exosomes released during prion disease is unknown. Here, we investigated the miRNA profile in exosomes released from prion-infected neuronal cells. We performed the first small RNA deep sequencing study of exosomes and demonstrated that neuronal exosomes contain a diverse range of RNA species including retroviral RNA repeat regions, messenger RNA fragments, transfer RNA fragments, non-coding RNA, small nuclear RNA, small nucleolar RNA, small cytoplasmic RNA, silencing RNA as well as known and novel candidate miRNA. Significantly, we show that exosomes released by prion-infected neuronal cells have increased let-7b, let-7i, miR-128a, miR-21, miR-222, miR-29b, miR-342-3p and miR-424 levels with decreased miR-146 a levels compared to non-infected exosomes. Overall, these results demonstrate that circulating exosomes released during prion infection have a distinct miRNA signature that can be utilized for diagnosis and understanding pathogenic mechanisms in prion disease.
Bellingham, Shayne A.; Coleman, Bradley M.; Hill, Andrew F.
?-amyloid oligomers and prion protein: Fatal attraction?
The relationship between Alzheimer disease (AD) and prion-related encephalopathies (TSE) has been proposed by different points of view. Recently, the scientific attention has been attracted by the results proposing the possibility that PrPc, the protein whose pathologic form is responsible of TSE, can mediated the toxic effect of ? amyloid (A?) oligomers. The oligomers are considered the culprit of the neurodegenerative process associated to AD, although the pathogenic mechanism activated by these small aggregates remain to be elucidated. In the initial study based on the binding screening PrPc was identified as ligand /receptor of A? oligomers, while long term potentiation (LTP) analysis in vitro and behavioural studies in vivo, demonstrated that the absence of PrPc abolished the damage induced by A? oligomers. The high affinity binding A? oligomers-PrPc has been confirmed, whereas a functional role of this association has been excluded by three different studies. We approached this issue by the direct application of A? oligomers in the brain followed by the behavioural examination of memory deficits. Our data using PrP knock-out mice suggest that A? 1-42 oligomers are responsible for cognitive impairment in AD but PrPc is not required for their effect. Similarly, in two other studies the LTP alterations induced by A? 1-42 oligomers was not influenced by the absence of PrP. Possible explanations of these contradictory results are discussed. PMID:21150333
Forloni, Gianluigi; Balducci, Claudia
Probing Structural Differences in Prion Protein Isoforms by Tyrosine Nitration�
Two conformational isomers of recombinant hamster prion protein (residues 90-232) have been probed by reaction with two tyrosine nitration reagents, peroxynitrite and tetranitromethane. Two conserved tyrosine residues (tyrosines 149 and 150) are not labeled by either reagent in the normal cellular form of the prion protein. These residues become reactive after the protein has been converted to the ?-oligomeric isoform, which is used as a model of the fibrillar form that causes disease. After conversion, a decrease in reactivity is noted for two other conserved residues, tyrosine 225 and tyrosine 226, whereas little to no effect was observed for other tyrosines. Thus, tyrosine nitration has identified two specific regions of the normal prion protein isoform that undergo a change in chemical environment upon conversion to a structure that is enriched in ?-sheet.
Lennon, Christopher W.; Cox, Holly D.; Hennelly, Scott P.; Chelmo, Sam J.; McGuirl, Michele A.
Probing structural differences in prion protein isoforms by tyrosine nitration.
Two conformational isomers of recombinant hamster prion protein (residues 90-232) have been probed by reaction with two tyrosine nitration reagents, peroxynitrite and tetranitromethane. Two conserved tyrosine residues (tyrosines 149 and 150) are not labeled by either reagent in the normal cellular form of the prion protein. These residues become reactive after the protein has been converted to the beta-oligomeric isoform, which is used as a model of the fibrillar form that causes disease. After conversion, a decrease in reactivity is noted for two other conserved residues, tyrosine 225 and tyrosine 226, whereas little to no effect was observed for other tyrosines. Thus, tyrosine nitration has identified two specific regions of the normal prion protein isoform that undergo a change in chemical environment upon conversion to a structure that is enriched in beta-sheet. PMID:17397138
Lennon, Christopher W; Cox, Holly D; Hennelly, Scott P; Chelmo, Sam J; McGuirl, Michele A
Prions Ex Vivo: What Cell Culture Models Tell Us about Infectious Proteins
Prions are unconventional infectious agents that are composed of misfolded aggregated prion protein. Prions replicate their conformation by template-assisted conversion of the endogenous prion protein PrP. Templated conversion of soluble proteins into protein aggregates is also a hallmark of other neurodegenerative diseases. Alzheimer's disease or Parkinson's disease are not considered infectious diseases, although aggregate pathology appears to progress in a stereotypical fashion reminiscent of the spreading behavior ofmammalian prions. While basic principles of prion formation have been studied extensively, it is still unclear what exactly drives PrP molecules into an infectious, self-templating conformation. In this review, we discuss crucial steps in the life cycle of prions that have been revealed in ex vivo models. Importantly, the persistent propagation of prions in mitotically active cells argues that cellular processes are in place that not only allow recruitment of cellular PrP into growing prion aggregates but also enable the multiplication of infectious seeds that are transmitted to daughter cells. Comparison of prions with other protein aggregates demonstrates that not all the characteristics of prions are equally shared by prion-like aggregates. Future experiments may reveal to which extent aggregation-prone proteins associated with other neurodegenerative diseases can copy the replication strategies of prions.
Krauss, Sybille
Inherited Prion Disease A117V Is Not Simply a Proteinopathy but Produces Prions Transmissible to Transgenic Mice Expressing Homologous Prion Protein
Prions are infectious agents causing fatal neurodegenerative diseases of humans and animals. In humans, these have sporadic, acquired and inherited aetiologies. The inherited prion diseases are caused by one of over 30 coding mutations in the human prion protein (PrP) gene (PRNP) and many of these generate infectious prions as evidenced by their experimental transmissibility by inoculation to laboratory animals. However, some, and in particular an extensively studied type of Gerstmann-Str�ussler-Scheinker syndrome (GSS) caused by a PRNP A117V mutation, are thought not to generate infectious prions and instead constitute prion proteinopathies with a quite distinct pathogenetic mechanism. Multiple attempts to transmit A117V GSS have been unsuccessful and typical protease-resistant PrP (PrPSc), pathognomonic of prion disease, is not detected in brain. Pathogenesis is instead attributed to production of an aberrant topological form of PrP, C-terminal transmembrane PrP (CtmPrP). Barriers to transmission of prion strains from one species to another appear to relate to structural compatibility of PrP in host and inoculum and we have therefore produced transgenic mice expressing human 117V PrP. We found that brain tissue from GSS A117V patients did transmit disease to these mice and both the neuropathological features of prion disease and presence of PrPSc was demonstrated in the brains of recipient transgenic mice. This PrPSc rapidly degraded during laboratory analysis, suggesting that the difficulty in its detection in patients with GSS A117V could relate to post-mortem proteolysis. We conclude that GSS A117V is indeed a prion disease although the relative contributions of CtmPrP and prion propagation in neurodegeneration and their pathogenetic interaction remains to be established.
Asante, Emmanuel A.; Linehan, Jacqueline M.; Smidak, Michelle; Tomlinson, Andrew; Grimshaw, Andrew; Jeelani, Asif; Jakubcova, Tatiana; Hamdan, Shyma; Powell, Caroline; Brandner, Sebastian; Wadsworth, Jonathan D. F.; Collinge, John
Marked increase of neuronal prion protein immunoreactivity in Alzheimer's disease and human prion diseases
In neurodegenerative disorders including Alzheimer's disease (AD), free radical damage to lipids, carbohydrates, proteins and DNA has been demonstrated to play a key pathogenetic role. In vitro studies have suggested a function of the cellular prion protein (PrPc) in the defense against oxidative stress. Therefore, we investigated the distribution of PrPc immunoreactivity in hippocampus (sectors CA4-CA1), subiculum (Sub), entorhinal (EC),
T. Voigtl�nder; S. Kl�ppel; P. Birner; C. Jarius; H. Flicker; S. Verghese-Nikolakaki; T. Sklaviadis; M. Guentchev; H. Budka
Prion Protein Accumulation in Lipid Rafts of Mouse Aging Brain
The cellular form of the prion protein (PrPC) is a normal constituent of neuronal cell membranes. The protein misfolding causes rare neurodegenerative disorders known as transmissible spongiform encephalopathies or prion diseases. These maladies can be sporadic, genetic or infectious. Sporadic prion diseases are the most common form mainly affecting aging people. In this work, we investigate the biochemical environment in which sporadic prion diseases may develop, focusing our attention on the cell membrane of neurons in the aging brain. It is well established that with aging the ratio between the most abundant lipid components of rafts undergoes a major change: while cholesterol decreases, sphingomyelin content rises. Our results indicate that the aging process modifies the compartmentalization of PrPC. In old mice, this change favors PrPC accumulation in detergent-resistant membranes, particularly in hippocampi. To confirm the relationship between lipid content changes and PrPC translocation into detergent-resistant membranes (DRMs), we looked at PrPC compartmentalization in hippocampi from acid sphingomyelinase (ASM) knockout (KO) mice and synaptosomes enriched in sphingomyelin. In the presence of high sphingomyelin content, we observed a significant increase of PrPC in DRMS. This process is not due to higher levels of total protein and it could, in turn, favor the onset of sporadic prion diseases during aging as it increases the PrP intermolecular contacts into lipid rafts. We observed that lowering sphingomyelin in scrapie-infected cells by using fumonisin B1 led to a 50% decrease in protease-resistant PrP formation. This may suggest an involvement of PrP lipid environment in prion formation and consequently it may play a role in the onset or development of sporadic forms of prion diseases.
Agostini, Federica; Dotti, Carlos G.; Perez-Canamas, Azucena; Ledesma, Maria Dolores; Benetti, Federico; Legname, Giuseppe
Construction and Characterization of an Anti?Prion scFv Fusion Protein Pair for Detection of Prion Protein on Antibody Chip
A pair of single chain Fv fragment (scFv) fusion proteins were constructed and characterized. Antibody chips using the pair were designed for sensitive detection of prion protein. Phage displayed antibody library was synthesized by immunizing mice with thioredoxin?mature bovine prion fusion protein (TrxA?bPrP). After five rounds of panning against recombinant bovine prion protein (rb?PrP) and ELISA test, two positive clones
Role of a Novel Topological Form of a Prion Protein in Prion Disease.
National Technical Information Service (NTIS)
Prion diseases are fatal neurological disorders of humans and other mammals. Prion diseases show an unusual etiology: they can arise from genetically, from infection through prion-contaminated food products, or sporadically. Most (but not all) cases of pr...
R. S. Stewart
Reversible symptoms and clearance of mutant prion protein in an inducible model of a genetic prion disease in Drosophila melanogaster.
Prion diseases are progressive disorders that affect the central nervous system leading to memory loss, personality changes, ataxia and neurodegeneration. In humans, these disorders include Creutzfeldt-Jakob disease, kuru and Gerstmann-Stra�ssler-Scheinker (GSS) syndrome, the latter being a dominantly inherited prion disease associated with missense mutations in the gene that codes for the prion protein. The exact mechanism by which mutant prion proteins affect the central nervous system and cause neurological disease is not well understood. We have generated an inducible model of GSS disease in Drosophila melanogaster by temporally expressing a misfolded form of the murine prion protein in cholinergic neurons. Flies accumulating this mutant protein develop motor abnormalities which are associated with electrophysiological defects in cholinergic neurons. We find that, upon blocking the expression of the mutant protein, both behavioral and electrophysiological defects can be reversed. This represents the first case of reversibility reported in a model of genetic prion disease. Additionally, we observe that endogenous mechanisms exist within Drosophila that are capable of clearing the accumulated prion protein. PMID:24686303
Murali, A; Maue, R A; Dolph, P J
Molecular Features of the Copper Binding Sites in the Octarepeat Domain of the Prion Protein �
Recent evidence suggests that the prion protein (PrP) is a copper binding protein. The N-terminal region of human PrP contains four sequential copies of the highly conserved octarepeat sequence PHGGGWGQ spanning residues 60-91. This region selectively binds Cu2+ in vivo. In a previous study using peptide design, EPR, and CD spectroscopy, we showed that the HGGGW segment within each octarepeat
Colin S. Burns; Eliah Aronoff-Spencer; Christine M. Dunham; Paula Lario; Nikolai I. Avdievich; William E. Antholine; Marilyn M. Olmstead; Alice Vrielink; Gary J. Gerfen; Jack Peisach; William G. Scott; Glenn L. Millhauser
Humic substances interfere with detection of pathogenic prion protein
Studies examining the persistence of prions (the etiological agent of transmissible spongiform encephalopathies) in soil require accurate quantification of pathogenic prion protein (PrPTSE) extracted from or in the presence of soil particles. Here, we demonstrate that natural organic matter (NOM) in soil impacts PrPTSE detection by immunoblotting. Methods commonly used to extract PrPTSE from soils release substantial amounts of NOM, and NOM inhibited PrPTSE immunoblot signal. The degree of immunoblot interference increased with increasing NOM concentration and decreasing NOM polarity. Humic substances affected immunoblot detection of prion protein from both deer and hamsters. We also establish that after interaction with humic acid, PrPTSE remains infectious to hamsters inoculated intracerebrally, and humic acid appeared to slow disease progression. These results provide evidence for interactions between PrPTSE and humic substances that influence both accurate measurement of PrPTSE in soil and disease transmission.
Smith, Christen B.; Booth, Clarissa J.; Wadzinski, Tyler J.; Legname, Giuseppe; Chappell, Rick; Johnson, Christopher J.; Pedersen, Joel A.
The presence of the cellular prion protein (PrPC) on the cell surface is critical for the neurotoxicity of prions. Although a number of biological activities have been attributed to PrPC, a definitive demonstration of its physiological function remains elusive. In this review, we will discuss some of the proposed functions of PrPC, focusing on recently suggested roles in cell adhesion, regulation of ionic currents at the cell membrane, and neuroprotection. We will also discuss recent evidence supporting the idea that PrPC may function as a receptor for soluble oligomers of the amyloid ? peptide and possibly other toxic protein aggregates. These data suggest surprising new connections between the physiological function of PrPC and its role in neurodegenerative diseases beyond those caused by prions.
Biasini, Emiliano; Turnbaugh, Jessie; Unterberger, Ursula; Harris, David A.
Heat stability of prion rods and recombinant prion protein in water, lipid and lipid-water mixtures
Prion rods, i.e. insoluble infectious aggregates of the N-terminally truncated form of the prion protein, PrP 27-30, and the corresponding recombinant protein, rPrP(90-231), were autoclaved in water, bovine lipid or lipid-water mixtures for 20 min at temperatures from 100 to 170SC. A protocol was developed for the quantitative precipitation of small amounts of protein from large excesses of lipid. PrP
Thomas Raul Appel; Michael Wolff; Friedrich von Rheinbaben; Michael Heinzel; Detlev Riesner
Increasing Prion Propensity by Hydrophobic Insertion
Prion formation involves the conversion of proteins from a soluble form into an infectious amyloid form. Most yeast prion proteins contain glutamine/asparagine-rich regions that are responsible for prion aggregation. Prion formation by these domains is driven primarily by amino acid composition, not primary sequence, yet there is a surprising disconnect between the amino acids thought to have the highest aggregation propensity and those that are actually found in yeast prion domains. Specifically, a recent mutagenic screen suggested that both aromatic and non-aromatic hydrophobic residues strongly promote prion formation. However, while aromatic residues are common in yeast prion domains, non-aromatic hydrophobic residues are strongly under-represented. Here, we directly test the effects of hydrophobic and aromatic residues on prion formation. Remarkably, we found that insertion of as few as two hydrophobic residues resulted in a multiple orders-of-magnitude increase in prion formation, and significant acceleration of in vitro amyloid formation. Thus, insertion or deletion of hydrophobic residues provides a simple tool to control the prion activity of a protein. These data, combined with bioinformatics analysis, suggest a limit on the number of strongly prion-promoting residues tolerated in glutamine/asparagine-rich domains. This limit may explain the under-representation of non-aromatic hydrophobic residues in yeast prion domains. Prion activity requires not only that a protein be able to form prion fibers, but also that these fibers be cleaved to generate new independently-segregating aggregates to offset dilution by cell division. Recent studies suggest that aromatic residues, but not non-aromatic hydrophobic residues, support the fiber cleavage step. Therefore, we propose that while both aromatic and non-aromatic hydrophobic residues promote prion formation, aromatic residues are favored in yeast prion domains because they serve a dual function, promoting both prion formation and chaperone-dependent prion propagation.
Petri, Michelina; Flores, Noe; Rogge, Ryan A.; Cascarina, Sean M.; Ross, Eric D.
Biological and Biochemical Characterization of Mice Expressing Prion Protein Devoid of the Octapeptide Repeat Region after Infection with Prions
Accumulating lines of evidence indicate that the N-terminal domain of prion protein (PrP) is involved in prion susceptibility in mice. In this study, to investigate the role of the octapeptide repeat (OR) region alone in the N-terminal domain for the susceptibility and pathogenesis of prion disease, we intracerebrally inoculated RML scrapie prions into tg(PrP?OR)/Prnp0/0 mice, which express mouse PrP missing only the OR region on the PrP-null background. Incubation times of these mice were not extended. Protease-resistant PrP?OR, or PrPSc?OR, was easily detectable but lower in the brains of these mice, compared to that in control wild-type mice. Consistently, prion titers were slightly lower and astrogliosis was milder in their brains. However, in their spinal cords, PrPSc?OR and prion titers were abundant and astrogliosis was as strong as in control wild-type mice. These results indicate that the role of the OR region in prion susceptibility and pathogenesis of the disease is limited. We also found that the PrPSc?OR, including the pre-OR residues 23�50, was unusually protease-resistant, indicating that deletion of the OR region could cause structural changes to the pre-OR region upon prion infection, leading to formation of a protease-resistant structure for the pre-OR region.
Yamaguchi, Yoshitaka; Miyata, Hironori; Uchiyama, Keiji; Ootsuyama, Akira; Inubushi, Sachiko; Mori, Tsuyoshi; Muramatsu, Naomi; Katamine, Shigeru; Sakaguchi, Suehiro
Prion protein in Alzheimer's pathogenesis: a hot and controversial issue
The role for cellular prion protein PrPc in ?-amyloid (A?) oligomer-induced synaptic impairment is a topic of great interest and some controversy. In this issue of EMBO Molecular Medicine Aguzzi and co-workers explore the contribution of PrPc to deficient long term potentiation (LTP) and soluble A? levels in an Alzheimer's disease mouse model and show that the role of prions in A? related toxicity is far from �black and white� suggesting complex interpretations of the data available thus far.
Benilova, Iryna; De Strooper, Bart
Yeast prions assembly and propagation
Yeast prions are self-perpetuating protein aggregates that are at the origin of heritable and transmissible non-Mendelian phenotypic traits. Among these, [PSI+], [URE3] and [PIN+] are the most well documented prions and arise from the assembly of Sup35p, Ure2p and Rnq1p, respectively, into insoluble fibrillar assemblies. Fibril assembly depends on the presence of N- or C-terminal prion domains (PrDs) which are not homologous in sequence but share unusual amino-acid compositions, such as enrichment in polar residues (glutamines and asparagines) or the presence of oligopeptide repeats. Purified PrDs form amyloid fibrils that can convert prion-free cells to the prion state upon transformation. Nonetheless, isolated PrDs and full-length prion proteins have different aggregation, structural and infectious properties. In addition, mutations in the �non-prion� domains (non-PrDs) of Sup35p, Ure2p and Rnq1p were shown to affect their prion properties in vitro and in vivo. Despite these evidences, the implication of the functional non-PrDs in fibril assembly and prion propagation has been mostly overlooked. In this review, we discuss the contribution of non-PrDs to prion assemblies, and the structure-function relationship in prion infectivity in the light of recent findings on Sup35p and Ure2p assembly into infectious fibrils from our laboratory and others.
NMR solution structure of the human prion protein
The NMR structures of the recombinant human prion protein, hPrP(23-230), and two C-terminal fragments, hPrP(90-230) and hPrP(121-230), include a globular domain extending from residues 125-228, for which a detailed structure was obtained, and an N-terminal flexibly disordered \\
Ralph Zahn; Aizhuo Liu; Thorsten L�hrs; Roland Riek; Christine von Schroetter; Francisco L�pez Garc�a; Martin Billeter; Luigi Calzolai; Gerhard Wider; Kurt W�thrich
Prion protein gene polymorphisms in natural goat scrapie
A total of 51 goats, including seven clinical cases, from the first herd in Greece reported to have scrapie was examined to discern an association between scrapie susceptibility and polymorphisms of the gene encoding the prion protein (PrP). Each animal was evaluated for clinical signs of the disease, histopathological lesions associated with scrapie, the presence of detectable protease- resistant PrP
Charalambos Billinis; Cynthia H. Panagiotidis; Vassilios Psychas; Stamatis Argyroudis; Anna Nicolaou; Sotirios Leontides; Orestis Papadopoulos; Theodoros Sklaviadis
Conserved Stress-protective Activity between Prion Protein and Shadoo*
Shadoo (Sho) is a neuronally expressed glycoprotein of unknown function. Although there is no overall sequence homology to the cellular prion protein (PrPC), both proteins contain a highly conserved internal hydrophobic domain (HD) and are tethered to the outer leaflet of the plasma membrane via a C-terminal glycosylphosphatidylinositol anchor. A previous study revealed that Sho can reduce toxicity of a PrP mutant devoid of the HD (PrP?HD). We have now studied the stress-protective activity of Sho in detail and identified domains involved in this activity. Like PrPC, Sho protects cells against physiological stressors such as the excitotoxin glutamate. Moreover, both PrPC and Sho required the N-terminal domain for this activity; the stress-protective capacity of PrP?N as well as Sho?N was significantly impaired. In both proteins, the HD promoted homodimer formation; however, deletion of the HD had different effects. Although Sho?HD lost its stress-protective activity, PrP?HD acquired a neurotoxic potential. Finally, we could show that the N-terminal domain of PrPC could be functionally replaced by that of Sho, suggesting a similar function of the N termini of Sho and PrPC. Our study reveals a conserved physiological activity between PrPC and Sho to protect cells from stress-induced toxicity and suggests that Sho and PrPC might act on similar signaling pathways.
Sakthivelu, Vignesh; Seidel, Ralf P.; Winklhofer, Konstanze F.; Tatzelt, Jorg
Prevalence of the prion protein gene E211K variant in U.S. cattle
BACKGROUND: In 2006, an atypical U.S. case of bovine spongiform encephalopathy (BSE) was discovered in Alabama and later reported to be polymorphic for glutamate (E) and lysine (K) codons at position 211 in the bovine prion protein gene (Prnp) coding sequence. A bovine E211K mutation is important because it is analogous to the most common pathogenic mutation in humans (E200K)
Michael P Heaton; John W Keele; Gregory P Harhay; J�rgen A Richt; Mohammad Koohmaraie; Tommy L Wheeler; Steven D Shackelford; Eduardo Casas; D Andy King; Tad S Sonstegard; Curtis P Van Tassell; Holly L Neibergs; Chad C Chase Jr; Theodore S Kalbfleisch; Timothy PL Smith; Michael L Clawson; William W Laegreid
Quaternary Structure of Pathological Prion Protein as a Determining Factor of Strain-Specific Prion Replication Dynamics
Prions are proteinaceous infectious agents responsible for fatal neurodegenerative diseases in animals and humans. They are essentially composed of PrPSc, an aggregated, misfolded conformer of the ubiquitously expressed host-encoded prion protein (PrPC). Stable variations in PrPSc conformation are assumed to encode the phenotypically tangible prion strains diversity. However the direct contribution of PrPSc quaternary structure to the strain biological information remains mostly unknown. Applying a sedimentation velocity fractionation technique to a panel of ovine prion strains, classified as fast and slow according to their incubation time in ovine PrP transgenic mice, has previously led to the observation that the relationship between prion infectivity and PrPSc quaternary structure was not univocal. For the fast strains specifically, infectivity sedimented slowly and segregated from the bulk of proteinase-K resistant PrPSc. To carefully separate the respective contributions of size and density to this hydrodynamic behavior, we performed sedimentation at the equilibrium and varied the solubilization conditions. The density profile of prion infectivity and proteinase-K resistant PrPSc tended to overlap whatever the strain, fast or slow, leaving only size as the main responsible factor for the specific velocity properties of the fast strain most infectious component. We further show that this velocity-isolable population of discrete assemblies perfectly resists limited proteolysis and that its templating activity, as assessed by protein misfolding cyclic amplification outcompetes by several orders of magnitude that of the bulk of larger size PrPSc aggregates. Together, the tight correlation between small size, conversion efficiency and duration of disease establishes PrPSc quaternary structure as a determining factor of prion replication dynamics. For certain strains, a subset of PrP assemblies appears to be the best template for prion replication. This has important implications for fundamental studies on prions.
Chapuis, Jerome; Sibille, Pierre; Herzog, Laetitia; Reine, Fabienne; Jaumain, Emilie; Laude, Hubert; Rezaei, Human; Beringue, Vincent
Similar folds with different stabilization mechanisms: the cases of prion and doppel proteins
Background Protein misfolding is the main cause of a group of fatal neurodegenerative diseases in humans and animals. In particular, in Prion-related diseases the normal cellular form of the Prion Protein PrP (PrPC) is converted into the infectious PrPSc through a conformational process during which it acquires a high ?-sheet content. Doppel is a protein that shares a similar native fold, but lacks the scrapie isoform. Understanding the molecular determinants of these different behaviours is important both for biomedical and biophysical research. Results In this paper, the dynamical and energetic properties of the two proteins in solution is comparatively analyzed by means of long time scale explicit solvent, all-atom molecular dynamics in different temperature conditions. The trajectories are analyzed by means of a recently introduced energy decomposition approach (Tiana et al, Prot. Sci. 2004) aimed at identifying the key residues for the stabilization and folding of the protein. Our analysis shows that Prion and Doppel have two different cores stabilizing the native state and that the relative contribution of the nucleus to the global stability of the protein for Doppel is sensitively higher than for PrP. Moreover, under misfolding conditions the Doppel core is conserved, while the energy stabilization network of PrP is disrupted. Conclusion These observations suggest that different sequences can share similar native topology with different stabilizing interactions and that the sequences of the Prion and Doppel proteins may have diverged under different evolutionary constraints resulting in different folding and stabilization mechanisms.
Colacino, Stefano; Tiana, Guido; Colombo, Giorgio
Spontaneous generation of rapidly transmissible prions in transgenic mice expressing wild-type bank vole prion protein
Currently, there are no animal models of the most common human prion disorder, sporadic Creutzfeldt-Jakob disease (CJD), in which prions are formed spontaneously from wild-type (WT) prion protein (PrP). Interestingly, bank voles (BV) exhibit an unprecedented promiscuity for diverse prion isolates, arguing that bank vole PrP (BVPrP) may be inherently prone to adopting misfolded conformations. Therefore, we constructed transgenic (Tg) mice expressing WT BVPrP. Tg(BVPrP) mice developed spontaneous CNS dysfunction between 108 and 340 d of age and recapitulated the hallmarks of prion disease, including spongiform degeneration, pronounced astrogliosis, and deposition of alternatively folded PrP in the brain. Brain homogenates of ill Tg(BVPrP) mice transmitted disease to Tg(BVPrP) mice in ?35 d, to Tg mice overexpressing mouse PrP in under 100 d, and to WT mice in ?185 d. Our studies demonstrate experimentally that WT PrP can spontaneously form infectious prions in vivo. Thus, Tg(BVPrP) mice may be useful for studying the spontaneous formation of prions, and thus may provide insight into the etiology of sporadic CJD.
Watts, Joel C.; Giles, Kurt; Stohr, Jan; Oehler, Abby; Bhardwaj, Sumita; Grillo, Sunny K.; Patel, Smita; DeArmond, Stephen J.; Prusiner, Stanley B.
High-resolution structure of infectious prion protein: the final frontier
Prions are the proteinaceous infectious agents responsible for the transmission of prion diseases. The main or sole component of prions is the misfolded prion protein (PrPSc), which is able to template the conversion of the host�s natively folded form of the protein (PrPC). The detailed mechanism of prion replication and the high-resolution structure of PrPSc are unknown. The currently available information on PrPSc structure comes mostly from low-resolution biophysical techniques, which have resulted in quite divergent models. Recent advances in the production of infectious prions, using very pure recombinant protein, offer new hope for PrPSc structural studies. This review highlights the importance of, challenges for and recent progress toward elucidating the elusive structure of PrPSc, arguably the major pending milestone to reach in understanding prions.
Diaz-Espinoza, Rodrigo; Soto, Claudio
Prion protein degradation by lichens of the genus Cladonia
It has recently been discovered that lichens contain a serine protease capable of degrading the pathogenic prion protein, the etiological agent of prion diseases such as sheep scrapie and cervid chronic wasting disease. Limited methods are available to degrade or inactivate prion disease agents, especially in the environment, and lichens or their serine protease could prove important for management of these diseases. Scant information is available regarding the presence or absence of the protease responsible for degrading prion protein (PrP) in lichen species and, in this study, we tested the hypothesis that PrP degradation activity in lichens is phylogenetically-based by testing 44 species of Cladonia lichens, a genus for which a significant portion of the phylogeny is well established. We categorized PrP degradation activity among the 44 species (high, moderate, low or none) and found that activity in Cladonia species did not correspond with phylogenetic position of the species. Degradation of PrP did correspond, however, with three classical taxonomic characters within the genus: species with brown apothecia, no usnic acid, and the presence of a cortex. Of the 44 species studied, 18 (41%) had either high or moderate PrP degradation activity, suggesting the protease may be frequent in this genus of lichens.
Bennett, James P.; Rodriguez, Cynthia M.; Johnson, Christopher J.
Sequestration of essential proteins causes prion associated toxicity in yeast
Summary Prions are infectious, aggregated proteins that cause diseases in mammals but are not normally toxic in fungi. Excess Sup35p, an essential yeast protein that can exist as the [PSI+] prion, inhibits growth of [PSI+] but not [psi?] cells. This toxicity is rescued by expressing the Sup35Cp domain of Sup35p, which is sufficient for cell viability but not prion propagation. We now show that rescue requires Sup35Cp levels to be proportional to Sup35p overexpression. Overexpression of Sup35p appeared to cause pre-existing [PSI+] aggregates to coalesce into larger aggregates, but these were not toxic per se because they formed even when Sup35Cp rescued growth. Overexpression of Sup45p, but not other tested essential Sup35p binding partners caused rescue. Sup45-GFPp formed puncta that co-localized with large [PSI+] Sup35-RFPp aggregates in cells overexpressing Sup35p, and the frequency of the Sup45-GFPp puncta was reduced by rescuing levels of Sup35Cp. In contrast, [PSI+] toxicity caused by a high excess of the Sup35p prion domain (Sup35NMp) was rescued by a single copy of Sup35Cp, was not rescued by Sup45p overexpression and was not associated with the appearance of Sup45-GFPp puncta. This suggests [PSI+] toxicity caused by excess Sup35p verses Sup35NMp is respectively through sequestration/inactivation of Sup45p verses Sup35p.
Vishveshwara, Namitha; Bradley, Michael E.; Liebman, Susan W.
Increased expression of p62/SQSTM1 in prion diseases and its association with pathogenic prion protein
Prion diseases are neurodegenerative disorders characterized by the aggregation of abnormally folded prion protein (PrPSc). In this study, we focused on the mechanism of clearance of PrPSc, which remains unclear. p62 is a cytosolic protein known to mediate both the formation and degradation of aggregates of abnormal proteins. The levels of p62 protein increased in prion-infected brains and persistently infected cell cultures. Upon proteasome inhibition, p62 co-localized with PrPSc, forming a large aggregate in the perinuclear region, hereafter referred to as PrPSc-aggresome. These aggregates were surrounded with autophagosome marker LC3 and lysosomes in prion-infected cells. Moreover, transient expression of the phosphomimic form of p62, which has enhanced ubiquitin-binding activity, reduced the amount of PrPSc in prion-infected cells, indicating that the activation of p62 could accelerate the clearance of PrPSc. Our findings would thus suggest that p62 could be a target for the therapeutic control of prion diseases.
Homma, Takujiro; Ishibashi, Daisuke; Nakagaki, Takehiro; Satoh, Katsuya; Sano, Kazunori; Atarashi, Ryuichiro; Nishida, Noriyuki
Functional Diversification of Hsp40: Distinct J-Protein Functional Requirements for Two Prions Allow for Chaperone-Dependent Prion Selection
Yeast prions are heritable amyloid aggregates of functional yeast proteins; their propagation to subsequent cell generations is dependent upon fragmentation of prion protein aggregates by molecular chaperone proteins. Mounting evidence indicates the J-protein Sis1 may act as an amyloid specificity factor, recognizing prion and other amyloid aggregates and enabling Ssa and Hsp104 to act in prion fragmentation. Chaperone interactions with prions, however, can be affected by variations in amyloid-core structure resulting in distinct prion variants or �strains�. Our genetic analysis revealed that Sis1 domain requirements by distinct variants of [PSI+] are strongly dependent upon overall variant stability. Notably, multiple strong [PSI+] variants can be maintained by a minimal construct of Sis1 consisting of only the J-domain and glycine/phenylalanine-rich (G/F) region that was previously shown to be sufficient for cell viability and [RNQ+] prion propagation. In contrast, weak [PSI+] variants are lost under the same conditions but maintained by the expression of an Sis1 construct that lacks only the G/F region and cannot support [RNQ+] propagation, revealing mutually exclusive requirements for Sis1 function between these two prions. Prion loss is not due to [PSI+]-dependent toxicity or dependent upon a particular yeast genetic background. These observations necessitate that Sis1 must have at least two distinct functional roles that individual prions differentially require for propagation and which are localized to the glycine-rich domains of the Sis1. Based on these distinctions, Sis1 plasmid-shuffling in a [PSI+]/[RNQ+] strain permitted J-protein-dependent prion selection for either prion. We also found that, despite an initial report to the contrary, the human homolog of Sis1, Hdj1, is capable of [PSI+] prion propagation in place of Sis1. This conservation of function is also prion-variant dependent, indicating that only one of the two Sis1-prion functions may have been maintained in eukaryotic chaperone evolution.
Patel, Milan J.; Sporn, Zachary A.; Hines, Justin K.
Characterization of the genomic region containing the Shadow of Prion Protein (SPRN) gene in sheep
Background TSEs are a group of fatal neurodegenerative diseases occurring in man and animals. They are caused by prions, alternatively folded forms of the endogenous prion protein, encoded by PRNP. Since differences in the sequence of PRNP can not explain all variation in TSE susceptibility, there is growing interest in other genes that might have an influence on this susceptibility. One of these genes is SPRN, a gene coding for a protein showing remarkable similarities with the prion protein. Until now, SPRN has not been described in sheep, a highly relevant species in prion matters. Results In order to characterize the genomic region containing SPRN in sheep, a BAC mini-contig was built, covering approximately 200,000 bp and containing the genes ECHS1, PAOX, MTG1, SPRN, LOC619207, CYP2E1 and at least partially SYCE1. FISH mapping of the two most exterior BAC clones of the contig positioned this contig on Oari22q24. A fragment of 4,544 bp was also sequenced, covering the entire SPRN gene and 1206 bp of the promoter region. In addition, the transcription profile of SPRN in 21 tissues was determined by RT-PCR, showing high levels in cerebrum and cerebellum, and low levels in testis, lymph node, jejunum, ileum, colon and rectum. Conclusion Annotation of a mini-contig including SPRN suggests conserved linkage between Oari22q24 and Hsap10q26. The ovine SPRN sequence, described for the first time, shows a high level of homology with the bovine, and to a lesser extent with the human SPRN sequence. In addition, transcription profiling in sheep reveals main expression of SPRN in brain tissue, as in rat, cow, man and mouse.
Lampo, Evelyne; Van Poucke, Mario; Hugot, Karine; Hayes, Helene; Van Zeveren, Alex; Peelman, Luc J
Canadian Association of Neurosciences Review: prion protein and prion diseases: the good and the bad.
In the 1700's a strange new disease affecting sheep was recognized in Europe. The disease later became known as "Scrapie" and was the first of a family of similar diseases affecting a number of species that are now known as the Transmissible Spongiform Encephalopathies (TSEs). The appearance of a new disease in humans linked to the consumption of meat products from infected cattle has stimulated widespread public concern and scientific interest in the prion protein and related diseases. Nearly 300 years after the first report, these diseases still merit the descriptor "strange". This family of diseases is characterized by a unique profile of histological changes, can be transmitted as inherited or acquired diseases, as well as apparent sporadic spontaneous generation of the disease. These diseases are believed by many, to be caused by a unique protein only infectious agent. The "prion protein" (PrPC), a term first coined by Stanley Prusiner in 1982 is crucial to the development of these diseases, apparently by acting as a substrate for an abnormal disease associated form. However, aside from being critical to the pathogenesis of the disease, the function of PrPC, which is expressed in all mammals, has defied definitive description. Several roles have been proposed on the basis of in vitro studies, however, thus far, in vivo confirmation has not been forthcoming. The biological features of PrPC also seem to be unusual. Numerous mouse models have been generated in an attempt to understand the pathogenesis of these diseases. This review summarizes the current state of histological features, the etiologic agent, the normal metabolism and the function of the prion protein, as well as the limitations of the mouse models. PMID:17598589
Gains, Malcolm J; LeBlanc, Andrea C
CELL BIOLOGY: Sowing the Protein Seeds of Prion Propagation
NSDL National Science Digital Library
Access to the article is free, however registration and sign-in are required. Ever since Prusiner first proposed his radical "protein-only" hypothesis to explain how certain infectious proteins (prions) are transmitted from one mammal to another in the absence of DNA or RNA, scientists have been trying to prove him right (or wrong). The study of mammalian prions, such as those causing Creutzfeldt-Jakob disease in humans, scrapie in sheep and mad cow disease in cattle, has been slow to yield answers. However, as Tuite discusses in his Perspective, the Sup35p and Ure2p proteins of yeast that exist in both normal and infectious forms are providing evidence that the "protein-only" hypothesis may be right (Sparrer et al.).
Mick F. Tuite (University of Kent;Department of Biosciences)
Scrapie prion protein accumulation by scrapie-infected neuroblastoma cells abrogated by exposure to a prion protein antibody.
Exposure of susceptible neuroblastoma N2a cells to mouse scrapie prions leads to infection, as evidenced by the continued presence of the scrapie form of the prion protein (PrP(Sc)) and infectivity after 300 or more cell doublings. We find that exposure to phosphatidylinositol-specific phospholipase C (PIPLC) or to the monoclonal anti-prion protein (PrP) antibody 6H4 not only prevents infection of susceptible N2a cells but also cures chronically scrapie-infected cultures, as judged by the long-term abrogation of PrP(Sc) accumulation after cessation of treatment. A nonpassaged, stationary infected culture rapidly loses PrP(Sc) when exposed to the antibody or PIPLC, indicating that the PrP(Sc) level is determined by steady state equilibrium between formation and degradation, and that depletion of the cellular form of PrP can interrupt the propagation of PrP(Sc). These findings encourage the belief that passive immunization may provide a therapeutic approach to prion disease. PMID:11470893
Enari, M; Flechsig, E; Weissmann, C
Searching for reliable premortem protein biomarkers for prion diseases: progress and challenges to date.
Prion diseases are a unique family of fatal neurodegenerative diseases caused by abnormal folding of normal cellular prion proteins in the brain. Due to the high risk of prion disease transmission and the lack of effective treatment to cure or delay the disease progression, prion diseases pose a serious threat to public health. To control and prevent prion diseases, an early diagnosis is urgently needed. Proteomic analysis has emerged as a powerful technology to decipher biological and pathophysiological processes and identify protein biomarkers indicative of disease. In this article, the authors review the use of the latest proteomic technologies for the identification of promising prion disease biomarkers, the challenges that exist in biomarker development pipelines and the new directions for utilizing proteomics for future biomarker discovery in the context of prion disease diagnostics. PMID:22809206
Ma, Di; Li, Lingjun
Structural and mechanistic commonalities of amyloid-? and the prion protein
Amyloid beta (A?) is a major causative agent of Alzheimer disease (AD). This neurotoxic peptide is generated as a result of the cleavage of the Amyloid-Precursor-Protein (APP) by the action of ?-secretase and ?-secretase. The neurotoxicity was previously thought to be the result of aggregation. However, recent studies suggest that the interaction of A? with numerous cell surface receptors such as N-methyl-D-aspartate (NMDA), receptor for advanced glycosylation end products (RAGE), P75 neurotrophin receptor (P75NTR) as well as cell surface proteins such as the cellular prion protein (PrPc) and heparan sulfate proteoglycans (HSPG) strongly enhances A? induced apoptosis and thereby contributes to neurotoxicity. This review focuses on the molecular mechanism resulting in A?-shedding as well as A?-induced apoptotic processes, genetic risk factors for familial AD and interactions of A? with cell surface receptors and proteins, with particular emphasis on the cellular prion protein. Furthermore, comparisons are drawn between AD and prion disorders and the role of laminin, an extracellular matrix protein, glycosaminoglycans and the 37 kDa/67 kDa laminin receptor (LRP/LR) have been highlighted with regards to both neurodegenerative diseases.
Da Costa Dias, Bianca; Jovanovic, Katarina; Gonsalves, Danielle
Neurotoxic Mutants of the Prion Protein Induce Spontaneous Ionic Currents in Cultured Cells*
The mechanisms by which prions kill neurons and the role of the cellular prion protein in this process are enigmatic. Insight into these questions is provided by the neurodegenerative phenotypes of transgenic mice expressing prion protein (PrP) molecules with deletions of conserved amino acids in the central region. We report here that expression in transfected cells of the most toxic of these PrP deletion mutants (?105�125) induces large, spontaneous ionic currents that can be detected by patch-clamping techniques. These currents are produced by relatively non-selective, cation-permeable channels or pores in the cell membrane and can be silenced by overexpression of wild-type PrP, as well as by treatment with a sulfated glycosaminoglycan. Similar currents are induced by PrP molecules carrying several different point mutations in the central region that cause familial prion diseases in humans. The ionic currents described here are distinct from those produced in artificial lipid membranes by synthetic peptides derived from the PrP sequence because they are induced by membrane-anchored forms of PrP that are synthesized by cells and that are found in vivo. Our results indicate that the neurotoxicity of some mutant forms of PrP is attributable to enhanced ion channel activity and that wild-type PrP possesses a channel-silencing activity. Drugs that block PrP-associated channels or pores may therefore represent novel therapeutic agents for treatment of patients with prion diseases.
Solomon, Isaac H.; Huettner, James E.; Harris, David A.
Transition-metal prion protein attachment: Competition with copper
NASA Astrophysics Data System (ADS)
Prion protein, PrP, is a protein capable of binding copper ions in multiple modes depending on their concentration. Misfolded PrP is implicated in a group of neurodegenerative diseases, which include ``mad cow disease'' and its human form, variant Creutzfeld-Jacob disease. An increasing amount of evidence suggests that attachment of non-copper metal ions to PrP triggers transformations to abnormal forms similar to those observed in prion diseases. In this work, we use hybrid Kohn-Sham/orbital-free density functional theory simulations to investigate copper replacement by other transition metals that bind to PrP, including zinc, iron and manganese. We consider all known copper binding modes in the N-terminal domain of PrP. Our calculations identify modes most susceptible to copper replacement and reveal metals that can successfully compete with copper for attachment to PrP.
Hodak, Miroslav; Bernholc, Jerry
The Cellular Concentration of the Yeast Ure2p Prion Protein Affects Its Propagation as a Prion
The [URE3] yeast prion is a self-propagating inactive form of the Ure2p protein. We show here that Ure2p from the species Saccharomyces paradoxus (Ure2pSp) can be efficiently converted into a prion form and propagate [URE3] when expressed in Saccharomyces cerevisiae at physiological level. We found however that Ure2pSp overexpression prevents efficient prion propagation. We have compared the aggregation rate and propagon numbers of Ure2pSp and of S. cerevisiae Ure2p (Ure2pSc) in [URE3] cells both at different expression levels. Overexpression of both Ure2p orthologues accelerates formation of large aggregates but Ure2pSp aggregates faster than Ure2pSc. Although the yeast cells that contain these large Ure2p aggregates do not transmit [URE3] to daughter cells, the corresponding crude extract retains the ability to induce [URE3] in wild-type [ure3-0] cells. At low expression level, propagon numbers are higher with Ure2pSc than with Ure2pSp. Overexpression of Ure2p decreases the number of [URE3] propagons with Ure2pSc. Together, our results demonstrate that the concentration of a prion protein is a key factor for prion propagation. We propose a model to explain how prion protein overexpression can produce a detrimental effect on prion propagation and why Ure2pSp might be more sensitive to such effects than Ure2pSc.
Crapeau, Myriam; Marchal, Christelle; Cullin, Christophe
Prion Protein Glycosylation Is Not Required for Strain-Specific Neurotropism? �
In this study, we tested the hypothesis that the glycosylation of the pathogenic isoform of the prion protein (PrPSc) might encode the selective neurotropism of prion strains. We prepared unglycosylated cellular prion protein (PrPC) substrate molecules from normal mouse brain by treatment with PNGase F and used reconstituted serial protein cyclic misfolding amplification reactions to produce RML and 301C mouse prions containing unglycosylated PrPSc molecules. Both RML- and 301C-derived prions containing unglycosylated PrPSc molecules were infectious to wild-type mice, and neuropathological analysis showed that mice inoculated with these samples maintained strain-specific patterns of PrPSc deposition and neuronal vacuolation. These results show that PrPSc glycosylation is not necessary for strain-dependent prion neurotropism.
Piro, Justin R.; Harris, Brent T.; Nishina, Koren; Soto, Claudio; Morales, Rodrigo; Rees, Judy R.; Supattapone, Surachai
Rapid cell-surface prion protein conversion revealed using a novel cell system
Prion diseases are fatal neurodegenerative disorders with unique transmissible properties. The infectious and pathological agent is thought to be a misfolded conformer of the prion protein. Little is known about the initial events in prion infection because the infecting prion source has been immunologically indistinguishable from normal cellular prion protein (PrPC). Here we develop a unique cell system in which epitope-tagged PrPC is expressed in a PrP knockdown (KD) neuroblastoma cell line. The tagged PrPC, when expressed in our PrP-KD cells, supports prion replication with the production of bona fide epitope-tagged infectious misfolded PrP (PrPSc). Using this epitope-tagged PrPSc, we study the earliest events in cellular prion infection and PrP misfolding. We show that prion infection of cells is extremely rapid occurring within 1 min of prion exposure, and we demonstrate that the plasma membrane is the primary site of prion conversion.
Goold, R.; Rabbanian, S.; Sutton, L.; Andre, R.; Arora, P.; Moonga, J.; Clarke, A.R.; Schiavo, G.; Jat, P.; Collinge, J.; Tabrizi, S.J.
Alzheimer Disease and the Prion Disorders Amyloid beta-Protein and Prion Protein Amyloidoses
Alzheimer disease and the prion disorders\\/spongiform encephalopathies share many common features. These chronic, progressive, sometimes familial diseases of the central nervous system are characterized by the presence of different types of amyloid deposits in the brain. This review provides a perspective on these two types of neurodegenerative disorders.
Donald L. Price; David R. Borchelt; Sangram S. Sisodia
Interaction between a recombinant prion protein and organo-mineral complexes as evidenced by CPMAS 13C-NMR
Prion proteins (PrP) are the main responsible for Transmissible Spongiform Encephalopathies (TSE). The TSE etiological agent is a misfolded form of the normal cellular prion protein. The amyloidal aggregates accumulated in the brain of infected animals and mainly composed of PrPSc exhibit resistance to protease attack and many conventional inactivating procedures. The prion protein diseases cause an environmental issue because
F. Russo; R. Scotti; L. Gianfreda; P. Conte; M. A. Rao
Visual detection of prion protein based on color complementarity principle.
Two complementary colors mixed in a proper proportion will produce a neutral color in the color theory. A novel colorimetric method on basis of the color complementarity principle has been well-established to detect recombinant prion protein (rPrP). We found that a colorless solution appeared after mixing orange CdTe quantum dots (QDs) with green-blue malachite green (MG) because of color complementarity. After the addition of rPrP into the mixed solution, the color changed from colorless to green-blue because rPrP could induce the aggregation of QDs, rapidly. And it could be observed by naked eyes. Based on this phenomenon, we developed a simple assay for visual detection of rPrP. At the same time, we obtained excellent correlation between absorption and concentrations of rPrP from 1 nmol L(-1) to 78 nmol L(-1) with the limit of detection of 0.3 nmol L(-1) (3?). Moreover, it can be applied to determine rPrP in human serum successfully. Importantly, this assay possesses the advantages of simplicity, rapidity, sensitivity, and selectivity, and shows the potential in the clinical diagnostic test of early prion disease and provides the possibility of preventing the spread of prion diseases. PMID:23827372
Liang, Liping; Long, Yijuan; Zhang, Haijie; Wang, Qinlong; Huang, Xiaoxiao; Zhu, Rui; Teng, Ping; Wang, Xiliang; Zheng, Huzhi
Manganese upregulates cellular prion protein and contributes to altered stabilization and proteolysis: relevance to role of metals in pathogenesis of prion disease.
Prion diseases are fatal neurodegenerative diseases resulting from misfolding of normal cellular prion (PrP(C)) into an abnormal form of scrapie prion (PrP(Sc)). The cellular mechanisms underlying the misfolding of PrP(C) are not well understood. Since cellular prion proteins harbor divalent metal-binding sites in the N-terminal region, we examined the effect of manganese on PrP(C) processing in in vitro models of prion disease. Exposure to manganese significantly increased PrP(C) levels both in cytosolic and in membrane-rich fractions in a time-dependent manner. Manganese-induced PrP(C) upregulation was independent of messenger RNA transcription or stability. Additionally, manganese treatment did not alter the PrP(C) degradation by either proteasomal or lysosomal pathways. Interestingly, pulse-chase analysis showed that the PrP(C) turnover rate was significantly altered with manganese treatment, indicating increased stability of PrP(C) with the metal exposure. Limited proteolysis studies with proteinase-K further supported that manganese increases the stability of PrP(C). Incubation of mouse brain slice cultures with manganese also resulted in increased prion protein levels and higher intracellular manganese accumulation. Furthermore, exposure of manganese to an infectious prion cell model, mouse Rocky Mountain Laboratory-infected CAD5 cells, significantly increased prion protein levels. Collectively, our results demonstrate for the first time that divalent metal manganese can alter the stability of prion proteins and suggest that manganese-induced stabilization of prion protein may play a role in prion protein misfolding and prion disease pathogenesis. PMID:20176619
Choi, Christopher J; Anantharam, Vellareddy; Martin, Dustin P; Nicholson, Eric M; Richt, J�rgen A; Kanthasamy, Arthi; Kanthasamy, Anumantha G
Manganese Upregulates Cellular Prion Protein and Contributes to Altered Stabilization and Proteolysis: Relevance to Role of Metals in Pathogenesis of Prion Disease
Prion diseases are fatal neurodegenerative diseases resulting from misfolding of normal cellular prion (PrPC) into an abnormal form of scrapie prion (PrPSc). The cellular mechanisms underlying the misfolding of PrPC are not well understood. Since cellular prion proteins harbor divalent metal-binding sites in the N-terminal region, we examined the effect of manganese on PrPC processing in in vitro models of prion disease. Exposure to manganese significantly increased PrPC levels both in cytosolic and in membrane-rich fractions in a time-dependent manner. Manganese-induced PrPC upregulation was independent of messenger RNA transcription or stability. Additionally, manganese treatment did not alter the PrPC degradation by either proteasomal or lysosomal pathways. Interestingly, pulse-chase analysis showed that the PrPC turnover rate was significantly altered with manganese treatment, indicating increased stability of PrPC with the metal exposure. Limited proteolysis studies with proteinase-K further supported that manganese increases the stability of PrPC. Incubation of mouse brain slice cultures with manganese also resulted in increased prion protein levels and higher intracellular manganese accumulation. Furthermore, exposure of manganese to an infectious prion cell model, mouse Rocky Mountain Laboratory�infected CAD5 cells, significantly increased prion protein levels. Collectively, our results demonstrate for the first time that divalent metal manganese can alter the stability of prion proteins and suggest that manganese-induced stabilization of prion protein may play a role in prion protein misfolding and prion disease pathogenesis.
Choi, Christopher J.; Anantharam, Vellareddy; Martin, Dustin P.; Nicholson, Eric M.; Richt, Jurgen A.; Kanthasamy, Arthi; Kanthasamy, Anumantha G.
Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding
Prions are the infectious agents responsible for transmissible spongiform encephalopathies. The principal component of prions is the glycoprotein PrPSc, which is a conformationally modified isoform of a normal cell-surface protein called PrPC (ref. 1). During the time between infection and the appearance of the clinical symptoms, minute amounts of PrPSc replicate by conversion of host PrPC, generating large amounts of
Gabriela P. Saborio; Bruno Permanne; Claudio Soto
Degradation of scrapie associated prion protein (PrP Sc ) by the gastrointestinal microbiota of cattle
A food-borne origin of the transmission of bovine spongiform encephalopathy (BSE) to cattle is commonly assumed. However, the fate of infectious prion protein during polygastric digestion remains unclear. It is unknown at present, whether infectious prion proteins, considered to be very stable, are degraded or inactivated by microbial processes in the gastrointestinal tract of cattle. In this study, rumen and
Christina Scherbel; Rohtraud Pichner; Martin H. Groschup; Simone Mueller-Hellwig; Siegfried Scherer; Richard Dietrich; Erwin Maertlbauer; Manfred Gareis
Instability of the Octarepeat Region of the Human Prion Protein Gene
Prion diseases are a family of unique fatal transmissible neurodegenerative diseases that affect humans and many animals. Sporadic Creutzfeldt-Jakob disease (sCJD) is the most common prion disease in humans, accounting for 85�90% of all human prion cases, and exhibits a high degree of diversity in phenotypes. The etiology of sCJD remains to be elucidated. The human prion protein gene has an octapeptide repeat region (octarepeats) that normally contains 5 repeats of 24�27 bp (1 nonapeptide and 4 octapeptide coding sequences). An increase of the octarepeat numbers to six or more or a decrease of the octarepeat number to three is linked to genetic prion diseases with heterogeneous phenotypes in humans. Here we report that the human octarepeat region is prone to either contraction or expansion when subjected to PCR amplification in vitro using Taq or Pwo polymerase and when replicated in wild type E. coli cells. Octarepeat insertion mutants were even less stable, and the mutation rate for the wild type octarepeats was much higher when replicated in DNA mismatch repair-deficient E.coli cells. All observed octarepeat mutants resulting from DNA replication in E.coli were contained in head-to-head plasmid dimers and DNA mfold analysis (http://mfold.rna.albany.edu/?q=mfold/DNA-Folding-Form) indicates that both DNA strands of the octarepeat region would likely form multiple stable hairpin structures, suggesting that the octarepeat sequence may form stable hairpin structures during DNA replication or repair to cause octarepeat instability. These results provide the first evidence supporting a somatic octarepeat mutation-based model for human sCJD etiology: 1) the instability of the octarepeat region leads to accumulation of somatic octarepeat mutations in brain cells during development and aging, 2) this instability is augmented by compromised DNA mismatch repair in aged cells, and 3) eventually some of the octarepeat mutation-containing brain cells start spontaneous de novo prion formation and replication to initiate sCJD.
Li, Baiya; Qing, Liuting; Yan, Jianqun; Kong, Qingzhong
Copper Coordination in the Full-Length, Recombinant Prion Protein�
The prion protein (PrP) binds divalent copper at physiologically relevant conditions and is believed to participate in copper regulation or act as a copper-dependent enzyme. Ongoing studies aim at determining the molecular features of the copper binding sites. The emerging consensus is that most copper binds in the octarepeat domain, which is composed of four or more copies of the fundamental sequence PHGGGWGQ. Previous work from our laboratory using PrP-derived peptides, in conjunction with EPR and X-ray crystallography, demonstrated that the HGGGW segment constitutes the fundamental binding unit in the octarepeat domain [Burns et al. (2002) Biochemistry 41, 3991�4001; Aronoff-Spencer et al. (2000) Biochemistry 39, 13760�13771]. Copper coordination arises from the His imidazole and sequential deprotonated glycine amides. In this present work, recombinant, full-length Syrian hamster PrP is investigated using EPR methodologies. Four copper ions are taken up in the octarepeat domain, which supports previous findings. However, quantification studies reveal a fifth binding site in the flexible region between the octarepeats and the PrP globular C-terminal domain. A series of PrP peptide constructs show that this site involves His96 in the PrP(92�96) segment GGGTH. Further examination by X-band EPR, S-band EPR, and electron spin�echo envelope spectroscopy, demonstrates coordination by the His96 imidazole and the glycine preceding the threonine. The copper affinity for this type of binding site is highly pH dependent, and EPR studies here show that recombinant PrP loses its affinity for copper below pH 6.0. These studies seem to provide a complete profile of the copper binding sites in PrP and support the hypothesis that PrP function is related to its ability to bind copper in a pH-dependent fashion.
Burns, Colin S.; Aronoff-Spencer, Eliah; Legname, Giuseppe; Prusiner, Stanley B.; Antholine, William E.; Gerfen, Gary J.; Peisach, Jack; Millhauser, Glenn L.
Molecular Interactions between Prions as Seeds and Recombinant Prion Proteins as Substrates Resemble the Biological Interspecies Barrier In Vitro
Prion diseases like Creutzfeldt-Jakob disease in humans, Scrapie in sheep or bovine spongiform encephalopathy are fatal neurodegenerative diseases, which can be of sporadic, genetic, or infectious origin. Prion diseases are transmissible between different species, however, with a variable species barrier. The key event of prion amplification is the conversion of the cellular isoform of the prion protein (PrPC) into the pathogenic isoform (PrPSc). We developed a sodiumdodecylsulfate-based PrP conversion system that induces amyloid fibril formation from soluble ?-helical structured recombinant PrP (recPrP). This approach was extended applying pre-purified PrPSc as seeds which accelerate fibrillization of recPrP. In the present study we investigated the interspecies coherence of prion disease. Therefore we used PrPSc from different species like Syrian hamster, cattle, mouse and sheep and seeded fibrillization of recPrP from the same or other species to mimic in vitro the natural species barrier. We could show that the in vitro system of seeded fibrillization is in accordance with what is known from the naturally occurring species barriers.
Panza, Giannantonio; Luers, Lars; Stohr, Jan; Nagel-Steger, Luitgard; Wei?, Jurgen; Riesner, Detlev; Willbold, Dieter; Birkmann, Eva
Molecular interactions between prions as seeds and recombinant prion proteins as substrates resemble the biological interspecies barrier in vitro.
Prion diseases like Creutzfeldt-Jakob disease in humans, Scrapie in sheep or bovine spongiform encephalopathy are fatal neurodegenerative diseases, which can be of sporadic, genetic, or infectious origin. Prion diseases are transmissible between different species, however, with a variable species barrier. The key event of prion amplification is the conversion of the cellular isoform of the prion protein (PrP(C)) into the pathogenic isoform (PrP(Sc)). We developed a sodiumdodecylsulfate-based PrP conversion system that induces amyloid fibril formation from soluble ?-helical structured recombinant PrP (recPrP). This approach was extended applying pre-purified PrP(Sc) as seeds which accelerate fibrillization of recPrP. In the present study we investigated the interspecies coherence of prion disease. Therefore we used PrP(Sc) from different species like Syrian hamster, cattle, mouse and sheep and seeded fibrillization of recPrP from the same or other species to mimic in vitro the natural species barrier. We could show that the in vitro system of seeded fibrillization is in accordance with what is known from the naturally occurring species barriers. PMID:21151607
Panza, Giannantonio; Luers, Lars; St�hr, Jan; Nagel-Steger, Luitgard; Weiss, J�rgen; Riesner, Detlev; Willbold, Dieter; Birkmann, Eva
Both Met(109) and Met(112) are utilized for Cu(II) coordination by the amyloidogenic fragment of the human prion protein at physiological pH
The prion protein is a ubiquitous neuronal membrane protein. Misfolding of the prion protein has been implicated in transmissible spongiform encephalopathies (prion diseases). It has been demonstrated that the human prion protein (PrP) is capable of coordinating at least five CuII ions under physiological conditions; four copper binding sites can be found in the octarepeat domain between residues 61 and
Jason Shearer; Pamela Soh; Stefanie Lentz
Both Met(109) and Met(112) are Utilized for Cu(II) Coordination to the Amyloidogenic Fragment of the Human Prion Protein
The prion protein is a ubiquitous neuronal membrane protein. Misfolding of the prion protein has been implicated in transmissible spongiform encephalopathies (prion diseases). It has been demonstrated that the human prion protein (PrP) is capable of coordinating at least five Cu{sup II} ions under physiological conditions; four copper binding sites can be found in the octarepeat domain between residues 61
J. Shearer; P Soh; S Lentz
High CJD infectivity remains after prion protein is destroyed.
The hypothesis that host prion protein (PrP) converts into an infectious prion form rests on the observation that infectivity progressively decreases in direct proportion to the decrease of PrP with proteinase K (PK) treatment. PrP that resists limited PK digestion (PrP-res, PrP(sc)) has been assumed to be the infectious form, with speculative types of misfolding encoding the many unique transmissible spongiform encephalopathy (TSE) agent strains. Recently, a PK sensitive form of PrP has been proposed as the prion. Thus we re-evaluated total PrP (sensitive and resistant) and used a cell-based assay for titration of infectious particles. A keratinase (NAP) known to effectively digest PrP was compared to PK. Total PrP in FU-CJD infected brain was reduced to ?0.3% in a 2 h PK digest, yet there was no reduction in titer. Remaining non-PrP proteins were easily visualized with colloidal gold in this highly infectious homogenate. In contrast to PK, NAP digestion left 0.8% residual PrP after 2 h, yet decreased titer by >2.5 log; few residual protein bands remained. FU-CJD infected cells with 10� the infectivity of brain by both animal and cell culture assays were also evaluated. NAP again significantly reduced cell infectivity (>3.5 log). Extreme PK digestions were needed to reduce cell PrP to <0.2%, yet a very high titer of 8 logs survived. Our FU-CJD brain results are in good accord with the only other report on maximal PrP destruction and titer. It is likely that one or more residual non-PrP proteins may protect agent nucleic acids in infectious particles. PMID:21793041
Miyazawa, Kohtaro; Emmerling, Kaitlin; Manuelidis, Laura
High CJD infectivity remains after prion protein is destroyed
The hypothesis that host prion protein (PrP) converts into an infectious prion form rests on the observation that infectivity progressively decreases in direct proportion to the decrease of PrP with proteinase K (PK) treatment. PrP that resists limited PK digestion (PrP-res, PrPsc) has been assumed to be the infectious form, with speculative types of misfolding encoding the many unique TSE agent strains. Recently, a PK sensitive form of PrP has been proposed as the prion. Thus we re-evaluated total PrP (sensitive and resistant) and used a cell-based assay for titration of infectious particles. A keratinase (NAP) known to effectively digest PrP was compared to PK. Total PrP in FU-CJD infected brain was reduced to ?0.3% in a 2hr PK digest, yet there was no reduction in titer. Remaining non-PrP proteins were easily visualized with colloidal gold in this highly infectious homogenate. In contrast to PK, NAP digestion left 0.8% residual PrP after 2hr, yet decreased titer by >2.5logs; few residual protein bands remained. FU-CJD infected cells with 10x the infectivity of brain by both animal and cell culture assays were also evaluated. NAP again significantly reduced cell infectivity (>3.5 logs). Extreme PK digestions were needed to reduce cell PrP to <0.2%, yet a very high titer of ?7.8 logs remained. Our FU-CJD brain results are in good accord with the only other report on maximal PrP digestion and titer. It is likely that one or more residual non-PrP proteins may protect agent nucleic acids in infectious particles.
Miyazawa, Kohtaro; Emmerling, Kaitlin; Manuelidis, Laura
Identification of scrapie prion protein-specific mRNA in scrapie-infected and uninfected brain
To date no nucleic acid has been found in the purified infectious agent which causes the spongiform encephalopathy known as scrapie. In an attempt to identify a unique scrapie virus-associated messenger RNA in tissues of infected animals, we have synthesized an oligonucleotide probe complementary to the mRNA sequence corresponding to the amino-acid sequence of the prion protein, PrP27-30 (ref. 1).
Bruce Chesebro; Richard Race; Kathy Wehrly; Jane Nishio; Marshall Bloom; David Lechner; Sven Bergstrom; Ken Robbins; Leonard Mayer; Jerry M. Keith; Claude Garon; Ashley Haase
Generation of human scFvs antibodies recognizing a prion protein epitope expressed on the surface of human lymphoblastoid cells
BACKGROUND: A hallmark of prion disease is the transformation of normal cellular prion protein (PrPc) into an infectious disease-associated isoform, (PrPsc). Anti-prion protein monoclonal antibodies are invaluable for structure-function studies of PrP molecules. Furthermore recent in vitro and in vivo studies indicate that anti-PrP monoclonal antibodies can prevent the incorporation of PrPc into propagating prions. In the present article, we
Michela Flego; Alessandro Ascione; Silvia Zamboni; Maria L Dupuis; Valentina Imperiale; Maurizio Cianfriglia
Modulation and elimination of yeast prions by protein chaperones and co-chaperones
The yeast system has provided considerable insight into the biology of amyloid and prions. Here we focus on how alterations in abundance or function of protein chaperones and co-chaperones affect propagation of yeast prions. In spite of a considerable amount of information, a clear understanding of the molecular mechanisms underlying these effects remains wanting.
Reidy, Michael
Ubiquitin Ligase gp78 Targets Unglycosylated Prion Protein PrP for Ubiquitylation and Degradation
Prion protein PrP is a central player in several devastating neurodegenerative disorders, including mad cow disease and Creutzfeltd-Jacob disease. Conformational alteration of PrP into an aggregation-prone infectious form PrPSc can trigger pathogenic events. How levels of PrP are regulated is poorly understood. Human PrP is known to be degraded by the proteasome, but the specific proteolytic pathway responsible for PrP destruction remains elusive. Here, we demonstrate that the ubiquitin ligase gp78, known for its role in protein quality control, is critical for unglycosylated PrP ubiquitylation and degradation. Furthermore, C-terminal sequences of PrP protein are crucial for its ubiquitylation and degradation. Our study reveals the first ubiquitin ligase specifically involved in prion protein PrP degradation and PrP sequences crucial for its turnover. Our data may lead to a new avenue to control PrP level and pathogenesis.
Cheng, Haili; Tsai, Yien Che; Weissman, Allan M.; Luo, Shiwen; Rao, Hai
Biological Roles of Prion Domains
In vivo amyloid formation is a widespread phenomenon in eukaryotes. Self-perpetuating amyloids provide a basis for the infectious or heritable protein isoforms (prions). At least for some proteins, amyloid-forming potential is conserved in evolution despite divergence of the amino acid (aa) sequences. In some cases, prion formation certainly represents a pathological process leading to a disease. However, there are several scenarios in which prions and other amyloids or amyloid-like aggregates are either shown or suspected to perform positive biological functions. Proven examples include self/nonself recognition, stress defense and scaffolding of other (functional) polymers. The role of prion-like phenomena in memory has been hypothesized. As an additional mechanism of heritable change, prion formation may in principle contribute to heritable variability at the population level. Moreover, it is possible that amyloid-based prions represent by-products of the transient feedback regulatory circuits, as normal cellular function of at least some prion proteins is decreased in the prion state.
Inge-Vechtomov, Sergey G; Zhouravleva, Galina A
Prion proteins: physiological functions and role in neurological disorders.
Stanley Prusiner was the first to promote the concept of misfolded proteins as a cause for neurological disease. It has since been shown by him and other investigators that the scrapie isoform of prion protein (PrP(Sc)) functions as an infectious agent in numerous human and non-human disorders of the central nervous system (CNS). Interestingly, other organ systems appear to be less affected, and do not appear to lead to major co-morbidities. The physiological function of the endogenous cellular form of the prion protein (PrP(C)) is much less clear. It is intriguing that PrP(c) is expressed on most tissues in mammals, suggesting not only biological functions outside the CNS, but also a role other than the propagation of its misfolded isotype. In this review, we summarize accumulating in vitro and in vivo evidence regarding the physiological functions of PrP(C) in the nervous system, as well as in lymphoid organs. PMID:17707411
Hu, Wei; Kieseier, Bernd; Frohman, Elliot; Eagar, Todd N; Rosenberg, Roger N; Hartung, Hans-Peter; St�ve, Olaf
Prion protein inhibits microtubule assembly by inducing tubulin oligomerization
A growing body of evidence points to an association of prion protein (PrP) with microtubular cytoskeleton. Recently, direct binding of PrP to tubulin has also been found. In this work, using standard light scattering measurements, sedimentation experiments, and electron microscopy, we show for First time the effect of a direct interaction between these proteins on tubulin polymerization. We demonstrate that full-length recombinant PrP induces a rapid increase in the turbidity of tubulin diluted below the critical concentration for microtubule assembly. This effect requires magnesium ions and is weakened by NaCl. Moreover, the PrP-induced light scattering structures of tubulin are cold-stable. In preparations of diluted tubulin incubated with PrP, electron microscopy revealed the presence of {approx}50 nm disc-shaped structures not reported so far. These unique tubulin oligomers may form large aggregates. The effect of PrP is more pronounced under the conditions promoting microtubule formation. In these tubulin samples, PrP induces formation of the above oligomers associated with short protofilaments and sheets of protofilaments into aggregates. Noticeably, this is accompanied by a significant reduction of the number and length of microtubules. Hence, we postulate that prion protein may act as an inhibitor of microtubule assembly by inducing formation of stable tubulin oligomers.
Nieznanski, Krzysztof [Nencki Institute of Experimental Biology, Department of Muscle Biochemistry, Warsaw (Poland)]. E-mail: email@example.com; Podlubnaya, Zoya A. [Institute of Theoretical and Experimental Biophysics, Laboratory of Structure and Function of Muscle Proteins, Pushchino (Russian Federation); Pushchino State University, Pushchino (Russian Federation); Nieznanska, Hanna [Nencki Institute of Experimental Biology, Department of Muscle Biochemistry, Warsaw (Poland)
Parameters that affect macromolecular self-assembly of prion protein.
Amyloidogenesis of prion protein (PrP) is closely associated with the pathobiology of prion diseases. To understand details on formation of PrP amyloids, we investigated various conditions that influence the process in vitro, using full length and truncated recombinant PrP. Disrupted agitation and fluctuated temperature resulted in prolongation of lag phase during PrP amyloid formation. With the same conditions and material for the assay, fluorescence microplate readers of different manufacturers, which are assumed to have incongruent level of mechanical performance, demonstrated variations for the length of lag phase and the level of fluorescence detection. Presence of preformed amyloid seeds accelerated PrP amyloid formation. Similarly, recombinant proteins of different species affected effectual generation of amyloids. This process was also influenced by the concentrations and truncation of recombinant PrP. By investigating several conditions to perform PrP amyloid formation assay, our study addresses the factors that determine how much and how rapidly PrP amyloids are formed. PMID:24671413
Kim, Seon-Gu; Lee, Hye-Mi; Ryou, Chongsuk
Prion protein expression in Chinese hamster ovary cells using a glutamine synthetase selection and amplification system.
Syrian hamster prion protein (PrPC) and a truncated Syrian hamster prion protein lacking the glycosylphosphatidylinositol (GPI) anchor C-terminal signal sequence (GPI-) were expressed in Chinese hamster ovary cells using a glutamine synthetase selection and amplification system. The CHO cell clones expressing the GPI- PrP secreted the majority of the protein into the media, whereas most of the PrP produced by clones expressing the full-length protein with the GPI anchor was located on the cell surface, as demonstrated by its release upon treatment with phosphatidylinositol-specific phospholipase C (PIPLC). A cell clone that expressed the highest levels of full length PrP was subcloned to obtain clone 30C3-1. PrP from clone 30C3-1 was shown to be sensitive to proteolysis by proteinase K and to react with monoclonal and polyclonal antibodies that recognize native PrPC. The recombinant PrP migrated as a diffuse band of 19-40 kDa but removal of the N-linked oligosaccharides with peptide N-glycosidase F (PNGase F) revealed three protein species of 19, 17 and 15 kDa. The 19 kDa band corresponding to deglycosylated full-length PrP was quantified and found to be expressed at a level approximately 14-fold higher than that of PrPC found in Syrian hamster brain. PMID:9543009
Blochberger, T C; Cooper, C; Peretz, D; Tatzelt, J; Griffith, O H; Baldwin, M A; Prusiner, S B
Stressing Out the ER: A Role of the Unfolded Protein Response in Prion-Related Disorders
Transmissible Spongiform Encephalopathies are fatal and infectious neurodegenerative diseases characterized by extensive neuronal apoptosis and the accumulation of an abnormally folded form of the cellular prion protein (PrP), denoted PrPSC. Compelling evidence suggests the involvement of several signaling pathways in prion pathogenesis, including proteasome dysfunction, alterations in the protein maturation pathways and the unfolded protein response. Recent reports indicate that endoplasmic reticulum stress due to the PrP misfolding may be a critical factor mediating neuronal dysfunction in prion diseases. These findings have applications for developing novel strategies for treatment and early diagnosis of transmissible spongiform encephalopathies and other neurodegenerative diseases.
Hetz, Claudio A.; Soto, Claudio
NSDL National Science Digital Library
Prion Links, provided by Eiso AB of the Department of Biochemistry at the University of Groningen (Netherlands), contains 39 diverse links related to prion diseases and research. Although prion research has been going on for over 25 years, the scientific and medical communities have only recently acknowledged the existence of prions and there remains serious debate over their role in a variety of neurological diseases. The name "prion" is derived from "proteinaceous infectious particles," and was coined by Dr. Stanley Prusiner, who discovered the agents and who recently received the Nobel Prize for Medicine for his work. Prions are thought to be the first transmissible and heritable disease-causing agents that lack DNA and RNA. They are composed solely of protein and appear to be the cause of such diseases as kuru and Creutzfeldt-Jakob disease in humans, and bovine spongiform encephalopathies, mad cow disease, and scrapie in sheep and goats.
Ab, Eiso.
Genetic and epigenetic control of the efficiency and fidelity of cross-species prion transmission
Summary Self-perpetuating amyloid-based protein isoforms (prions) transmit neurodegenerative diseases in mammals and phenotypic traits in yeast. Although mechanisms that control species-specificity of prion transmission are poorly understood, studies of closely related orthologs of yeast prion protein Sup35 demonstrate that cross-species prion transmission is modulated by both genetic (specific sequence elements) and epigenetic (prion variants, or �strains�) factors. Depending on the prion variant, the species barrier could be controlled at the level of either heterologous coaggregation or conversion of the aggregate-associated heterologous protein into a prion polymer. Sequence divergence influences cross-species transmission of different prion variants in opposing ways. The ability of a heterologous prion domain to either faithfully reproduce or irreversibly switch the variant-specific prion patterns depends on both sequence divergence and the prion variant. Sequence variations within different modules of prion domains contribute to transmission barriers in different cross-species combinations. Individual amino acid substitutions within short amyloidogenic stretches drastically alter patterns of cross-species prion conversion, implicating these stretches as major determinants of species specificity.
Chen, Buxin; Bruce, Kathryn L.; Newnam, Gary P.; Gyoneva, Stefka; Romanyuk, Andrey V.; Chernoff, Yury O.
Enhancement of protein misfolding cyclic amplification by using concentrated cellular prion protein source
Protein misfolding cyclic amplification (PMCA) is a cell-free assay mimicking the prion replication process. However, constraints affecting PMCA have not been well-defined. Although cellular prion protein (PrPC) is required for prion replication, the influence of PrPC abundance on PMCA has not been assessed. Here, we show that PMCA was enhanced by using mouse brain material in which PrPC was overexpressed. Tg(MoPrP)4112 mice overexpressing PrPC supported more sensitive and efficient PMCA than wild type mice. As brain homogenate of Tg(MoPrP)4112 mice was diluted with PrPC-deficient brain material, PMCA became less robust. Our studies suggest that abundance of PrPC is a determinant that directs enhancement of PMCA. PMCA established here will contribute to optimizing conditions to enhance PrPSc amplification by using concentrated PrPC source and expands the use of this methodology.
Mays, Charles E.; Titlow, William; Seward, Tanya; Telling, Glenn C.; Ryou, Chongsuk
Characterization of Conformation-dependent Prion Protein Epitopes*
Whereas prion replication involves structural rearrangement of cellular prion protein (PrPC), the existence of conformational epitopes remains speculative and controversial, and PrP transformation is monitored by immunoblot detection of PrP(27�30), a protease-resistant counterpart of the pathogenic scrapie form (PrPSc) of PrP. We now describe the involvement of specific amino acids in conformational determinants of novel monoclonal antibodies (mAbs) raised against randomly chimeric PrP. Epitope recognition of two mAbs depended on polymorphisms controlling disease susceptibility. Detection by one, referred to as PRC5, required alanine and asparagine at discontinuous mouse PrP residues 132 and 158, which acquire proximity when residues 126�218 form a structured globular domain. The discontinuous epitope of glycosylation-dependent mAb PRC7 also mapped within this domain at residues 154 and 185. In accordance with their conformational dependence, tertiary structure perturbations compromised recognition by PRC5, PRC7, as well as previously characterized mAbs whose epitopes also reside in the globular domain, whereas conformation-independent epitopes proximal or distal to this region were refractory to such destabilizing treatments. Our studies also address the paradox of how conformational epitopes remain functional following denaturing treatments and indicate that cellular PrP and PrP(27�30) both renature to a common structure that reconstitutes the globular domain.
Kang, Hae-Eun; Weng, Chu Chun; Saijo, Eri; Saylor, Vicki; Bian, Jifeng; Kim, Sehun; Ramos, Laylaa; Angers, Rachel; Langenfeld, Katie; Khaychuk, Vadim; Calvi, Carla; Bartz, Jason; Hunter, Nora; Telling, Glenn C.
Isolation and characterization of a polymerized prion protein.
A polymerized form of recombinant mouse prion protein (mPrP) domain 23-231 [mPrP-(23-231)], designated mPrP-z, was generated at acidic pH (pH 2-5) in the presence of selected concentrations of denaturant (2 M guanidinium chloride or 5 M urea). This isoform of mPrP is stable in acidic solution after removal of denaturant. It can be isolated and purified using reversed-phase HPLC or size-exclusion HPLC. mPrP-z bears structural properties that partially resemble those of scrapie prion. Unlike the native mPrP-(23-231) (mPrP-N), mPrP-z exhibits a high content of beta-sheet structure, as shown by CD spectroscopy, and exists as an oligomer with an approximate molecular mass of 340000 Da, as measured by light scattering. However, similarly to mPrP-N, mPrP-z contains the intact disulphide bond and is sensitive to digestion by proteinase K.
Lu, Bao-Yuan; Chang, Jui-Yoa
NMR structure of the mouse prion protein domain PrP(121-231)
THE 'protein only' hypothesis1 states that a modified form of normal prion protein triggers infectious neurodegenerative diseases, such as bovine spongiform encephalopathy (BSE), or Creutzfeldt-Jakob disease (CJD) in humans2-4. Prion proteins are thought to exist in two different conformations5: the 'benign' PrPC form, and the infectious 'scrapie form', PrPSc. Knowledge of the three-dimensional structure of PrPC is essential for understanding
Roland Riek; Simone Hornemann; Gerhard Wider; Martin Billeter; Rudi Glockshuber; Kurt W�thrich
Functional disruption of the prion protein gene in cloned goats.
The cellular prion protein (PrPC), a membrane glycoprotein anchored to the outer surface of neurons, lymphocytes and other cells, is associated directly with the pathogenesis of the transmissible spongiform encephalopathies (TSEs) occurring mainly in humans, cattle, sheep and goats. Although mice lacking PrPC develop and reproduce normally and are resistant to scrapie infection, large animals lacking PrPC, especially those species in which TSE occurs naturally, are currently not available. Here, five live PRNP+/- goats cloned by gene targeting are reported. Detailed RNA-transcription and protein-expression analysis of one PRNP+/- goat showed that one allele of the caprine PRNP gene had been disrupted functionally. No gross abnormal development or behaviour could be seen in these PRNP+/- goats up to at least 3 months of age. These heterozygous PRNP+/- goats are ready to be used in producing homozygous PRNP-/- goats in which no PrPC should be expressed. PMID:16528053
Yu, Guohua; Chen, Jianquan; Yu, Huiqing; Liu, Siguo; Chen, Juan; Xu, Xujun; Sha, Hongying; Zhang, Xufeng; Wu, Guoxiang; Xu, Shaofu; Cheng, Guoxiang
The Prion Protein Preference of Sporadic Creutzfeldt-Jakob Disease Subtypes*
Sporadic Creutzfeldt-Jakob disease (CJD) is the most prevalent manifestation of the transmissible spongiform encephalopathies or prion diseases affecting humans. The disease encompasses a spectrum of clinical phenotypes that have been correlated with molecular subtypes that are characterized by the molecular mass of the protease-resistant fragment of the disease-related conformation of the prion protein and a polymorphism at codon 129 of the gene encoding the prion protein. A cell-free assay of prion protein misfolding was used to investigate the ability of these sporadic CJD molecular subtypes to propagate using brain-derived sources of the cellular prion protein (PrPC). This study confirmed the presence of three distinct sporadic CJD molecular subtypes with PrPC substrate requirements that reflected their codon 129 associations in vivo. However, the ability of a sporadic CJD molecular subtype to use a specific PrPC substrate was not determined solely by codon 129 as the efficiency of prion propagation was also influenced by the composition of the brain tissue from which the PrPC substrate was sourced, thus indicating that nuances in PrPC or additional factors may determine sporadic CJD subtype. The results of this study will aid in the design of diagnostic assays that can detect prion disease across the diversity of sporadic CJD subtypes.
Klemm, Helen M. J.; Welton, Jeremy M.; Masters, Colin L.; Klug, Genevieve M.; Boyd, Alison; Hill, Andrew F.; Collins, Steven J.; Lawson, Victoria A.
SUMMARY Prion protein (PrP) prevents Bax-mediated cell death by inhibiting the initial Bax conformational change that converts cytosolic Bax into a pro-apoptotic protein. PrP is mostly a glycophosphatidylinositol-anchored cell surface protein but it is also retrotranslocated into cytosolic PrP (CyPrP) or can become a type 1 or type 2 transmembrane protein. To determine the form and subcellular location of the PrP that has anti-Bax function, we co-expressed various Syrian hamster PrP (SHaPrP) mutants that favour specific PrP topologies and subcellular localization with N-terminally green fluorescent protein tagged pro-apoptotic Bax (EGFP-Bax) in MCF-7 cells and primary human neurons. Mutants that generate both CyPrP and secreted PrP (SecPrP) or only CyPrP have anti-Bax activity. Mutants that produce CtmPrP or NtmPrP lose the anti-Bax activity, despite their ability to also make SecPrP. Transmembrane generating mutants do not produce CyPrP and both normal and cognate mutant forms of CyPrP rescue against the loss of anti-Bax activity. SecPrP generating constructs also produce non-membrane attached SecPrP. However, this form of PrP has minimal anti-Bax activity. We conclude that CyPrP is the predominant form of PrP with anti-Bax function. These results imply that the retro-translocation of PrP encompasses a survival function and is not merely a pathway for the proteasomal degradation of misfolded protein.
Lin, David T. S.; Jodoin, Julie; Baril, Michael; Goodyer, Cynthia G.; LeBlanc, Andrea C.
Prion Protein Expression and Functional Importance in Skeletal Muscle
Abstract Skeletal muscle expresses prion protein (PrP) that buffers oxidant activity in neurons. Aims We hypothesize that PrP deficiency would increase oxidant activity in skeletal muscle and alter redox-sensitive functions, including contraction and glucose uptake. We used real-time polymerase chain reaction and Western blot analysis to measure PrP mRNA and protein in human diaphragm, five murine muscles, and muscle-derived C2C12 cells. Effects of PrP deficiency were tested by comparing PrP-deficient mice versus wild-type mice and morpholino-knockdown versus vehicle-treated myotubes. Oxidant activity (dichlorofluorescin oxidation) and specific force were measured in murine diaphragm fiber bundles. Results PrP content differs among mouse muscles (gastrocnemius>extensor digitorum longus, EDL>tibialis anterior, TA; soleus>diaphragm) as does glycosylation (di-, mono-, nonglycosylated; gastrocnemius, EDL, TA=60%, 30%, 10%; soleus, 30%, 40%, 30%; diaphragm, 30%, 30%, 40%). PrP is predominantly di-glycosylated in human diaphragm. PrP deficiency decreases body weight (15%) and EDL mass (9%); increases cytosolic oxidant activity (fiber bundles, 36%; C2C12 myotubes, 7%); and depresses specific force (12%) in adult (8�12?mos) but not adolescent (2?mos) mice. Innovation This study is the first to directly assess a role of prion protein in skeletal muscle function. Conclusions PrP content varies among murine skeletal muscles and is essential for maintaining normal redox homeostasis, muscle size, and contractile function in adult animals. Antioxid. Redox Signal. 15, 2465�2475.
Smith, Jeffrey D.; Moylan, Jennifer S.; Hardin, Brian J.; Chambers, Melissa A.; Estus, Steven; Telling, Glenn C.
Detection of the Prion Protein in a Liquid Phase Capture Assay Using Magnetic Beads Coupled to Protein A
Detection of the abnormal prion protein in blood and cerebral spinal fluid of transmissible spongiform encephalophathy (TSE) infected individuals has not been possible by Western blot, immunohistochemistry or the present ELISA tests. We used an analytical approach in conjugation with a fluorescence immunoassay to develop methods to measure the abnormal prion in blood and cerebral spinal fluid. Monoclonal antibodies (mabs)
Wen-Chu Yang; Edward Yeung; Mary Schmerr; Walter Bodemer
Crystallographic Studies of Prion Protein (PrP) Segments Suggest How Structural Changes Encoded by Polymorphism at Residue 129 Modulate Susceptibility to Human Prion Disease
A single nucleotide polymorphism (SNP) in codon 129 of the human prion gene, leading to a change from methionine to valine at residue 129 of prion protein (PrP), has been shown to be a determinant in the susceptibility to prion disease. However, the molecular basis of this effect remains unexplained. In the current study, we determined crystal structures of prion segments having either Met or Val at residue 129. These 6-residue segments of PrP centered on residue 129 are 'steric zippers,' pairs of interacting {beta}-sheets. Both structures of these 'homozygous steric zippers' reveal direct intermolecular interactions between Met or Val in one sheet and the identical residue in the mating sheet. These two structures, plus a structure-based model of the heterozygous Met-Val steric zipper, suggest an explanation for the previously observed effects of this locus on prion disease susceptibility and progression.
Apostol, Marcin I.; Sawaya, Michael R.; Cascio, Duilio; Eisenberg, David (UCLA)
Rational targeting for prion therapeutics
Prions � pathogens that are lethal to humans and other animals � are thought to be conformational isomers of the cellular prion protein. Their unique biology, and the potential for a wider pathobiological significance of prion-like mechanisms, has motivated much research into understanding prion neurodegeneration. Moreover, concerns that extensive dietary exposure to bovine spongiform encephalopathy (BSE) prions might have infected
Giovanna Mallucci; John Collinge
Molecular Cross-talk between Misfolded Proteins in Animal Models of Alzheimer's and Prion Diseases
The central event in Protein Misfolding Disorders (PMDs) is the accumulation of a misfolded form of a naturally expressed protein. Despite the diversity of clinical symptoms associated to different PMDs, many similarities in their mechanism suggest that distinct pathologies may cross-talk at the molecular level. The main goal of this study was to analyze the interaction of the protein misfolding processes implicated in Alzheimer�s and prion diseases. For this purpose we inoculated prions in an Alzheimer�s transgenic mouse model that develop typical amyloid plaques and followed the progression of pathological changes over time. Our findings show a dramatic acceleration and exacerbation of both pathologies. The onset of prion disease symptoms in transgenic mice appeared significantly faster with a concomitant increase on the level of misfolded prion protein in the brain. A striking increase in amyloid plaque deposition was observed in prion infected mice compared with their non-inoculated counterparts. Histological and biochemical studies showed the association of the two misfolded proteins in the brain and in vitro experiments showed that protein misfolding can be enhanced by a cross-seeding mechanism. These results suggest a profound interaction between Alzheimer�s and prion pathologies, indicating that one protein misfolding process may be an important risk factor for the development of a second one. Our findings may have important implications to understand the origin and progression of PMDs.
Morales, Rodrigo; Estrada, Lisbell D.; Diaz-Espinoza, Rodrigo; Morales-Scheihing, Diego; Jara, Maria C.; Castilla, Joaquin; Soto, Claudio
Three hamster species with different scrapie incubation times and neuropathological features encode distinct prion proteins.
Given the critical role of the prion protein (PrP) in the transmission and pathogenesis of experimental scrapie, we investigated the PrP gene and its protein products in three hamster species, Chinese (CHa), Armenian (AHa), and Syrian (SHa), each of which were found to have distinctive scrapie incubation times. Passaging studies demonstrated that the host species, and not the source of scrapie prions, determined the incubation time for each species, and histochemical studies of hamsters with clinical signs of scrapie revealed characteristic patterns of neuropathology. Northern (RNA) analysis showed the size of PrP mRNA from CHa, AHa, and SHa hamsters to be 2.5, 2.4, and 2.1 kilobases, respectively. Immunoblotting demonstrated that the PrP isoforms were of similar size (33 to 35 kilodaltons); however, the monoclonal antibody 13A5 raised against SHa PrP did not react with the CHa or AHa PrP molecules. Comparison of the three predicted amino acid sequences revealed that each is distinct. Furthermore, differences within the PrP open reading frame that uniquely distinguish the three hamster species are within a hydrophilic segment of 11 amino acids that includes polymorphisms linked to scrapie incubation times in inbred mice and an inherited prion disease of humans. Single polymorphisms in this region correlate with the presence or absence of amyloid plaques for a given hamster species or mouse inbred strain. Our findings demonstrate distinctive molecular, pathological, and clinical characteristics of scrapie in three related species and are consistent with the hypothesis that molecular properties of the host PrP play a pivotal role in determining the incubation time and neuropathological features of scrapie. Images
Lowenstein, D H; Butler, D A; Westaway, D; McKinley, M P; DeArmond, S J; Prusiner, S B
Observation of intermediate states of the human prion protein by high pressure NMR spectroscopy
BACKGROUND: Prions as causative agents of transmissible spongiform encephalopathies (TSEs) in humans and animals are composed of the infectious isomer, PrPSc, of the cellular prion protein, PrPC. The conversion and thus the propensity of PrPC to adopt alternative folds leads to the species-specific propagation of the disease. High pressure is a powerful tool to study the physico-chemical properties of proteins
Norman Kachel; Werner Kremer; Ralph Zahn; Hans Robert Kalbitzer
A novel insertional mutation in the prion protein gene: clinical and bio-molecular findings
A young man, presenting with early onset of personality and behavioural changes followed by slowly progressive cognitive impairment associated with marked bi-parietal cerebral atrophy, was found to carry a novel seven extra-repeat insertional mutation in the prion protein gene (PRNP). In vitro, the mutated recombinant prion protein (PrP) showed biochemical properties that were consistent with pathological PrP variants. Our results
C Mauro; G Giaccone; G Piscosquito; A Lavorgna; M Nigro; G Di Fede; A Leonardi; C Coppola; S Formisano; F Tagliavini; R Cotrufo; G Puoti
Interaction between Prion Protein and A? Amyloid Fibrils Revisited.
Recent studies indicate that the pathogenesis of Alzheimer disease may be related to the interaction between prion protein (PrP) and certain oligomeric species of A? peptide. However, the mechanism of this interaction remains unclear and controversial. Here we provide direct experimental evidence that, in addition to previously demonstrated binding to A? oligomers, PrP also interacts with mature A? fibrils. However, contrary to the recent claim that PrP causes fragmentation of A? fibrils into oligomeric species, no evidence for such a disassembly could be detected in the present study. In contrast, our data indicate that the addition of PrP to preformed A? fibrils results in a lateral association of individual fibrils into larger bundles. These findings have potentially important implications for understanding the mechanism by which PrP might impact A? toxicity as well as for the emerging efforts to use PrP-derived compounds as inhibitors of A?-induced neurodegeneration. PMID:24669873
Nieznanski, Krzysztof; Surewicz, Krystyna; Chen, Shugui; Nieznanska, Hanna; Surewicz, Witold K
Prion protein codon 129 polymorphism and risk of Alzheimer disease.
The authors investigated the PRNP Met129Val polymorphism in 1,393 subjects including 482 patients with Alzheimer disease (AD) and two independent control groups. In patients, PRNP Met homozygosity conferred increasing risk with decreasing age at onset (onset: 61 to 70 years, n = 151, p = 0.02, odds ratio [OR] = 1.72, 95% CI = 1.2 to 2.53; onset: < or =60 years, n = 138, p = 0.013, OR = 1.92, 95% CI = 1.31 to 2.87), whereas no association was obtained in patients with onset at older than 70 years. The results suggest involvement of the prion protein in the pathogenesis of early-onset AD. PMID:15277640
Riemenschneider, M; Klopp, N; Xiang, W; Wagenpfeil, S; Vollmert, C; M�ller, U; F�rstl, H; Illig, T; Kretzschmar, H; Kurz, A
Prion protein detection in serum using micromechanical resonator arrays.
Prion proteins that have transformed from their normal cellular counterparts (PrP(c)) into infectious form (PrP(res)) are responsible for causing progressive neurodegenerative diseases in numerous species, such as bovine spongiform encephalopathy (BSE) in cattle (also known as mad cow disease), scrapie in sheep, and Creutzfeldt-Jakob disease (CJD) in humans. Due to a possible link between BSE and CJD it is highly desirable to develop non-invasive and ante mortem tests for the detection of prion proteins in bovine samples. Such ante mortem tests of all cows prior to slaughter will help to prevent the introduction of PrP(res) into the human food supply. Furthermore, detection of PrP(res) in donated blood will also help to prevent the transmission of CJD among humans through blood transfusion. In this study, we have continued development of a micromechanical resonator array that is capable of detecting PrP(c) in bovine blood serum. The sensitivity of the resonators for the detection of PrP(c) is further enhanced by the use of secondary mass labels. A pair of antibodies is used in a sandwich immunoassay format to immobilize PrP(c) on the surface of resonators and attach nanoparticles as secondary mass labels to PrP(c). Secondary mass labeling is optimized in terms of incubation time to maximize the frequency shifts that correspond to the presence of PrP(c) on the surface of resonators. Our results show that a minimum of 200 pg mL(-1) of PrP(c) in blood serum can be detected using micromechanical resonator arrays. PMID:19836525
Varshney, Madhukar; Waggoner, Philip S; Montagna, Richard A; Craighead, Harold G
Lactoferrin from bovine colostrum regulates prolyl hydroxylase 2 activity and prevents prion protein-mediated neuronal cell damage via cellular prion protein.
Prion disorders are associated with the conversion of normal cellular prion protein (PrPc) to the abnormal scrapie isoform of prion protein (PrPsc). Recent studies have shown that expression of normal PrPc is regulated by hypoxia-inducible factor-1 alpha (HIF-1?), and that lactoferrin increases full-length PrPc on the cell surface. Lactoferrin is an 80-kDa iron-binding glycoprotein with various biological activities, including iron-chelating ability. HIF-1? and the associated ubiquitin-proteasome pathway are regulated by HIF prolyl-hydroxylases 2 (PHD2). We hypothesized that lactoferrin regulates PHD2 expression and enzymatic activity, and the PHD2 regulation promotes HIF-1? stability and prevention of neuronal cell death mediated by prion protein (PrP) residues (106-126). Lactoferrin prevented PrP (106-126)-induced neurotoxicity by the induction of PrPc expression via promoting HIF-1? stability in neuronal cells. Our results demonstrated that lactoferrin prevented PrP (106-126)-induced neurotoxicity via the up-regulation of HIF-1? stability determined by PHD2 expression and enzymatic activity. These findings suggest that possible therapies such as PHD2 inhibition, or promotion of lactoferrin secretion, may have clinical benefits in neurodegenerative diseases, including prion disease. PMID:24875174
Park, Y-G; Moon, J-H; Park, S-Y
Aggregation of prion protein with insertion mutations is proportional to the number of inserts
Mutation in the prion gene, PRNP, accounts for approx. 10�15% of human prion diseases. However, little is known about the mechanisms by which a mutant prion protein (PrP) causes disease. We compared the biochemical properties of a wild-type human prion protein, rPrPC (recombinant wild-type PrP), which has five octapeptide-repeats, with two recombinant human prion proteins with insertion mutations, one with three more octapeptide repeats, rPrP8OR, and the other with five more octapeptide repeats, rPrP10OR. We found that the insertion mutant proteins are more prone to aggregate, and the degree and kinetics of aggregation are proportional to the number of inserts. The octapeptide-repeat and ?-helix 1 regions are important in aggregate formation, because aggregation is inhibited with monoclonal antibodies that are specific for epitopes in these regions. We also showed that a small amount of mutant protein could enhance the formation of mixed aggregates that are composed of mutant protein and wild-type rPrPC. Accordingly, rPrP10OR is also more efficient in promoting the aggregation of rPrPC than rPrP8OR. These findings provide a biochemical explanation for the clinical observations that the severity of the disease in patients with insertion mutations is proportional to the number of inserts, and thus have implications for the pathogenesis of inherited human prion disease.
Yu, Shuiliang; Yin, Shaoman; Li, Chaoyang; Wong, Poki; Chang, Binggong; Xiao, Fan; Kang, Shin-Chung; Yan, Huimin; Xiao, Gengfu; Tien, Po; Sy, Man-Sun
Antemortem Detection and Conformational Switches of Prion Proteins.
National Technical Information Service (NTIS)
Blood from animals with prion disease contain low levels of prion infectivity, which primarily resides in the white blood cells (WBCs). We have developed a method that combines isolation of WBCs and cell blotting of PrPsc to detect individual cells that c...
D. Schubert
Diagnosing prion diseases: needs, challenges and hopes
Prion diseases are among the most intriguing infectious diseases and are associated with unconventional proteinaceous infectious agents known as prions. Prions seem to lack nucleic acid and propagate by transmission of protein misfolding. The nature of prions and their unique mode of transmission present challenges for early diagnosis of prion diseases. In this article, state-of-the-art prion diagnostic techniques, together with
Claudio Soto
Structural characterization of ?-sheeted oligomers formed on the pathway of oxidative prion protein aggregation in vitro
The pathology of transmissible spongiform encephalopathies (TSEs) is strongly associated with the structural conversion of the cellular prion protein (PrPC) into a misfolded isoform (PrPSc) that assembles into amyloid fibrils. Since increased levels of oxidative stress have been linked to prion diseases, we investigated the metal-induced oxidation of human PrP (90�231). A novel in vitro conversion assay based on aerobic
Lars Redecke; Martin von Bergen; Joachim Clos; Peter V. Konarev; Dimitri I. Svergun; Ursula E. A. Fittschen; Jos� A. C. Broekaert; Oliver Bruns; Dessislava Georgieva; Eckhard Mandelkow; Nicolay Genov; Christian Betzel
Altered circadian activity rhythms and sleep in mice devoid of prion protein
THERE is a wealth of data supporting a central role for the prion protein (PrP) in the neurodegenerative prion diseases of both humans and other species1, yet the normal function of PrP, which is expressed at the cell surface of neurons and glial cells2,3, is unknown. It has been speculated that neuropathology may be due to loss of normal function
I. Tobler; S. E. Gaus; T. Deboer; P. Achermann; M. Fischer; T. R�licke; M. Moser; B. Oesch; P. A. McBride; J. C. Manson
The Comprehensive Native Interactome of a Fully Functional Tagged Prion Protein
The enumeration of the interaction partners of the cellular prion protein, PrPC, may help clarifying its elusive molecular function. Here we added a carboxy proximal myc epitope tag to PrPC. When expressed in transgenic mice, PrPmyc carried a GPI anchor, was targeted to lipid rafts, and was glycosylated similarly to PrPC. PrPmyc antagonized the toxicity of truncated PrP, restored prion
Dorothea Rutishauser; Kirsten D. Mertz; Rita Moos; Erich Brunner; Thomas R�licke; Anna Maria Calella; Adriano Aguzzi
NSDL National Science Digital Library
Prion Diseases is one of a set of lecture notes for Virology 335 by Shaun Heaphy of Leicester University (UK). It contains detailed information on its topic, along with selected links. Although prion research has been going on for over 25 years, the scientific and medical communities have only recently acknowledged the existence of prions and there remains serious debate over their role in a variety of neurological diseases. The name "prion" is derived from "proteinaceous infectious particles," and was coined by Dr. Stanley Prusiner, who discovered the agents and who recently received the Nobel Prize for Medicine for his work. Prions are thought to be the first transmissible and heritable disease-causing agents that lack DNA and RNA. They are composed solely of protein and appear to be the cause of such diseases as kuru and Creutzfeldt-Jakob disease in humans, and bovine spongiform encephalopathies, mad cow disease, and scrapie in sheep and goats.
Heaphy, Shaun.
Conformational polymorphism of the amyloidogenic peptide homologous to residues 113-127 of the prion protein.
Conformational transitions are thought to be the prime mechanism of amyloid formation in prion diseases. The prion proteins are known to exhibit polymorphic behavior that explains their ability of "conformation switching" facilitated by structured "seeds" consisting of transformed proteins. Oligopeptides containing prion sequences showing the polymorphism are not known even though amyloid formation is observed in these fragments. In this work, we have observed polymorphism in a 15-residue peptide PrP (113-127) that is known to form amyloid fibrils on aging. To see the polymorphic behavior of this peptide in different solvent environments, circular dichroism (CD) spectroscopic studies on an aqueous solution of PrP (113-127) in different trifluoroethanol (TFE) concentrations were carried out. The results show that PrP (113-127) have sheet preference in lower TFE concentration whereas it has more helical conformation in higher TFE content (>40%). The structural transitions involved in TFE solvent were studied using interval-scan CD and FT-IR studies. It is interesting to note that the alpha-helical structure persists throughout the structural transition process involved in amyloid fibril formation implicating the involvement of both N- and C-terminal sequences. To unravel the role of the N-terminal region in the polymorphism of the PrP (113-127), CD studies on another synthetic peptide, PrP (113-120) were carried out. PrP(113-120) exhibits random coil conformation in 100% water and helical conformation in 100% TFE, indicating the importance of full-length sequence for beta-sheet formation. Besides, the influence of different chemico-physical conditions such as concentration, pH, ionic strength, and membrane like environment on the secondary structure of the peptide PrP (113-127) has been investigated. At higher concentration, PrP (113-127) shows features of sheet conformation even in 100% TFE suggesting aggregation. In the presence of 5% solution of sodium dodecyl sulfate, PrP (113-127) takes high alpha-helical propensity. The environment-dependent conformational polymorphism of PrP (113-127) and its marked tendency to form stable beta-sheet structure at acidic pH could account for its conformation switching behavior from alpha-helix to beta-sheet. This work emphasizes the coordinative involvement of N-terminal and C-terminal sequences in the self-assembly of PrP (113-127). PMID:12829502
Satheeshkumar, K S; Jayakumar, R
Unusual cerebral vascular prion protein amyloid distribution in scrapie-infected transgenic mice expressing anchorless prion protein
Background In some prion diseases, misfolded aggregated protease-resistant prion protein (PrPres) is found in brain as amyloid, which can cause cerebral amyloid angiopathy. Small diffusible precursors of PrPres amyloid might flow with brain interstitial fluid (ISF), possibly accounting for the perivascular and intravascular distribution of PrPres amyloid. We previously reported that PrPres amyloid in scrapie-infected transgenic mice appeared to delay clearance of microinjected brain ISF tracer molecules. Results Here we studied distribution of PrPres amyloid on capillaries, arteries and veins to test whether vascular specificity of PrPres corresponded to distribution of ISF tracer molecules. To distinguish PrPres-positive arteries from veins and capillaries, scrapie-infected mouse brains were studied by immunodetection of alpha smooth muscle actin. ISF was studied using fluorescein-labeled ovalbumin microinjected into brain as a tracer. In infected preclinical or clinical mice, PrPres was found mostly on capillaries (73-78%). Lower levels were found on arteries (11-14%) and veins (11-13%). Compared to PrPres, ISF tracer was found at higher levels on capillaries (96-97%), and the remaining tracer was found at a skewed ratio of 4 to 1 on arteries and veins respectively. Conclusions PrPres association with blood vessels suggested that ISF flow might transport diffusible PrPres precursor molecules to perivascular sites. However, the different vascular specificity of PrPres and ISF tracer indicated that ISF flow did not alone control PrPres dissemination. Possibly blood vessel basement membrane (BM) components, such as glucosaminoglycans, might concentrate small PrPres aggregates and serve as scaffolds for PrP conversion on multiple vessel types.
Interactions of Prion Protein With Intracellular Proteins: So Many Partners and no Consequences?
Prion protein (PrP) plays a key role in the pathogenesis of transmissible spongiform encephalopathies (TSEs)�fatal diseases\\u000a of the central nervous system. Its physiological function as well as exact role in neurodegeneration remain unclear, hence\\u000a screens for proteins interacting with PrP seem to be the most promising approach to elucidating these issues. PrP is mostly\\u000a a plasma membrane-anchored extracellular glycoprotein and
Krzysztof Nieznanski
N-terminal peptides from unprocessed prion proteins enter cells by macropinocytosis
A peptide derived from the N-terminus of the unprocessed bovine prion protein (bPrPp), incorporating the hydrophobic signal sequence (residues 1-24) and a basic domain (KKRPKP, residues 25-30), internalizes into mammalian cells, even when coupled to a sizeable cargo, and therefore functions as a cell-penetrating peptide (CPP). Confocal microscopy and co-localization studies indicate that the internalization of bPrPp is mainly through macropinocytosis, a fluid-phase endocytosis process, initiated by binding to cell-surface proteoglycans. Electron microscopy studies show internalized bPrPp-DNA-gold complexes residing in endosomal vesicles. bPrPp induces expression of a complexed luciferase-encoding DNA plasmid, demonstrating the peptide's ability to transport the cargo across the endosomal membrane and into the cytosol and nucleus. The novel CPP activity of the unprocessed N-terminal domain of PrP could be important for the retrotranslocation of partly processed PrP and for PrP trafficking inside or between cells, with implications for the infectivity associated with prion diseases.
Magzoub, Mazin [Department of Biochemistry and Biophysics, Stockholm University (Sweden); Sandgren, Staffan [Department of Clinical Sciences, Section for Oncology, Lund University (Sweden); Lundberg, Pontus [Department of Neurochemistry, Stockholm University (Sweden); Oglecka, Kamila [Department of Biochemistry and Biophysics, Stockholm University (Sweden); Lilja, Johanna [Department of Clinical Sciences, Section for Oncology, Lund University (Sweden); Wittrup, Anders [Department of Clinical Sciences, Section for Oncology, Lund University (Sweden); Goeran Eriksson, L.E. [Department of Biochemistry and Biophysics, Stockholm University (Sweden); Langel, Ulo [Department of Neurochemistry, Stockholm University (Sweden); Belting, Mattias [Department of Clinical Sciences, Section for Oncology, Lund University (Sweden)]. E-mail: email@example.com; Graeslund, Astrid [Department of Biochemistry and Biophysics, Stockholm University (Sweden)]. E-mail: firstname.lastname@example.com
Copper attachment to a non-octarepeat site in prion protein
NASA Astrophysics Data System (ADS)
Prion protein, PrP, plays a causative role in several neurodegenerative diseases, including mad cow disease in cattle and Creutzfeldt-Jakob disease in humans. The PrP is known to efficiently bind copper ions and this ability has been linked to its function. PrP contains up to six binding sites, four of which are located in the so-called octarepeat region and are now well known. The binding sites outside this region are still largely undetermined, despite evidence of their relevance to prion diseases. Using a hybrid DFT/DFT, which combines Kohn-Sham DFT with orbital-free DFT to achieve accurate and efficient description of solvent effects in ab initio calculations, we have investigated copper attachment to the sequence GGGTH, which represents the copper binding site located at His96. We have considered both NNNN and NNNO types of copper coordination, as suggested by experiments. Our calculations have determined the geometry of copper attachment site and its energetics. Comparison to the already known binding sites provides insight into the process of copper uptake in PrP.
Hodak, Miroslav; Bernholc, Jerry
Aggregation and amyloid fibril formation of the prion protein is accelerated in the presence of glycogen.
Prion diseases like Creutzfeldt-Jakob disease in humans or scrapie in sheep and goats are infectious neurodegenerative diseases. Their infectious agent, called prion, is composed mainly of aggregated and misfolded prion protein and non-proteinaceous components. An example of such a common non-proteinaceous secondary component of natural prions is the polysaccharide scaffold. We studied the influence of such a polysaccharide on the conformational transition of PrP applying an in vitro conversion system. Here we report that glycogen supports and accelerates PrP amorphous aggregation similar to seeded aggregation and leads to co-aggregates. Furthermore, PrP fibril formation was highly accelerated in the presence of glycogen. PMID:18341429
Panza, Giannantonio; St�hr, Jan; Birkmann, Eva; Riesner, Detlev; Willbold, Dieter; Baba, Otto; Terashima, Tatsuo; Dumpitak, Christian
Origins and Evolution of the HET-s Prion-Forming Protein: Searching for Other Amyloid-Forming Solenoids
The HET-s prion-forming domain from the filamentous fungus Podospora anserina is gaining considerable interest since it yielded the first well-defined atomic structure of a functional amyloid fibril. This structure has been identified as a left-handed beta solenoid with a triangular hydrophobic core. To delineate the origins of the HET-s prion-forming protein and to discover other amyloid-forming proteins, we searched for all homologs of the HET-s protein in a database of protein domains and fungal genomes, using a combined application of HMM, psi-blast and pGenThreader techniques, and performed a comparative evolutionary analysis of the N-terminal alpha-helical domain and the C-terminal prion-forming domain of HET-s. By assessing the tandem evolution of both domains, we observed that the prion-forming domain is restricted to Sordariomycetes, with a marginal additional sequence homolog in Arthroderma otae as a likely case of horizontal transfer. This suggests innovation and rapid evolution of the solenoid fold in the Sordariomycetes clade. In contrast, the N-terminal domain evolves at a slower rate (in Sordariomycetes) and spans many diverse clades of fungi. We performed a full three-dimensional protein threading analysis on all identified HET-s homologs against the HET-s solenoid fold, and present detailed structural annotations for identified structural homologs to the prion-forming domain. An analysis of the physicochemical characteristics in our set of structural models indicates that the HET-s solenoid shape can be readily adopted in these homologs, but that they are all less optimized for fibril formation than the P. anserina HET-s sequence itself, due chiefly to the presence of fewer asparagine ladders and salt bridges. Our combined structural and evolutionary analysis suggests that the HET-s shape has �limited scope� for amyloidosis across the wider protein universe, compared to the �generic� left-handed beta helix. We discuss the implications of our findings on future identification of amyloid-forming proteins sharing the solenoid fold.
Gendoo, Deena M. A.; Harrison, Paul M.
Altered toxicity of the prion protein peptide PrP106-126 carrying the Ala(117)-->Val mutation.
The inherited prion diseases such as Gerstmann-Str�ussler-Scheinker syndrome (GSS) are linked to point mutations in the gene coding for the cellular isoform of the prion protein (PrP(C)). One particular point mutation A117V (Ala(117)-->Val) is linked to a variable pathology that usually includes deposition of neurofibrillary tangles. A prion protein peptide carrying this point mutation [PrP106-126(117V)] was generated and compared with a peptide based on the normal human sequence [PrP106-126(117A)]. The inclusion of this point mutation increased the toxicity of PrP106-126 which could be linked to an increased beta-sheet content. An assay of microtubule formation in the presence of tau indicated that PrP106-126 decreased the rate of microtubule formation that could be related to the displacement of tau. PrP106-126 carrying the 117 mutation was more efficient at inhibiting microtubule formation. These results suggest a possible mechanism of toxicity for protein carrying this mutation via destabilization of the cytoskeleton and deposition of tau in filaments, as observed in GSS.
Brown, D R
Combined copper/zinc attachment to prion protein
NASA Astrophysics Data System (ADS)
Misfolding of prion protein (PrP) is responsible for diseases such as ``mad-cow disease'' in cattle and Creutzfeldt-Jacob in humans. Extensive experimental investigation has established that this protein strongly interacts with copper ions, and this ability has been linked to its still unknown function. Attachment of other metal ions (zinc, iron, manganese) have been demonstrated as well, but none of them could outcompete copper. Recent finding, however, indicates that at intermediate concentrations both copper and zinc ions can attach to the PrP at the octarepeat region, which contains high affinity metal binding sites. Based on this evidence, we have performed density functional theory simulations to investigate the combined Cu/Zn attachment. We consider all previously reported binding modes of copper at the octarepeat region and examine a possibility simultaneous Cu/Zn attachment. We find that this can indeed occur for only one of the known binding sites, when copper changes its coordination mode to allow for attachment of zinc ion. The implications of the simultaneous attachment on neural function remain to be explored.
Hodak, Miroslav; Bernholc, Jerry
Amyloid-? nanotubes are associated with prion protein-dependent synaptotoxicity
Growing evidence suggests water-soluble, non-fibrillar forms of amyloid-? protein (A?) have important roles in Alzheimer�s disease with toxicities mimicked by synthetic A?1�42. However, no defined toxic structures acting via specific receptors have been identified and roles of proposed receptors, such as prion protein (PrP), remain controversial. Here we quantify binding to PrP of A?1�42 after different durations of aggregation. We show PrP-binding and PrP-dependent inhibition of long-term potentiation (LTP) correlate with the presence of protofibrils. Globular oligomers bind less avidly to PrP and do not inhibit LTP, whereas fibrils inhibit LTP in a PrP-independent manner. That only certain transient A? assemblies cause PrP-dependent toxicity explains conflicting reports regarding the involvement of PrP in A?-induced impairments. We show that these protofibrils contain a defined nanotubular structure with a previously unidentified triple helical conformation. Blocking the formation of A? nanotubes or their interaction with PrP might have a role in treatment of Alzheimer�s disease.
Nicoll, Andrew J.; Panico, Silvia; Freir, Darragh B.; Wright, Daniel; Terry, Cassandra; Risse, Emmanuel; Herron, Caroline E.; O'Malley, Tiernan; Wadsworth, Jonathan D. F.; Farrow, Mark A.; Walsh, Dominic M.; Saibil, Helen R.; Collinge, John
Regulation of amyloid-? production by the prion protein
Alzheimer disease (AD) is characterized by the amyloidogenic processing of the amyloid precursor protein (APP), culminating in the accumulation of amyloid-? peptides in the brain. The enzymatic action of the ?-secretase, BACE1 is the rate-limiting step in this amyloidogenic processing of APP. BACE1 cleavage of wild-type APP (APPWT) is inhibited by the cellular prion protein (PrPC). Our recent study has revealed the molecular and cellular mechanisms behind this observation by showing that PrPC directly interacts with the pro-domain of BACE1 in the trans-Golgi network (TGN), decreasing the amount of BACE1 at the cell surface and in endosomes where it cleaves APPWT, while increasing BACE1 in the TGN where it preferentially cleaves APP with the Swedish mutation (APPSwe). PrPC deletion in transgenic mice expressing the Swedish and Indiana familial mutations (APPSwe,Ind) failed to affect amyloid-? accumulation, which is explained by the differential subcellular sites of action of BACE1 toward APPWT and APPSwe. This, together with our observation that PrPC is reduced in sporadic but not familial AD brain, suggests that PrPC plays a key protective role against sporadic AD. It also highlights the need for an APPWT transgenic mouse model to understand the molecular and cellular mechanisms underlying sporadic AD.
Griffiths, Heledd H.; Whitehouse, Isobel J.; Hooper, Nigel M.
Molecular docking of thiamine reveals similarity in binding properties between the prion protein and other thiamine-binding proteins.
Prion-induced diseases are a global health concern. The lack of effective therapy and 100% mortality rates for such diseases have made the prion protein an important target for drug discovery. Previous NMR experimental work revealed that thiamine and its derivatives bind the prion protein in a pocket near the N-terminal loop of helix 1, and conserved intermolecular interactions were noted between thiamine and other thiamine-binding proteins. Furthermore, water-mediated interactions were observed in all of the X-ray crystallographic structures of thiamine-binding proteins, but were not observed in the thiamine-prion NMR study. To better understand the potential role of water in thiamine-prion binding, a docking study was employed using structural X-ray solvent. Before energy minimization, docked thiamine assumed a "V" shape similar to some of the known thiamine-dependent proteins. Following minimization with NMR-derived restraints, the "F" conformation was observed. Our findings confirmed that water is involved in ligand stabilization and phosphate group interaction. The resulting refined structure of thiamine bound to the prion protein allowed the 4-aminopyrimidine ring of thiamine to ?-stack with Tyr150, and facilitated hydrogen bonding between Asp147 and the amino group of 4-aminopyrimidine. Investigation of the ?-stacking interaction through mutation of the tyrosine residue further revealed its importance in ligand placement. The resulting refined structure is in good agreement with previous experimental restraints, and is consistent with the pharmacophore model of thiamine-binding proteins. PMID:24126825
Pagadala, Nataraj S; Bjorndahl, Trent C; Blinov, Nikolay; Kovalenko, Andriy; Wishart, David S
Glycosaminoglycan Sulphation Affects the Seeded Misfolding of a Mutant Prion Protein
Background The accumulation of protease resistant conformers of the prion protein (PrPres) is a key pathological feature of prion diseases. Polyanions, including RNA and glycosaminoglycans have been identified as factors that contribute to the propagation, transmission and pathogenesis of prion disease. Recent studies have suggested that the contribution of these cofactors to prion propagation may be species specific. Methodology/Principal Finding In this study a cell-free assay was used to investigate the molecular basis of polyanion stimulated PrPres formation using brain tissue or cell line derived murine PrP. Enzymatic depletion of endogenous nucleic acids or heparan sulphate (HS) from the PrPC substrate was found to specifically prevent PrPres formation seeded by mouse derived PrPSc. Modification of the negative charge afforded by the sulphation of glycosaminoglycans increased the ability of a familial PrP mutant to act as a substrate for PrPres formation, while having no effect on PrPres formed by wildtype PrP. This difference may be due to the observed differences in the binding of wild type and mutant PrP for glycosaminoglycans. Conclusions/Significance Cofactor requirements for PrPres formation are host species and prion strain specific and affected by disease associated mutations of the prion protein. This may explain both species and strain dependent propagation characteristics and provide insights into the underlying mechanisms of familial prion disease. It further highlights the challenge of designing effective therapeutics against a disease which effects a range of mammalian species, caused by range of aetiologies and prion strains.
Lawson, Victoria A.; Lumicisi, Brooke; Welton, Jeremy; Machalek, Dorothy; Gouramanis, Katrina; Klemm, Helen M.; Stewart, James D.; Masters, Colin L.; Hoke, David E.; Collins, Steven J.; Hill, Andrew F.
Influence of prion protein genotypes on milk production traits in Spanish Churra sheep.
The aim of this work was to analyze the possible relationships between milk production traits and prion protein genotypes in Spanish Churra sheep. For this purpose, 2 analyses were carried out. First, an association study was performed of the prion protein genotypes of 12,533 Churra ewes and their milk yield, protein percentage, fat percentage, and somatic cell score as phenotypes, followed by a quantitative trait loci screening on the chromosome where the prion protein gene was located in this population. The latter analysis was carried out using 8 genetic markers (7 microsatellites and the prion protein genotypes) spanning ovine chromosome 13 using a daughter design. Regarding genotype frequencies, the most frequent allele was ARQ (75.90%), which linked with a high susceptibility to scrapie, followed by the resistant haplotype, ARR (18.16 %). The frequency of the most susceptible allele, VRQ, was around 1%. No evidence of association or linkage between prion protein genotypes and milk traits has been detected in Churra sheep. These results indicate that increasing the ARR frequency in Churra population will not have an adverse effect on selection for milk traits included in the breeding objectives. However, the low allele frequencies for ARR should be considered in the initial stages to prevent possible bottlenecks in future genetic progress. PMID:16606750
Alvarez, L; Guti�rrez-Gil, B; San Primitivo, F; de la Fuente, L F; Arranz, J J
NMR characterization of the full-length recombinant murine prion protein, mPrP(23�231)
The recombinant murine prion protein, mPrP(23�231), was expressed in E. coli with uniform 15N-labeling. NMR experiments showed that the previously determined globular three-dimensional structure of the C-terminal domain mPrP(121�231) is preserved in the intact protein, and that the N-terminal polypeptide segment 23�120 is flexibly disordered. This structural information is based on nearly complete sequence-specific assignments for the backbone amide nitrogens,
Roland Riek; Simone Hornemann; Gerhard Wider; Rudi Glockshuber; Kurt W�thrich
Accumulation and dissemination of prion protein in experimental sheep scrapie in the natural host
Background In order to study the sites of uptake and mechanisms of dissemination of scrapie prions in the natural host under controlled conditions, lambs aged 14 days and homozygous for the VRQ allele of the PrP gene were infected by the oral route. Infection occurred in all lambs with a remarkably short and highly consistent incubation period of approximately 6 months. Challenge of lambs at approximately eight months of age resulted in disease in all animals, but with more variable incubation periods averaging significantly longer than those challenged at 14 days. This model provides an excellent system in which to study the disease in the natural host by virtue of the relatively short incubation period and close resemblance to natural infection. Results Multiple sites of prion uptake were identified, of which the most important was the Peyer's patch of the distal ileum. Neuroinvasion was detected initially in the enteric nervous system prior to infection of the central nervous system. At end stage disease prion accumulation was widespread throughout the entire neuraxis, but vacuolar pathology was absent in most animals that developed disease at 6�7 months of age. Conclusion Initial spread of detectable PrP was consistent with drainage in afferent lymph to dependent lymph nodes. Subsequent accumulation of prions in lymphoid tissue not associated with the gut is consistent with haematogenous spread. In addition to macrophages and follicular dendritic cells, prion containing cells consistent with afferent lymph dendritic cells were identified and are suggested as a likely vehicle for carriage of prions from initial site of uptake to the lymphoreticular system, and as potential carriers of prion protein in blood. It is apparent that spongiform change, the characteristic lesion of scrapie and other prion diseases, is not responsible for the clinical signs in sheep, but may develop in an age dependent manner.
Ryder, Stephen J; Dexter, Glenda E; Heasman, Lindsay; Warner, Richard; Moore, S Jo
Transport of the Pathogenic Prion Protein through Soils
Transmissible spongiform encephalopathies (TSEs) are progressive neurodegenerative diseases and include bovine spongiform encephalopathy of cattle, chronic wasting disease (CWD) of deer and elk, scrapie in sheep and goats, and Creutzfeldt-Jakob disease in humans. An abnormally folded form of the prion protein (designated PrPTSE) is typically associated with TSE infectivity and may constitute the major, if not sole, component of the infectious agent. Transmission of CWD and scrapie is mediated in part by an environmental reservoir of infectivity. Soil appears to be a plausible candidate for this reservoir. TSE agent transport through soil is expected to influence the accessibility of the pathogen to animals after deposition and must be understood to assess the risks associated with burial of infected carcasses. We report results of saturated column experiments designed to evaluate PrPTSE transport through five soils with relatively high sand or silt contents. Protease-treated TSE-infected brain homogenate was used as a model for PrPTSE present in decomposing infected tissue. Synthetic rainwater was used as the eluent. PrPTSE was retained by all five soils; no detectable PrPTSE was eluted over more than 40 pore volumes of flow. Lower bound apparent attachment coefficients were estimated for each soil. Our results suggest that TSE agent released from decomposing tissues would remain near the site of initial deposition. In the case of infected carcasses deposited on the land surface, this may result in local sources of infectivity to other animals.
Jacobson, Kurt H.; Lee, Seunghak; Somerville, Robert A.; McKenzie, Debbie; Benson, Craig H.; Pedersen, Joel A.
Cellular Prion Protein Promotes Brucella Infection into Macrophages
The products of the Brucella abortus virB gene locus, which are highly similar to conjugative DNA transfer system, enable the bacterium to replicate within macrophage vacuoles. The replicative phagosome is thought to be established by the interaction of a substrate of the VirB complex with macrophages, although the substrate and its host cellular target have not yet been identified. We report here that Hsp60, a member of the GroEL family of chaperonins, of B. abortus is capable of interacting directly or indirectly with cellular prion protein (PrPC) on host cells. Aggregation of PrPC tail-like formation was observed during bacterial swimming internalization into macrophages and PrPC was selectively incorporated into macropinosomes containing B. abortus. Hsp60 reacted strongly with serum from human brucellosis patients and was exposed on the bacterial surface via a VirB complex�associated process. Under in vitro and in vivo conditions, Hsp60 of B. abortus bound to PrPC. Hsp60 of B. abortus, expressed on the surface of Lactococcus lactis, promoted the aggregation of PrPC but not PrPC tail formation on macrophages. The PrPC deficiency prevented swimming internalization and intracellular replication of B. abortus, with the result that phagosomes bearing the bacteria were targeted into the endocytic network. These results indicate that signal transduction induced by the interaction between bacterial Hsp60 and PrPC on macrophages contributes to the establishment of B. abortus infection.
Watarai, Masahisa; Kim, Suk; Erdenebaatar, Janchivdorj; Makino, Sou-ichi; Horiuchi, Motohiro; Shirahata, Toshikazu; Sakaguchi, Suehiro; Katamine, Shigeru
Prion protein accumulation in the spinal cords of patients with sporadic and growth hormone associated Creutzfeldt-Jakob disease
An immunohistological study of the spinal cord in 20 cases of sporadic and 4 iatrogenic (growth hormone) cases of Creutzfeldt-Jakob (CJD) disease patients was performed to detect the presence of disease specific prion protein using a number of different antisera. Prion protein was present in all the growth hormone recipients and in 11 of the 20 sporadic CJD cases. Plaque-like
I. A. Goodbrand; J. W. Ironside; D. Nicolson; J. E. Bell
Fatal Familial Insomnia and Familial Creutzfeldt-Jakob Disease: Different Prion Proteins Determined by a DNA Polymorphism
Fatal familial insomnia and a subtype of Creutzfeldt-Jakob disease, two clinically and pathologically distinct diseases, are linked to the same mutation at codon 178 (Asp-178 --> Asn) but segregate with different genotypes determined by this mutation and the methionine-valine polymorphism at codon 129 of the prion protein gene. The abnormal isoforms of the prion protein in these two diseases were
Lucia Monari; Shu G. Chen; Paul Brown; Piero Parchi; Robert B. Petersen; Jacqueline Mikol; Franscoise Gray; Pietro Cortelli; Pasquale Montagna; Bernardino Ghetti; Lev G. Goldfarb; D. Carleton Gajdusek; Elio Lugaresi; Pierluigi Gambetti; Lucila Autilio-Gambetti
Elongation of Mouse Prion Protein Amyloid-Like Fibrils: Effect of Temperature and Denaturant Concentration
Prion protein is known to have the ability to adopt a pathogenic conformation, which seems to be the basis for protein-only infectivity. The infectivity is based on self-replication of this pathogenic prion structure. One of possible mechanisms for such replication is the elongation of amyloid-like fibrils. We measured elongation kinetics and thermodynamics of mouse prion amyloid-like fibrils at different guanidine hydrochloride (GuHCl) concentrations. Our data show that both increases in temperature and GuHCl concentration help unfold monomeric protein and thus accelerate elongation. Once the monomers are unfolded, further increases in temperature raise the rate of elongation, whereas the addition of GuHCl decreases it. We demonstrated a possible way to determine different activation energies of amyloid-like fibril elongation by using folded and unfolded protein molecules. This approach separates thermodynamic data for fibril-assisted monomer unfolding and for refolding and formation of amyloid-like structure.
Milto, Katazyna; Michailova, Ksenija; Smirnovas, Vytautas
Molecular Features of the Copper Binding Sites in the Octarepeat Domain of the Prion Protein�
Recent evidence suggests that the prion protein (PrP) is a copper binding protein. The N-terminal region of human PrP contains four sequential copies of the highly conserved octarepeat sequence PHGGGWGQ spanning residues 60�91. This region selectively binds Cu2+ in vivo. In a previous study using peptide design, EPR, and CD spectroscopy, we showed that the HGGGW segment within each octarepeat comprises the fundamental Cu2+ binding unit [Aronoff-Spencer et al. (2000) Biochemistry 40, 13760�13771]. Here we present the first atomic resolution view of the copper binding site within an octarepeat. The crystal structure of HGGGW in a complex with Cu2+ reveals equatorial coordination by the histidine imidazole, two deprotonated glycine amides, and a glycine carbonyl, along with an axial water bridging to the Trp indole. Companion S-band EPR, X-band ESEEM, and HYSCORE experiments performed on a library of 15N-labeled peptides indicate that the structure of the copper binding site in HGGGW and PHGGGWGQ in solution is consistent with that of the crystal structure. Moreover, EPR performed on PrP(23�28, 57�91) and an 15N-labeled analogue demonstrates that the identified structure is maintained in the full PrP octarepeat domain. It has been shown that copper stimulates PrP endocytosis. The identified Gly�Cu linkage is unstable below pH ?6.5 and thus suggests a pH-dependent molecular mechanism by which PrP detects Cu2+ in the extracellular matrix or releases PrP-bound Cu2+ within the endosome. The structure also reveals an unusual complementary interaction between copper-structured HGGGW units that may facilitate molecular recognition between prion proteins, thereby suggesting a mechanism for transmembrane signaling and perhaps conversion to the pathogenic form.
Burns, Colin S.; Aronoff-Spencer, Eliah; Dunham, Christine M.; Lario, Paula; Avdievich, Nikolai I.; Antholine, William E.; Olmstead, Marilyn M.; Vrielink, Alice; Gerfen, Gary J.; Peisach, Jack; Scott, William G.; Millhauser, Glenn L.
Flexibility damps macromolecular crowding effects on protein folding dynamics: Application to the murine prion protein (121-231)
NASA Astrophysics Data System (ADS)
A model of protein folding kinetics is applied to study the combined effects of protein flexibility and macromolecular crowding on protein folding rate and stability. It is found that the increase in stability and folding rate promoted by macromolecular crowding is damped for proteins with highly flexible native structures. The model is applied to the folding dynamics of the murine prion protein (121-231). It is found that the high flexibility of the native isoform of the murine prion protein (121-231) reduces the effects of macromolecular crowding on its folding dynamics. The relevance of these findings for the pathogenic mechanism are discussed.
Bergasa-Caceres, Fernando; Rabitz, Herschel A.
Monitoring prion protein expression in complex biological samples by SERS for diagnostic applications
NASA Astrophysics Data System (ADS)
Surface-enhanced Raman spectroscopy (SERS) allows a new insight into the analysis of cell physiology. In this work, the difficulty of producing suitable substrates that, besides permitting the amplification of the Raman signal, do not interact with the biological material causing alteration, has been overcome by a combined method of hydrothermal green synthesis and thermal annealing. The SERS analysis of the cell membrane has been performed with special attention to the cellular prion protein PrPC. In addition, SERS has also been used to reveal the prion protein-Cu(II) interaction in four different cell models (B104, SH-SY5Y, GN11, HeLa), expressing PrPC at different levels. A significant implication of the current work consists of the intriguing possibility of revealing and quantifying prion protein expression in complex biological samples by a cheap SERS-based method, replacing the expensive and time-consuming immuno-assay systems commonly employed.
Manno, D.; Filippo, E.; Fiore, R.; Serra, A.; Urso, E.; Rizzello, A.; Maffia, M.
SIRP? polymorphisms, but not the prion protein, control phagocytosis of apoptotic cells
Prnp?/? mice lack the prion protein PrPC and are resistant to prion infections, but variable phenotypes have been reported in Prnp?/? mice and the physiological function of PrPC remains poorly understood. Here we examined a cell-autonomous phenotype, inhibition of macrophage phagocytosis of apoptotic cells, previously reported in Prnp?/? mice. Using formal genetic, genomic, and immunological analyses, we found that the regulation of phagocytosis previously ascribed to PrPC is instead controlled by a linked locus encoding the signal regulatory protein ? (Sirpa). These findings indicate that control of phagocytosis was previously misattributed to the prion protein and illustrate the requirement for stringent approaches to eliminate confounding effects of flanking genes in studies modeling human disease in gene-targeted mice. The plethora of seemingly unrelated functions attributed to PrPC suggests that additional phenotypes reported in Prnp?/? mice may actually relate to Sirpa or other genetic confounders.
Nuvolone, Mario; Kana, Veronika; Hutter, Gregor; Sakata, Daiji; Mortin-Toth, Steven M.; Russo, Giancarlo
Chronic wasting disease (CWD) was first identified in Wisconsin (USA) in white- tailed deer (Odocoileus virginianus) in February 2002. To determine if prion protein gene (Prnp) allelic variability was associated with CWD in white-tailed deer from Wisconsin, we sequenced Prnp from 26 CWD-positive and 100 CWD-negative deer. Sequence analysis of Prnp suggests that at least 86-96% of the white-tailed deer
Chad Johnson; Jody Johnson; Murray Clayton; Debbie McKenzie; Judd Aiken
Protocol for aerosol-free recombinant production and NMR analysis of prion proteins.
The central hallmark of prion diseases is the misfolding of cellular prion protein (PrP(C)) into a disease-associated aggregated isoform known as scrapie prion protein (PrP(Sc)). NMR spectroscopy has made many essential contributions to the characterization of recombinant PrP in its folded, unfolded and aggregated states. Recent studies reporting on de novo generation of prions from recombinant PrP and infection of animals using prion aerosols suggest that adjustment of current biosafety measures may be necessary, particularly given the relatively high protein concentrations required for NMR applications that favor aggregation. We here present a protocol for the production of recombinant PrP under biosafety level 2 conditions that avoids entirely exposure of the experimenter to aerosols that might contain harmful PrP aggregates. In addition, we introduce an NMR sample tube setup that allows for safe handling of PrP samples at the spectrometer that usually is not part of a dedicated biosafety level 2 laboratory. PMID:24771297
Rehbein, Peter; Saxena, Krishna; Schlepckow, Kai; Schwalbe, Harald
Enhancement of protein misfolding cyclic amplification by using concentrated cellular prion protein source.
Protein misfolding cyclic amplification (PMCA) is a cell-free assay mimicking the prion replication process. However, constraints affecting PMCA have not been well-defined. Although cellular prion protein (PrP(C)) is required for prion replication, the influence of PrP(C) abundance on PMCA has not been assessed. Here, we show that PMCA was enhanced by using mouse brain material in which PrP(C) was overexpressed. Tg(MoPrP)4112 mice overexpressing PrP(C) supported more sensitive and efficient PMCA than wild type mice. As brain homogenate of Tg(MoPrP)4112 mice was diluted with PrP(C)-deficient brain material, PMCA became less robust. Our studies suggest that abundance of PrP(C) is a determinant that directs enhancement of PMCA. PMCA established here will contribute to optimizing conditions to enhance PrP(Sc) amplification by using concentrated PrP(C) source and expands the use of this methodology. PMID:19664595
Mays, Charles E; Titlow, William; Seward, Tanya; Telling, Glenn C; Ryou, Chongsuk
Neuroimmunoendocrine Regulation of the Prion Protein in Neutrophils*
The prion protein (PrPC) is a cell surface protein expressed mainly in the nervous system. In addition to the role of its abnormal conformer in transmissible spongiform encephalopathies, normal PrPC may be implicated in other degenerative conditions often associated with inflammation. PrPC is also present in cells of hematopoietic origin, including T cells, dendritic cells, and macrophages, and it has been shown to modulate their functions. Here, we investigated the impact of inflammation and stress on the expression and function of PrPC in neutrophils, a cell type critically involved in both acute and chronic inflammation. We found that systemic injection of LPS induced transcription and translation of PrPC in mouse neutrophils. Up-regulation of PrPC was dependent on the serum content of TGF-? and glucocorticoids (GC), which, in turn, are contingent on the activation of the hypothalamic-pituitary-adrenal axis in response to systemic inflammation. GC and TGF-?, either alone or in combination, directly up-regulated PrPC in neutrophils, and accordingly, the blockade of GC receptors in vivo curtailed the LPS-induced increase in the content of PrPC. Moreover, GC also mediated up-regulation of PrPC in neutrophils following noninflammatory restraint stress. Finally, neutrophils with up-regulated PrPC presented enhanced peroxide-dependent cytotoxicity to endothelial cells. The data demonstrate a novel interplay of the nervous, endocrine, and immune systems upon both the expression and function of PrPC in neutrophils, which may have a broad impact upon the physiology and pathology of various organs and systems.
Mariante, Rafael M.; Nobrega, Alberto; Martins, Rodrigo A. P.; Areal, Romulo B.; Bellio, Maria; Linden, Rafael
Prion protein polymorphisms in white-tailed deer influence susceptibility to chronic wasting disease.
The primary sequence of the prion protein affects susceptibility to transmissible spongiform encephalopathies, or prion diseases, in mice, sheep and humans. The Prnp gene sequence of free-ranging, Wisconsin white-tailed deer was determined and the Prnp genotypes of chronic wasting disease (CWD)-positive and CWD-negative deer were compared. Six amino acid changes were identified, two of which were located in pseudogenes. Two alleles, a Q-->K polymorphism at codon 226 and a single octapeptide repeat insertion into the pseudogene, have not been reported previously. The predominant alleles--wild-type (Q95, G96 and Q226) and a G96S polymorphism--comprised almost 98% of the Prnp alleles in the Wisconsin white-tailed deer population. Comparison of the allelic frequencies in the CWD-positive and CWD-negative deer suggested that G96S and a Q95H polymorphism were linked to a reduced susceptibility to CWD. The G96S allele did not, however, provide complete resistance, as a CWD-positive G96S/G96S deer was identified. The G96S allele was also linked to slower progression of the disease in CWD-positive deer based on the deposition of PrP(CWD) in the obex region of the medulla oblongata. Although the reduced susceptibility of deer with at least one copy of the Q95H or G96S allele is insufficient to serve as a genetic barrier, the presence of these alleles may modulate the impact of CWD on white-tailed deer populations. PMID:16760415
Johnson, Chad; Johnson, Jody; Vanderloo, Joshua P; Keane, Delwyn; Aiken, Judd M; McKenzie, Debbie
A comparative molecular dynamics study on thermostability of human and chicken prion proteins
To compare the thermostabilities of human and chicken normal cellular prion proteins (HuPrP{sup C} and CkPrP{sup C}), molecular dynamics (MD) simulations were performed for both proteins at an ensemble level (10 parallel simulations at 400 K and 5 parallel simulations at 300 K as a control). It is found that the thermostability of HuPrP{sup C} is comparable with that of CkPrP{sup C}, which implicates that the non-occurrence of prion diseases in non-mammals cannot be completely attributed to the thermodynamic properties of non-mammalian PrP{sup C}.
Ji, Hong-Fang [Shandong Provincial Research Center for Bioinformatic Engineering and Technique, Center for Advanced Study, Shandong University of Technology, Zibo 255049 (China); Zhang, Hong-Yu [Shandong Provincial Research Center for Bioinformatic Engineering and Technique, Center for Advanced Study, Shandong University of Technology, Zibo 255049 (China)]. E-mail: email@example.com
Comparative analysis of essential collective dynamics and NMR-derived flexibility profiles in evolutionarily diverse prion proteins
Collective motions on ns-�s time scales are known to have a major impact on protein folding, stability, binding and enzymatic efficiency. It is also believed that these motions may have an important role in the early stages of prion protein misfolding and prion disease. In an effort to accurately characterize these motions and their potential influence on the misfolding and prion disease transmissibility we have conducted a combined analysis of molecular dynamic simulations and NMR-derived flexibility measurements over a diverse range of prion proteins. Using a recently developed numerical formalism, we have analyzed the essential collective dynamics (ECD) for prion proteins from eight different species including human, cow, elk, cat, hamster, chicken, turtle and frog. We also compared the numerical results with flexibility profiles generated by the random coil index (RCI) from NMR chemical shifts. Prion protein backbone flexibility derived from experimental NMR data and from theoretical computations show strong agreement with each other, demonstrating that it is possible to predict the observed RCI profiles employing the numerical ECD formalism. Interestingly, flexibility differences in the loop between second b strand (S2) and the second a helix (HB) appear to distinguish prion proteins from species that are susceptible to prion disease and those that are resistant. Our results show that the different levels of flexibility in the S2-HB loop in various species are predictable via the ECD method, indicating that ECD may be used to identify disease resistant variants of prion proteins, as well as the influence of prion proteins mutations on disease susceptibility or misfolding propensity.
Santo, Kolattukudy P; Berjanskii, Mark; Wishart, David S
Pathogenic prion protein is degraded by a manganese oxide mineral found in soils
Prions, the aetiological agents of transmissible spongiform encephalopathies, exhibit extreme resistance to degradation. Soil can retain prion infectivity in the environment for years. Reactive soil components may, however, contribute to the inactivation of prions in soil. Members of the birnessite family of manganese oxides (MnO2) rank among the strongest natural oxidants in soils. Here, we report the abiotic degradation of pathogenic prion protein (PrPTSE) by a synthetic analogue of naturally occurring birnessite minerals. Aqueous MnO2 suspensions degraded the PrPTSE as evidenced by decreased immunoreactivity and diminished ability to seed protein misfolding cyclic amplification reactions. Birnessite-mediated PrPTSE degradation increased as a solution's pH decreased, consistent with the pH-dependence of the redox potential of MnO2. Exposure to 5.6 mg MnO2 ml-1 (PrPTSE:MnO2=1 : 110) decreased PrPTSE levels by ???4 orders of magnitude. Manganese oxides may contribute to prion degradation in soil environments rich in these minerals. ?? 2009 SGM.
Russo, F.; Johnson, C. J.; Johnson, C. J.; McKenzie, D.; Aiken, J. M.; Pedersen, J. A.
Cellular prion protein promotes Brucella infection into macrophages.
The products of the Brucella abortus virB gene locus, which are highly similar to conjugative DNA transfer system, enable the bacterium to replicate within macrophage vacuoles. The replicative phagosome is thought to be established by the interaction of a substrate of the VirB complex with macrophages, although the substrate and its host cellular target have not yet been identified. We report here that Hsp60, a member of the GroEL family of chaperonins, of B. abortus is capable of interacting directly or indirectly with cellular prion protein (PrPC) on host cells. Aggregation of PrPC tail-like formation was observed during bacterial swimming internalization into macrophages and PrPC was selectively incorporated into macropinosomes containing B. abortus. Hsp60 reacted strongly with serum from human brucellosis patients and was exposed on the bacterial surface via a VirB complex-associated process. Under in vitro and in vivo conditions, Hsp60 of B. abortus bound to PrPC. Hsp60 of B. abortus, expressed on the surface of Lactococcus lactis, promoted the aggregation of PrPC but not PrPC tail formation on macrophages. The PrPC deficiency prevented swimming internalization and intracellular replication of B. abortus, with the result that phagosomes bearing the bacteria were targeted into the endocytic network. These results indicate that signal transduction induced by the interaction between bacterial Hsp60 and PrPC on macrophages contributes to the establishment of B. abortus infection. PMID:12847134
Watarai, Masahisa; Kim, Suk; Erdenebaatar, Janchivdorj; Makino, Sou-ichi; Horiuchi, Motohiro; Shirahata, Toshikazu; Sakaguchi, Suehiro; Katamine, Shigeru
Shaking Alone Induces De Novo Conversion of Recombinant Prion Proteins to ?-Sheet Rich Oligomers and Fibrils
The formation of ?-sheet rich prion oligomers and fibrils from native prion protein (PrP) is thought to be a key step in the development of prion diseases. Many methods are available to convert recombinant prion protein into ?-sheet rich fibrils using various chemical denaturants (urea, SDS, GdnHCl), high temperature, phospholipids, or mildly acidic conditions (pH 4). Many of these methods also require shaking or another form of agitation to complete the conversion process. We have identified that shaking alone causes the conversion of recombinant PrP to ?-sheet rich oligomers and fibrils at near physiological pH (pH 5.5 to pH 6.2) and temperature. This conversion does not require any denaturant, detergent, or any other chemical cofactor. Interestingly, this conversion does not occur when the water-air interface is eliminated in the shaken sample. We have analyzed shaking-induced conversion using circular dichroism, resolution enhanced native acidic gel electrophoresis (RENAGE), electron microscopy, Fourier transform infrared spectroscopy, thioflavin T fluorescence and proteinase K resistance. Our results show that shaking causes the formation of ?-sheet rich oligomers with a population distribution ranging from octamers to dodecamers and that further shaking causes a transition to ?-sheet fibrils. In addition, we show that shaking-induced conversion occurs for a wide range of full-length and truncated constructs of mouse, hamster and cervid prion proteins. We propose that this method of conversion provides a robust, reproducible and easily accessible model for scrapie-like amyloid formation, allowing the generation of milligram quantities of physiologically stable ?-sheet rich oligomers and fibrils. These results may also have interesting implications regarding our understanding of prion conversion and propagation both within the brain and via techniques such as protein misfolding cyclic amplification (PMCA) and quaking induced conversion (QuIC).
Ladner-Keay, Carol L.; Griffith, Bethany J.; Wishart, David S.
Human prion diseases are classified into 3 categories according to etiologies: idiopathic of unknown cause, acquired of infectious origin, and genetic by PRNP mutation. The surveillance committee have analyzed 2,494 cases and identified 1,402 as prion diseases. Most of them are idiopathic, namely sporadic CJD (77%) with less genetic and acquired prion diseases (17% and 5%, respectively). The number of patients identified by the surveillance committee in these years is about 120 which are less than the number of annual death of prion disease. The difference might be due to partly the fact our surveillance need the consent from patients' family and is not complete. The mean age at onset of prion disease is late 60s while the range is fairly wide. Brain MRIs and increase of CSF 14-3-3 and tau protein levels are very characteristic. Classical sporadic CJD could show completely normal T1WI with patchy high signals in the cerebral cortex and basal ganglia on DWI. In Japan, classical sporadic CJD (MM1) is most popular but there are some rare atypical subtypes. Among them, MM2-thalamic CJD is hardest to diagnose because it shows no high intensity signals on DWI, in addition to frequent absence of CSF and EEG characteristics. In this case, CBF decrease in the thalamus on SPECT is very helpful. Genetic prion diseases in Japan are quite distinct from those in Europe. V180I and M232R mutations are unique to Japan and show sporadic CJD phenotype. Dura graft-associated CJD (dCJD) are composed of 67% of classical sporadic CJD phenotype and 33% of atypical phenotype showing slower progression with amyloid plaques. Trace-back experiments suggested the PrP(sc) of the atypical dCJD was likely to be modified from infection of abnormal VV2 protein. Although there are some atypical forms of prion diseases as mentioned above, almost all prion cases could be diagnosed with EEG, MRI, genetic test, CSF test and SPECT. We also have some incidents in which brain surgery was done before the diagnosis of prion disease and many other patients were operated using the same operating instruments before their sterilization against prion disease had been done. The explanation of possibility of prion disease infection to the patients and their follow-up was started by the incident committee. It is very important for all the nations to cooperate with each other in order to overcome this intractable disease. PMID:21921445
Mizusawa, Hidehiro
Motor behavioral and neuropathological deficits in mice deficient for normal prion protein expression
Summary It has been difficult to reconcile the absence of pathology and apparently normal behavior of mice lacking prion protein (PrP), referred to as Prnp0/0 mice, with a mechanism of prion pathogenesis involving progressive loss of PrPC-mediated neuroprotection. However, here we report that Prnp0/0 mice exhibit significant age-related defects in motor coordination and balance compared with mice expressing wild type Prnp on a syngeneic background, and that the brains of behaviorally-impaired Prnp0/0 mice display the cardinal neuropathological hallmarks of spongiform pathology and reactive astrocytic gliosis that normally accompany prion disease. Consistent with the appearance of cerebellar ataxia as an early symptom in an patients with Gerstmann-Str�ussler-Scheinker syndrome (GSS), an inherited form of human prion disease, motor coordination and balance defects manifested in a transgenic (Tg) mouse model of GSS considerably earlier than the onset of end-stage neurodegenerative disease. Our results are consistent with a mechanism in which loss of normal PrPC function is an important pathological component of prion diseases.
Nazor, Karah E.; Seward, Tanya; Telling, Glenn C.
Compartment-Restricted Biotinylation Reveals Novel Features of Prion Protein Metabolism in Vivo
Proteins are often made in more than one form, with alternate versions sometimes residing in different cellular compartments than the primary species. The mammalian prion protein (PrP), a cell surface GPI-anchored protein, is a particularly noteworthy example for which minor cytosolic and transmembrane forms have been implicated in disease pathogenesis. To study these minor species, we used a selective labeling strategy in which spatially restricted expression of a biotinylating enzyme was combined with asymmetric engineering of the cognate acceptor sequence into PrP. Using this method, we could show that even wild-type PrP generates small amounts of the CtmPrP transmembrane form. Selective detection of CtmPrP allowed us to reveal its N-terminal processing, long half-life, residence in both intracellular and cell surface locations, and eventual degradation in the lysosome. Surprisingly, some human disease-causing mutants in PrP selectively stabilized CtmPrP, revealing a previously unanticipated mechanism of CtmPrP up-regulation that may contribute to disease. Thus, spatiotemporal tagging has uncovered novel aspects of normal and mutant PrP metabolism and should be readily applicable to the analysis of minor topologic isoforms of other proteins.
Emerman, Amy B.; Zhang, Zai-Rong; Chakrabarti, Oishee
Prion propagation in mice expressing human and chimeric PrP transgenes implicates the interaction of cellular PrP with another protein
Transgenic (Tg) mice expressing human (Hu) and chimeric prion protein (PrP) genes were inoculated with brain extracts from humans with inherited or sporadic prion disease to investigate the mechanism by which PrPc is transformed into PrPSc. Although Tg(HuPrP) mice expressed high levels of HuPrPc, they were resistant to human prions. They became susceptible to human prions upon ablation of the
Glenn C. Telling; Michael Scott; James Mastrianni; Ruth Gabizon; Marilyn Torchia; Fred E. Cohen; Stephen J. DeArmond
Detection of prion protein particles in blood plasma of scrapie infected sheep.
Prion diseases are transmissible neurodegenerative diseases affecting humans and animals. The agent of the disease is the prion consisting mainly, if not solely, of a misfolded and aggregated isoform of the host-encoded prion protein (PrP). Transmission of prions can occur naturally but also accidentally, e.g. by blood transfusion, which has raised serious concerns about blood product safety and emphasized the need for a reliable diagnostic test. In this report we present a method based on surface-FIDA (fluorescence intensity distribution analysis), that exploits the high state of molecular aggregation of PrP as an unequivocal diagnostic marker of the disease, and show that it can detect infection in blood. To prepare PrP aggregates from blood plasma we introduced a detergent and lipase treatment to separate PrP from blood lipophilic components. Prion protein aggregates were subsequently precipitated by phosphotungstic acid, immobilized on a glass surface by covalently bound capture antibodies, and finally labeled with fluorescent antibody probes. Individual PrP aggregates were visualized by laser scanning microscopy where signal intensity was proportional to aggregate size. After signal processing to remove the background from low fluorescence particles, fluorescence intensities of all remaining PrP particles were summed. We detected PrP aggregates in plasma samples from six out of ten scrapie-positive sheep with no false positives from uninfected sheep. Applying simultaneous intensity and size discrimination, ten out of ten samples from scrapie sheep could be differentiated from uninfected sheep. The implications for ante mortem diagnosis of prion diseases are discussed. PMID:22567169
Bannach, Oliver; Birkmann, Eva; Reinartz, Elke; Jaeger, Karl-Erich; Langeveld, Jan P M; Rohwer, Robert G; Gregori, Luisa; Terry, Linda A; Willbold, Dieter; Riesner, Detlev
Detection of Prion Protein Particles in Blood Plasma of Scrapie Infected Sheep
Prion diseases are transmissible neurodegenerative diseases affecting humans and animals. The agent of the disease is the prion consisting mainly, if not solely, of a misfolded and aggregated isoform of the host-encoded prion protein (PrP). Transmission of prions can occur naturally but also accidentally, e.g. by blood transfusion, which has raised serious concerns about blood product safety and emphasized the need for a reliable diagnostic test. In this report we present a method based on surface-FIDA (fluorescence intensity distribution analysis), that exploits the high state of molecular aggregation of PrP as an unequivocal diagnostic marker of the disease, and show that it can detect infection in blood. To prepare PrP aggregates from blood plasma we introduced a detergent and lipase treatment to separate PrP from blood lipophilic components. Prion protein aggregates were subsequently precipitated by phosphotungstic acid, immobilized on a glass surface by covalently bound capture antibodies, and finally labeled with fluorescent antibody probes. Individual PrP aggregates were visualized by laser scanning microscopy where signal intensity was proportional to aggregate size. After signal processing to remove the background from low fluorescence particles, fluorescence intensities of all remaining PrP particles were summed. We detected PrP aggregates in plasma samples from six out of ten scrapie-positive sheep with no false positives from uninfected sheep. Applying simultaneous intensity and size discrimination, ten out of ten samples from scrapie sheep could be differentiated from uninfected sheep. The implications for ante mortem diagnosis of prion diseases are discussed.
Reinartz, Elke; Jaeger, Karl-Erich; Langeveld, Jan P. M.; Rohwer, Robert G.; Gregori, Luisa; Terry, Linda A.; Willbold, Dieter; Riesner, Detlev
New Structural Approaches to Understanding the Disease Related Forms of the Prion Protein.
National Technical Information Service (NTIS)
The mouse pron protein peptide (residues 89-143 with the substitution of Leu for Pro atresidue 101) induces prion disease in sensitized mice. Samp%es of this peptide isotope%abe%ed with I 5N were prepared by expression of a fusion in E.co%i c%eaved to yie...
D. E. Wemmer
New Structural Approaches to Understanding the Disease Related Forms of the Prion Protein.
National Technical Information Service (NTIS)
The mouse pron protein peptide (residues 89-143 with the substitution of Leu for Pro atresidue 101) induces prion disease in sensitized mice. Samples of this peptide, isotope labeled with 15N, have been prepared by expression of a fusion in E. coli, cleav...
D. E. Wemmer
Ultra-Sensitive Detection of Prion Protein in Blood Using Isothermal Amplification Technology.
National Technical Information Service (NTIS)
The detection of the pathologic prion protein that is Implicated in transmissible spongiform encephalopathies (TSEs) is necessary to diagnose the disease. Presently, the Western Blot or ELISA are used to test the brain stem in cattle for the presence of p...
N. T. Constantine
Prion Protein M129V Polymorphism Affects Retrieval-Related Brain Activity
ERIC Educational Resources Information Center
The prion protein Met129Val polymorphism has recently been related to human long-term memory with carriers of either the 129[superscript MM] or the 129[superscript MV] genotype recalling 17% more words than 129[superscript VV] carriers at 24 h following learning. Here, we sampled genotype differences in retrieval-related brain activity at 30 min�
Buchmann, Andreas; Mondadori, Christian R. A.; Hanggi, Jurgen; Aerni, Amanda; Vrticka, Pascal; Luechinger, Roger; Boesiger, Peter; Hock, Christoph; Nitsch, Roger M.; de Quervain, Dominique J.-F.; Papassotiropoulos, Andreas; Henke, Katharina
A bipolar functionality of Q/N-rich proteins: Lsm4 amyloid causes clearance of yeast prions
Prions are epigenetic modifiers that cause partially loss-of-function phenotypes of the proteins in Saccharomyces cerevisiae. The molecular chaperone network that supports prion propagation in the cell has seen a great progress in the last decade. However, the cellular machinery to activate or deactivate the prion states remains an enigma, largely due to insufficient knowledge of prion-regulating factors. Here, we report that overexpression of a [PSI+]-inducible Q/N-rich protein, Lsm4, eliminates the three major prions [PSI+], [URE3], and [RNQ+]. Subcloning analysis revealed that the Q/N-rich region of Lsm4 is responsible for the prion loss. Lsm4 formed an amyloid in vivo, which seemed to play a crucial role in the prion elimination. Fluorescence correlation spectroscopy analysis revealed that in the course of the Lsm4-driven [PSI+] elimination, the [PSI+] aggregates undergo a size increase, which ultimately results in the formation of conspicuous foci in otherwise [psi?]-like mother cells. We also found that the antiprion activity is a general property of [PSI+]-inducible factors. These data provoked a novel �unified� model that explains both prion induction and elimination by a single scheme.
Oishi, Keita; Kurahashi, Hiroshi; Pack, Chan-Gi; Sako, Yasushi; Nakamura, Yoshikazu
A bipolar functionality of Q/N-rich proteins: Lsm4 amyloid causes clearance of yeast prions.
Prions are epigenetic modifiers that cause partially loss-of-function phenotypes of the proteins in Saccharomyces cerevisiae. The molecular chaperone network that supports prion propagation in the cell has seen a great progress in the last decade. However, the cellular machinery to activate or deactivate the prion states remains an enigma, largely due to insufficient knowledge of prion-regulating factors. Here, we report that overexpression of a [PSI(+) ]-inducible Q/N-rich protein, Lsm4, eliminates the three major prions [PSI(+) ], [URE3], and [RNQ(+) ]. Subcloning analysis revealed that the Q/N-rich region of Lsm4 is responsible for the prion loss. Lsm4 formed an amyloid in vivo, which seemed to play a crucial role in the prion elimination. Fluorescence correlation spectroscopy analysis revealed that in the course of the Lsm4-driven [PSI(+) ] elimination, the [PSI(+) ] aggregates undergo a size increase, which ultimately results in the formation of conspicuous foci in otherwise [psi(-) ]-like mother cells. We also found that the antiprion activity is a general property of [PSI(+) ]-inducible factors. These data provoked a novel "unified" model that explains both prion induction and elimination by a single scheme. PMID:23512891
Oishi, Keita; Kurahashi, Hiroshi; Pack, Chan-Gi; Sako, Yasushi; Nakamura, Yoshikazu
Role of Lipid Rafts and GM1 in the Segregation and Processing of Prion Protein
The prion protein (PrPC) is highly expressed within the nervous system. Similar to other GPI-anchored proteins, PrPC is found in lipid rafts, membrane domains enriched in cholesterol and sphingolipids. PrPC raft association, together with raft lipid composition, appears essential for the conversion of PrPC into the scrapie isoform PrPSc, and the development of prion disease. Controversial findings were reported on the nature of PrPC-containing rafts, as well as on the distribution of PrPC between rafts and non-raft membranes. We investigated PrPC/ganglioside relationships and their influence on PrPC localization in a neuronal cellular model, cerebellar granule cells. Our findings argue that in these cells at least two PrPC conformations coexist: in lipid rafts PrPC is present in the native folding (?-helical), stabilized by chemico-physical condition, while it is mainly present in other membrane compartments in a PrPSc-like conformation. We verified, by means of antibody reactivity and circular dichroism spectroscopy, that changes in lipid raft-ganglioside content alters PrPC conformation and interaction with lipid bilayers, without modifying PrPC distribution or cleavage. Our data provide new insights into the cellular mechanism of prion conversion and suggest that GM1-prion protein interaction at the cell surface could play a significant role in the mechanism predisposing to pathology.
Botto, Laura; Cunati, Diana; Coco, Silvia; Sesana, Silvia; Bulbarelli, Alessandra; Biasini, Emiliano; Colombo, Laura; Negro, Alessandro; Chiesa, Roberto; Masserini, Massimo; Palestini, Paola
Caspase-12 and endoplasmic reticulum stress mediate neurotoxicity of pathological prion protein
Prion diseases are characterized by accumulation of misfolded prion protein (PrPSc), and neuronal death by apoptosis. Here we show that nanomolar concentrations of purified PrPSc from mouse scrapie brain induce apoptosis of N2A neuroblastoma cells. PrPSc toxicity was associated with an increase of intracellular calcium released from endoplasmic reticulum (ER) and up-regulation of several ER chaperones. Caspase-12 activation was detected in cells treated with PrPSc, and cellular death was inhibited by overexpression of a catalytic mutant of caspase-12 or an ER-targeted Bcl-2 chimeric protein. Scrapie-infected N2A cells were more susceptible to ER-stress and to PrPSc toxicity than non-infected cells. In scrapie-infected mice a correlation between caspase-12 activation and neuronal loss was observed in histological and biochemical analyses of different brain areas. The extent of prion replication was closely correlated with the up-regulation of ER-stress chaperone proteins. Similar results were observed in humans affected with sporadic and variant Creutzfeldt�Jakob disease, implicating for the first time the caspase-12 dependent pathway in a neurodegenerative disease in vivo, and thus offering novel potential targets for the treatment of prion disorders.
Hetz, Claudio; Russelakis-Carneiro, Milene; Maundrell, Kinsey; Castilla, Joaquin; Soto, Claudio
Linkage of prion protein and scrapie incubation time genes
A single gene (Prn-i) that affects scrapie incubation period in mice has been identified. I\\/LnJ mice have a very long incubation period after inoculation of scrapie prions (200-385 days) and NZW\\/LacJ mice have a short one (113 +\\/- 2.8 days). (NZW X I\\/Ln)F1 hybrid mice had incubation times of 223 +\\/- 2.8 days indicating longer incubation times were dominant. Incubation
G A Carlson; D T Kingsbury; P A Goodman; S Coleman; S T Marshall; S DeArmond; D Westaway; S B Prusiner
Development of techniques in magnetic resonance and structural studies of the prion protein
Magnetic resonance is the most powerful analytical tool used by chemists today. Its applications range from determining structures of large biomolecules to imaging of human brains. Nevertheless, magnetic resonance remains a relatively young field, in which many techniques are currently being developed that have broad applications. In this dissertation, two new techniques are presented, one that enables the determination of torsion angles in solid-state peptides and proteins, and another that involves imaging of heterogenous materials at ultra-low magnetic fields. In addition, structural studies of the prion protein via solid-state NMR are described. More specifically, work is presented in which the dependence of chemical shifts on local molecular structure is used to predict chemical shift tensors in solid-state peptides with theoretical ab initio surfaces. These predictions are then used to determine the backbone dihedral angles in peptides. This method utilizes the theoretical chemicalshift tensors and experimentally determined chemical-shift anisotropies (CSAs) to predict the backbone and side chain torsion angles in alanine, leucine, and valine residues. Additionally, structural studies of prion protein fragments are described in which conformationally-dependent chemical-shift measurements were made to gain insight into the structural differences between the various conformational states of the prion protein. These studies are of biological and pathological interest since conformational changes in the prion protein are believed to cause prion diseases. Finally, an ultra-low field magnetic resonance imaging technique is described that enables imaging and characterization of heterogeneous and porous media. The notion of imaging gases at ultra-low fields would appear to be very difficult due to the prohibitively low polarization and spin densities as well as the low sensitivities of conventional Faraday coil detectors. However, Chapter 5 describes how gas imaging at ultra-low fields is realized by incorporating the high sensitivities of a dc superconducting quantum interference device (SQUID) with the high polarizations attainable through optica11y pumping {sup 129}Xe gas.
Bitter, Hans-Marcus L.
Comparison of DNA Sequences with Protein Sequences
The FASTA package of sequence comparison programs has been expanded to include FASTX and FASTY, which compare a DNA sequence to a protein sequence database, translating the DNA sequence in three frames and aligning the translated DNA sequence to each sequence in the protein database, allowing gaps and frameshifts. Also new are TFASTX and TFASTY, which compare a protein sequence
William R. Pearson; Todd Wood; Zheng Zhang; Webb Miller
Hyperbaric Oxygen Enhances the Expression of Prion Protein and Heat Shock Protein 70 in a Mouse Neuroblastoma Cell Line
1. Cellular prion protein, PrPC, is a ubiquitous glycoprotein strongly expressed in neurons with an as yet unknown biological function. In previous studies, we demonstrated that PrPC could be regulated by heat shock stress, implying that it might be a stress-responsive protein. Hyperbaric oxygen (HBO) administration is a well-defined model for the study of oxidative stress.
Woei-Cherng Shyu; Shinn-Zong Lin; Keiichi Saeki; Astsutaka Kubosaki; Yoshitsugu Matsumoto; Takashi Onodera; Ming-Fu Chiang; Peterus Thajeb; Hung Li
Caffeine prevents human prion protein-mediated neurotoxicity through the induction of autophagy.
The human prion protein (PrP) fragment PrP(106?126) possesses the majority of the pathogenic properties associated with the infectious scrapie isoform of PrP, known as PrPSc. The accumulation of PrPSc in the brain of humans and animals affects the central nervous system. Recent epidemiological studies have suggested that caffeine, one of the major components of coffee, exerts protective effects against the development of neurodegeneration. However, the protective effects of caffeine against prion disease have not been reported to date. In this study, we therefore investigated the effects of caffeine on PrP-mediated neurotoxicity. The protein expression of the autophagosomal marker, LC3-II, was increased by caffeine in a dose-dependent manner, and the autophagy induced by caffeine protected the neuronal cells against PrP(106?126)?induced cell death. On the contrary, the downregulation of LC3-II using the autophagy inhibitors, 3-methyladenine (3-??) and wortmannin, prevented the caffeine-mediated neuroprotective effects. To the best of our knowledge, the present study provides the first evidence that treatment with caffeine protects human neuronal cells against prion?mediated neurotoxicity and these neuroprotective effects are mediated by caffeine-induced autophagy signals. Our data suggest that treatment with caffeine may be a novel therapeutic strategy for prion peptide?induced apoptosis. PMID:24938171
Moon, Ji-Hong; Lee, Ju-Hee; Park, Jin-Young; Kim, Sung-Wook; Lee, You-Jin; Kang, Seog-Jin; Seol, Jae-Won; Ahn, Dong-Choon; Park, Sang-Youel
Lentivector-mediated RNAi efficiently suppresses prion protein and prolongs survival of scrapie-infected mice
Prion diseases are fatal neurodegenerative diseases characterized by the accumulation of PrPSc, the infectious and protease-resistant form of the cellular prion protein (PrPC). We generated lentivectors expressing PrPC-specific short hairpin RNAs (shRNAs) that efficiently silenced expression of the prion protein gene (Prnp) in primary neuronal cells. Treatment of scrapie-infected neuronal cells with these lentivectors resulted in an efficient and stable suppression of PrPSc accumulation. After intracranial injection, lentiviral shRNA reduced PrPC expression in transgenic mice carrying multiple copies of Prnp. To test the therapeutic potential of lentiviral shRNA, we used what we believe to be a novel approach in which the clinical situation was mimicked. We generated chimeric mice derived from lentivector-transduced embryonic stem cells. Depending on the degree of chimerism, these animals carried the lentiviral shRNAs in a certain percentage of brain cells and expressed reduced levels of PrPC. Importantly, in highly chimeric mice, survival after scrapie infection was significantly extended. Taken together, these data suggest that lentivector-mediated RNA interference could be an approach for the treatment of prion disease.
Pfeifer, Alexander; Eigenbrod, Sabina; Al-Khadra, Saba; Hofmann, Andreas; Mitteregger, Gerda; Moser, Markus; Bertsch, Uwe; Kretzschmar, Hans
Early Detection of Abnormal Prion Protein in Genetic Human Prion Diseases Now Possible Using Real-Time QUIC Assay
Introduction The definitive diagnosis of genetic prion diseases (gPrD) requires pathological confirmation. To date, diagnosis has relied upon the finding of the biomarkers 14-3-3 protein and total tau (t-tau) protein in the cerebrospinal fluid (CSF), but many researchers have reported that these markers are not sufficiently elevated in gPrD, especially in Gerstmann-Str�ussler-Scheinker syndrome (GSS). We recently developed a new in vitro amplification technology, designated �real-time quaking-induced conversion (RT-QUIC)�, to detect the abnormal form of prion protein in CSF from sporadic Creutzfeldt-Jakob disease (sCJD) patients. In the present study, we aimed to investigate the presence of biomarkers and evaluate RT-QUIC assay in patients with gPrD, as the utility of RT-QUIC as a diagnostic tool in gPrD has yet to be determined. Method/Principal Findings 56 CSF samples were obtained from gPrD patients, including 20 cases of GSS with P102L mutation, 12 cases of fatal familial insomnia (FFI; D178N), and 24 cases of genetic CJD (gCJD), comprising 22 cases with E200K mutation and 2 with V203I mutation. We subjected all CSF samples to RT-QUIC assay, analyzed 14-3-3 protein by Western blotting, and measured t-tau protein using an ELISA kit. The detection sensitivities of RT-QUIC were as follows: GSS (78%), FFI (100%), gCJD E200K (87%), and gCJD V203I (100%). On the other hand the detection sensitivities of biomarkers were considerably lower: GSS (11%), FFI (0%), gCJD E200K (73%), and gCJD V203I (67%). Thus, RT-QUIC had a much higher detection sensitivity compared with testing for biomarkers, especially in patients with GSS and FFI. Conclusion/Significance RT-QUIC assay is more sensitive than testing for biomarkers in gPrD patients. RT-QUIC method would thus be useful as a diagnostic tool when the patient or the patient's family does not agree to genetic testing, or to confirm the diagnosis in the presence of a positive result for genetic testing.
Sano, Kazunori; Satoh, Katsuya; Atarashi, Ryuichiro; Takashima, Hiroshi; Iwasaki, Yasushi; Yoshida, Mari; Sanjo, Nobuo; Murai, Hiroyuki; Mizusawa, Hidehiro; Schmitz, Matthias; Zerr, Inga; Kim, Yong-Sun; Nishida, Noriyuki
Normal levels of the antiprion proteins Btn2 and Cur1 cure most newly formed [URE3] prion variants.
[URE3] is an amyloid prion of the Saccharomyces cerevisiae Ure2p, a regulator of nitrogen catabolism. Overproduction of Btn2p, involved in late endosome to Golgi protein transport, or its paralog Cur1p, cures [URE3]. Btn2p, in curing, is colocalized with Ure2p in a single locus, suggesting sequestration of Ure2p amyloid filaments. We find that most [URE3] variants generated in a btn2 cur1 double mutant are cured by restoring normal levels of Btn2p and Cur1p, with both proteins needed for efficient curing. The [URE3] variants cured by normal levels of Btn2p and Cur1p all have low seed number, again suggesting a seed sequestration mechanism. Hsp42 overproduction also cures [URE3], and Hsp42p aids Btn2 overproduction curing. Cur1p is needed for Hsp42 overproduction curing of [URE3], but neither Btn2p nor Cur1p is needed for overproduction curing by the other. Although hsp42? strains stably propagate [URE3-1], hsp26? destabilizes this prion. Thus, Btn2p and Cur1p are antiprion system components at their normal levels, acting with Hsp42. Btn2p is related in sequence to human Hook proteins, involved in aggresome formation and other transport activities. PMID:24938787
Wickner, Reed B; Bezsonov, Evgeny; Bateman, David A
Identification and removal of proteins that co-purify with infectious prion protein improves the analysis of its secondary structure.
Prion diseases are neurodegenerative disorders associated with the accumulation of an abnormal isoform of the mammalian prion protein (PrP). Fourier transform infrared spectroscopy (FTIR) has previously been used to show that the conformation of aggregated, infectious PrP (PrP(Sc) ) varies between prion strains and these unique conformations may determine strain-specific disease phenotypes. However, the relative amounts of ?-helix, ?-sheet and other secondary structures have not always been consistent between studies, suggesting that other proteins might be confounding the analysis of PrP(Sc) secondary structure. We have used FTIR and LC-MS/MS to analyze enriched PrP(Sc) from mouse and hamster prion strains both before and after the removal of protein contaminants that commonly co-purify with PrP(Sc) . Our data show that non-PrP proteins do contribute to absorbances that have been associated with ?-helical, loop, turn and ?-sheet structures attributed to PrP(Sc) . The major contaminant, the ?-helical protein ferritin, absorbs strongly at 1652 cm(-1) in the FTIR spectrum associated with PrP(Sc) . However, even the removal of more than 99% of the ferritin from PrP(Sc) did not completely abolish absorbance at 1652 cm(-1) . Our results show that contaminating proteins alter the FTIR spectrum attributed to PrP(Sc) and suggest that the ?-helical, loop/turn and ?-sheet secondary structure that remains following their removal are derived from PrP(Sc) itself. PMID:21805638
Moore, Roger A; Timmes, Andrew G; Wilmarth, Phillip A; Safronetz, David; Priola, Suzette A
Distinct prion proteins in short and long scrapie incubation period mice
The Prn-i gene, controlling scrapie incubation period, is linked to or congruent with the murine prion protein (PrP) gene, Prn-p. In prototypic mouse strains with long (l\\/Ln) and short (NZW) incubation periods, Prn-p transcription is initiated at similar multiple sites. The predicted NZW and l\\/Ln PrP proteins differ at codons 108 and 189. Codon 189, highly conserved in mammals, lies
D Westaway; P A Goodman; C A Mirenda; M P McKinley; G A Carlson; S B Prusiner
Altered Prion Protein Expression Pattern in CSF as a Biomarker for Creutzfeldt-Jakob Disease
Creutzfeldt-Jakob disease (CJD) is the most frequent human Prion-related disorder (PrD). The detection of 14-3-3 protein in the cerebrospinal fluid (CSF) is used as a molecular diagnostic criterion for patients clinically compatible with CJD. However, there is a pressing need for the identification of new reliable disease biomarkers. The pathological mechanisms leading to accumulation of 14-3-3 protein in CSF are
Mauricio Torres; Luis Cartier; Jos� Manuel Matamala; Nancy Hern�ndez; Ute Woehlbier; Claudio Hetz
GFP-tagged mutant prion protein forms intra-axonal aggregates in transgenic mice
A nine-octapeptide insertional mutation in the prion protein (PrP) causes a fatal neurodegenerative disorder in both humans and transgenic mice. To determine the precise cellular localization of this mutant PrP (designated PG14), we have generated transgenic mice expressing PG14-EGFP, a fluorescent fusion protein that can be directly visualized in vivo. Tg(PG14-EGFP) mice develop an ataxic neurological illness characterized by astrogliosis,
Andrea Z. Medrano; Sami J. Barmada; Emiliano Biasini; David A. Harris
Two amyloid states of the prion protein display significantly different folding patterns
Summary It has been well established that a single amino acid sequence can give rise to several conformationally distinct amyloid states. The extent to which amyloid structures formed within the same sequence are different, however, remains unclear. To address this question we studied two amyloid states (referred to as R- and S-fibrils) produced in vitro from highly purified full-length recombinant prion protein (PrP). Several biophysical techniques including X-ray diffraction, CD, FTIR, hydrogen-deuterium exchange, proteinase K-digestion, and binding of a conformation-sensitive fluorescence dye revealed that R- and S-fibrils have substantially different secondary, tertiary and quaternary structures. While both states displayed a 4.8 � meridional X-ray diffraction typical for amyloid cross-? spines, they showed markedly different equatorial profiles suggesting different folding pattern of ?-strands. The experiments on hydrogen-deuterium exchange monitored by FTIR revealed that only small fractions of amide protons were protected in R- or S-fibrils, an argument for the dynamic nature of their cross-? structure. Despite this fact, both amyloid states were found to be very stable conformationally as judged from temperature-induced denaturation monitored by FTIR and the conformation-sensitive dye. Upon heating to 80 �C, only local unfolding was revealed, while individual state-specific cross-? features were preserved. The current studies demonstrated that the two amyloid states formed by the same amino acid sequence exhibited significantly different folding patterns that presumably reflect two different architectures of cross-? structure. Both S- and R-fibrils, however, shared high conformational stability arguing that the energy landscape for protein folding and aggregation can contain several deep free energy minima.
Ostapchenko, Valeriy G.; Sawaya, Michael R.; Makarava, Natallia; Savtchenko, Regina; Nilsson, K. Peter R.; Eisenberg, David; Baskakov, Ilia V.
Spongiform Pathology in Mouse CNS Lacking 'Neuropathy Target Esterase' and Cellular Prion Protein
Conditional inactivation of the �neuropathy target esterase� (NTE) gene in mouse nerve cells was previously shown to result in CNS pathology comparable to the spongiform encephalopathy characteristic of prion diseases. To determine whether cellular prion protein (PrPc) is essential for development of this pathology we examined hippocampi of mice lacking NTE alone, PrPc alone or both NTE and PrPc. Light microscopic survey showed clear-cut spongiform changes in a majority of NTE-/- and NTE/PrP-/- double knockout mice but in only one PrP-/- mouse. EM analysis of spongiform lesions from NTE-/- and NTE/PrP-/- mice, and from the one affected PrP-/- mouse, revealed patches of branching tubular inclusions, comparable to the �tubulovesicular inclusions� described previously in prion diseases. We conclude that spongiform pathology in conditional NTE knockout mice is not mediated by PrPc, and that tubulovesicular inclusions can be seen in spongiform encephalopathy of other etiologies and are not pathognomonic of prion disease.
Rosenbluth, Jack; Schiff, Rolf; Lam, Pokman; Nuriel, Tal; Chao, Moses V.
The prion infection is a conversion of host encoded prion protein (PrP) from its cellular isoform PrPC into the pathological and infectious isoform PrPSc; the conversion process was investigated by in vitro studies using recombinant and cellular PrP and natural PrPSc. We present a brief summary of the results determined with our in vitro conversion system and the derived mechanistic models. We describe well characterized intermediates and precursor states during the conversion process, kinetic studies of spontaneous and seeded fibrillogenesis and the impact of the membrane environment.
Birkmann, Eva
The prion protein gene in humans revisited: Lessons from a worldwide resequencing study
Ample evidence has accumulated showing that different coding variants of the PRNP gene confer differential susceptibility for prion diseases. Here we evaluate the patterns of nucleotide variation in PRNP exon 2, which includes all the protein-coding sequence, by resequencing a worldwide sample of 174 humans for 2378 bp. In line with previous studies, we found two main haplotypes differentiated by nonsynonymous substitution in codon 129. Our analyses reveal the worldwide pattern of variation at the PRNP gene to be inconsistent with neutral expectations, indicating instead an excess of low-frequency variants, a footprint of the action of either positive or purifying selection. A comparison of neutrality test statistics for PRNP with other human genes indicates that the signal of positive selection on PRNP is stronger than expected from a possible confounding genome-wide background signal of population expansion. Two main conclusions arise from our analysis. First, the existence of an ancient, stable, balanced polymorphism that has been claimed in a previous study and related to cannibalism can be rejected and is shown to be due to ascertainment bias. Second, our results are consistent with a complex history of selection including mainly positive selection, even if short local periods of balancing selection (Kuru-like episodes), or even a weak purifying selection model, are consistent with our data.
Soldevila, Marta; Andres, Aida M.; Ramirez-Soriano, Anna; Marques-Bonet, Tomas; Calafell, Francesc; Navarro, Arcadi; Bertranpetit, Jaume
The POM Monoclonals: A Comprehensive Set of Antibodies to Non-Overlapping Prion Protein Epitopes
PrPSc, a misfolded and aggregated form of the cellular prion protein PrPC, is the only defined constituent of the transmissible agent causing prion diseases. Expression of PrPC in the host organism is necessary for prion replication and for prion neurotoxicity. Understanding prion diseases necessitates detailed structural insights into PrPC and PrPSc. Towards this goal, we have developed a comprehensive collection of monoclonal antibodies denoted POM1 to POM19 and directed against many different epitopes of mouse PrPC. Three epitopes are located within the N-terminal octarepeat region, one is situated within the central unstructured region, and four epitopes are discontinuous within the globular C-proximal domain of PrPC. Some of these antibodies recognize epitopes that are resilient to protease digestion in PrPSc. Other antibodies immunoprecipitate PrPC, but not PrPSc. A third group was found to immunoprecipitate both PrP isoforms. Some of the latter antibodies could be blocked with epitope-mimicking peptides, and incubation with an excess of these peptides allowed for immunochromatography of PrPC and PrPSc. Amino-proximal antibodies were found to react with repetitive PrPC epitopes, thereby vastly increasing their avidity. We have also created functional single-chain miniantibodies from selected POMs, which retained the binding characteristics despite their low molecular mass. The POM collection, thus, represents a unique set of reagents allowing for studies with a variety of techniques, including western blotting, ELISA, immunoprecipitation, conformation-dependent immunoassays, and plasmon surface plasmon resonance-based assays.
Polymenidou, Magdalini; Moos, Rita; Scott, Mike; Sigurdson, Christina; Shi, Yong-zhong; Yajima, Bill; Hafner-Bratkovic, Iva; Jerala, Roman; Hornemann, Simone; Wuthrich, Kurt; Bellon, Anne; Vey, Martin; Garen, Graciela; James, Michael N. G.; Kav, Nat; Aguzzi, Adriano
Immunization with Recombinant Prion Protein Leads to Partial Protection in a Murine Model of TSEs through a Novel Mechanism
Transmissible spongiform encephalopathies are neurodegenerative diseases, which despite fervent research remain incurable. Immunization approaches have shown great potential at providing protection, however tolerance effects hamper active immunization protocols. In this study we evaluated the antigenic potential of various forms of recombinant murine prion protein and estimated their protective efficacy in a mouse model of prion diseases. One of the forms tested provided a significant elongation of survival interval. The elongation was mediated via an acute depletion of mature follicular dendritic cells, which are associated with propagation of the prion infectious agent in the periphery and in part to the development of humoral immunity against prion protein. This unprecedented result could offer new strategies for protection against transmissible encephalopathies as well as other diseases associated with follicular dendritic cells.
Xanthopoulos, Konstantinos; Lagoudaki, Rosa; Kontana, Anastasia; Kyratsous, Christos; Panagiotidis, Christos; Grigoriadis, Nikolaos; Yiangou, Minas; Sklaviadis, Theodoros
FTY720 protects neuronal cells from damage induced by human prion protein by inactivating the JNK pathway.
Prion diseases affect the central nervous system (CNS) in humans and animals, and are associated with the conversion of the cellular prion protein (PrPC) to the misfolded isoform (PrPSc). FTY720, an immune modulator and synthetic analogue of sphingosine-1-phosphate (S1P), activates S1P receptors and has been shown to be effective in experimental models of transplantation and autoimmunity, including multiple sclerosis. Whereas the immune modulatory functions of FTY720 have been extensively investigated, the other functions of FTY720 are not yet well understood. In this study, we investigated the effects of FTY720 phosphate (FTY720-p) on prion protein-mediated neuronal cell death, as well as its effects on intracellular apoptotic pathways. Treatment with FTY720-p protected neuronal cells from synthetic human prion protein peptide [PrP�(106?126)]-mediated damage and prevented mitochondrial dysfunction by inhibiting the activation of c-jun N-terminal kinase. Moreover, FTY720-p prevented the PrP�(106?126)-induced reduction in mitochondrial potential, the translocation of Bax to the mitochondria and the release of cytochrome�c. To the best of our knowledge, this study is the first to demonstrate the effects of FTY720 on prion protein-mediated neurotoxicity and to suggest that FTY720 has therapeutic potential in prion diseases. PMID:24142108
Moon, Myung-Hee; Jeong, Jae-Kyo; Lee, You-Jin; Park, Sang-Youel
?-Helical to ?-Helical Conformation Change in the C-Terminal of the Mammalian Prion Protein
NASA Astrophysics Data System (ADS)
We employ all-atom structure-based models with mixed basis contact maps to explore whether there are any significant geometric or energetic constraints limiting conjectured conformational transitions between the alpha-helical (?H) and the left handed beta helical (LHBH) conformations for the C-terminal (residues 166-226) of the mammalian prion protein. The LHBH structure has been proposed to describe infectious oligomers and one class of in vitro grown fibrils, as well as possibly self- templating the conversion of normal cellular prion protein to the infectious form. Our results confirm that the kinetics of the conformation change are not strongely limited by large scale geometry modification and there exists an overall preference for the LHBH conformation.
Singh, Jesse; Whitford, Paul; Hayre, Natha; Cox, Daniel; Onuchic, Jos�.
A nine-octapeptide insertional mutation in the prion protein (PrP) causes a fatal neurodegenerative disorder in both humans and transgenic mice. To determine the precise cellular localization of this mutant PrP (designated PG14), we have generated transgenic mice expressing PG14-EGFP, a fluorescent fusion protein that can be directly visualized in vivo. Tg(PG14-EGFP) mice develop an ataxic neurological illness characterized by astrogliosis, PrP aggregation, and accumulation of a partially protease-resistant form of the mutant PrP. Strikingly, PG14-EGFP forms numerous fluorescent aggregates in the neuropil and white matter of multiple brain regions. These aggregates are particularly prominent along axonal tracts in both brain and peripheral nerve, and similar intracellular deposits are visible along the processes of cultured neurons. Our results reveal intra-axonal aggregates of a mutant PrP, which could contribute to the pathogenesis of familial prion disease by disrupting axonal transport.
Medrano, Andrea Z.; Barmada, Sami J.; Biasini, Emiliano; Harris, David A.
In vitro prion protein conversion suggests risk of bighorn sheep (Ovis canadensis) to transmissible spongiform encephalopathies
Our results indicate the in vitro conversion assay used in our study does mimic the species barrier of mice to the TSE agents that we tested. Based on Prnp genotype and results from conversion assays, BHS are likely to be susceptible to infection by classical scrapie. Despite mismatches in amino acids thought to modulate prion protein conversion, our data indicate that A136R154Q171 genotype sheep prion protein is misfolded by CWD agent, suggesting that these animals could be susceptible to CWD. Further investigation of TSE transmissibility to BHS, including animal studies, is warranted. The lack of reported TSEs in BHS may be attributable to other host factors or a lack of TSE surveillance in this species.
Johnson, Christopher J.; Morawski, A. R.; Carlson, C. M.; Chang, H.
Spongiform encephalopathy in siblings with no evidence of protease-resistant prion protein or a mutation in the prion protein gene.
We discuss relevant aspects in two siblings with a neurodegenerative process of unclear aetiology who developed progressive dementia with global aphasia and hyperoral behaviour at the ages of 39 and 46 years and who died 6 and 5 years after disease onset. The cases were reported to the National Reference Center for TSE Surveillance in G�ttingen, Germany. Detailed clinical examinations, CSF, blood samples, and copies of the important diagnostic tests (magnetic resonance imaging, electroencephalogram, laboratory tests) were obtained. Further neuropathological and genetic analyses were performed. Cerebral magnetic resonance imaging of both siblings showed prominent changes in signal intensity, especially in the left medial temporal cortex, but also the hippocampal formation. Neuropathological examination revealed spongiform changes, neuronal loss, and astrocytic gliosis, which are typical in Creutzfeldt-Jakob disease. However, no prion protein deposits were detectable by immunohistochemical analysis, Western blot, or PET blot, though abundant tau protein deposits were observed. A mutation in the coding region of the prion protein genes of both siblings was excluded. A detailed search of the literature revealed no other cases with a similar clinical and neuropathological appearance. While the disease aetiology remains unclear, the findings point to a neurodegenerative process and most likely a genetic disease. PMID:23546304
Varges, Daniela; Schulz-Schaeffer, Walter J; Wemheuer, Wiebke M; Damman, Insa; Schmitz, Matthias; Cramm, Maria; Kallenberg, Kai; Shirneshan, Katayoon; Elkenani, Manar; Markwort, Susanne; Faist, Michael; Kohlhase, J�rgen; Windl, Otto; Zerr, Inga
Naturally prion resistant mammals
Each known abnormal prion protein (PrPSc) is considered to have a specific range and therefore the ability to infect some species and not others. Consequently, some species have been assumed to be prion disease resistant as no successful natural or experimental challenge infections have been reported. This assumption suggested that, independent of the virulence of the PrPSc strain, normal prion protein (PrPC) from these �resistant� species could not be induced to misfold. Numerous in vitro and in vivo studies trying to corroborate the unique properties of PrPSc have been undertaken. The results presented in the article �Rabbits are not resistant to prion infection� demonstrated that normal rabbit PrPC, which was considered to be resistant to prion disease, can be misfolded to PrPSc and subsequently used to infect and transmit a standard prion disease to leporids. Using the concept of species resistance to prion disease, we will discuss the mistake of attributing species specific prion disease resistance based purely on the absence of natural cases and incomplete in vivo challenges. The BSE epidemic was partially due to an underestimation of species barriers. To repeat this error would be unacceptable, especially if present knowledge and techniques can show a theoretical risk. Now that the myth of prion disease resistance has been refuted it is time to re-evaluate, using the new powerful tools available in modern prion laboratories, whether any other species could be at risk.
Fernandez-Borges, Natalia; Chianini, Francesca; Erana, Hasier; Vidal, Enric; Eaton, Samantha L; Pintado, Belen; Finlayson, Jeanie; Dagleish, Mark P.; Castilla, Joaquin
Cotranslational Partitioning of Nascent Prion Protein into Multiple Populations at the Translocation Channel
The decisive events that direct a single polypeptide such as the prion protein (PrP) to be synthesized at the endoplasmic reticulum in both fully translocated and transmembrane forms are poorly understood. In this study, we demonstrate that the topological heterogeneity of PrP is determined cotranslationally, while at the translocation channel. By evaluating sequential inter- mediates during PrP topogenesis, we find
Soo Jung Kim; Ramanujan S. Hegde
Dissociation of Prion Protein Amyloid Seeding from Transmission of a Spongiform Encephalopathy
Misfolding and aggregation of proteins are common pathogenic mechanisms of a group of diseases called proteinopathies. The formation and spread of proteinaceous lesions within and between individuals were first described in prion diseases and proposed as the basis of their infectious nature. Recently, a similar �prion-like� mechanism of transmission has been proposed in other neurodegenerative diseases such as Alzheimer's disease. We investigated if misfolding and aggregation of corrupted prion protein (PrPTSE) are always associated with horizontal transmission of disease. Knock-in transgenic mice (101LL) expressing mutant PrP (PrP-101L) that are susceptible to disease but do not develop any spontaneous neurological phenotype were inoculated with (i) brain extracts containing PrPTSE from healthy 101LL mice with PrP plaques in the corpus callosum or (ii) brain extracts from mice overexpressing PrP-101L with neurological disease, severe spongiform encephalopathy, and formation of proteinase K-resistant PrPTSE. In all instances, 101LL mice developed PrP plaques in the area of inoculation and vicinity in the absence of clinical disease or spongiform degeneration of the brain. Importantly, 101LL mice did not transmit disease on serial passage, ruling out the presence of subclinical infection. Thus, in both experimental models the formation of PrPTSE is not infectious. These results have implications for the interpretation of tests based on the detection of protein aggregates and suggest that de novo formation of PrPTSE in the host does not always result in a transmissible prion disease. In addition, these results question the validity of assuming that all diseases due to protein misfolding can be transmitted between individuals.
Piccardo, Pedro; King, Declan; Telling, Glenn; Manson, Jean C.
Subcellular Colocalization of the Cellular and Scrapie Prion Proteins in Caveolae-Like Membranous Domains
Results of transgenetic studies argue that the scrapie isoform of the prion protein (PrPSc) interacts with the substrate cellular PrP (PrPC) during conversion into nascent PrPSc. While PrPSc appears to accumulate primarily in lysosomes, caveolae-like domains (CLDs) have been suggested to be the site where PrPC is converted into PrPSc. We report herein that CLDs isolated from scrapie-infected neuroblastoma (ScN2a)
Martin Vey; Susanne Pilkuhn; Holger Wille; Randal Nixon; Stephen J. Dearmond; Eric J. Smart; Richard G. W. Anderson; Albert Taraboulos
Deposition of disease-associated prion protein involves the peripheral nervous system in experimental scrapie
There is some evidence that the peripheral nervous system (PNS) is involved in the pathogenesis of transmissible spongiform\\u000a encephalopathies (TSEs). The TSE-specific abnormal prion protein (PrPsc) is considered as surrogate marker for infectivity. We traced the deposition of PrPsc by immunocytochemistry in sheep and hamsters inoculated intraperitoneally with scrapie. The trigeminal, dorsal root, celiac,\\u000a thoracic, and nodose ganglia contained ganglion
Martin H. Groschup; Michael Beekes; Patricia A. McBride; Michael Hardt; Johannes A. Hainfellner; Herbert Budka
Pronounced cytosolic aggregation of cellular prion protein in pancreatic ?-cells in response to hyperglycemia
Cellular prion protein (PrPC), an N-linked glycoprotein, is expressed in a variety of tissues, but its functions remain unclear. PrPC is abundantly expressed in the endocrine pancreas, which regulates blood glucose homeostasis. Therefore, we investigated whether the expression of PrPC was altered in islets of Langerhans in a model of spontaneous type 1 diabetes, the diabetes-prone BioBreeding (BBdp) rat and
Alexander Strom; Gen-Sheng Wang; Rudolph Reimer; Diane T Finegood; Fraser W Scott
A Comparative Study of Immunohistochemical Methods for Detecting Abnormal Prion Protein with Monoclonal and Polyclonal Antibodies
Transmissible spongiform encephalopathies are associated with the accumulation of abnormal prion protein (PrPSc) in the central nervous system which can be detected immunohistochemically. Using a monoclonal antibody (L42) to an epitope on the first ?-helix of ruminant PrP, we compared previously reported immunohistochemical antigen unmasking and �visualization� systems. In addition, a variety of polyclonal and monoclonal antibodies to other epitopes
M Hardt; T Baron; MH Groschup
The Crystal Structure of the Globular Domain of Sheep Prion Protein
The prion protein PrP is a naturally occurring polypeptide that becomes transformed from a normal conformation to that of an aggregated form, characteristic of pathological states in fatal transmissible spongiform conditions such as Creutzfeld�Jacob Disease and Bovine Spongiform Encephalopathy. We report the crystal structure, at 2� resolution, of residues 123�230 of the C-terminal globular domain of the ARQ allele of
L. F. Haire; S. M. Whyte; N. Vasisht; A. C. Gill; C. Verma; E. J. Dodson; G. G. Dodson; P. M. Bayley
BSE prions propagate as either variant CJD-like or sporadic CJD-like prion strains in transgenic mice expressing human prion protein
Variant Creutzfeldt�Jakob disease (vCJD) has been recognized to date only in individuals homozygous for methionine at PRNP codon 129. Here we show that transgenic mice expressing human PrP methionine 129, inoculated with either bovine spongiform encephalopathy (BSE) or variant CJD prions, may develop the neuropathological and molecular phenotype of vCJD, consistent with these diseases being caused by the same prion strain. Surprisingly, however, BSE transmission to these transgenic mice, in addition to producing a vCJD-like phenotype, can also result in a distinct molecular phenotype that is indistinguishable from that of sporadic CJD with PrPSc type�2. These data suggest that more than one BSE-derived prion strain might infect humans; it is therefore possible that some patients with a phenotype consistent with sporadic CJD may have a disease arising from BSE exposure.
Asante, Emmanuel A.; Linehan, Jacqueline M.; Desbruslais, Melanie; Joiner, Susan; Gowland, Ian; Wood, Andrew L.; Welch, Julie; Hill, Andrew F.; Lloyd, Sarah E.; Wadsworth, Jonathan D.F.; Collinge, John
A role for His155 in binding of human prion peptide144-167 to immobilised prion protein.
The interactions and conformational changes that lead to the conversion of the normal prion protein (PrP(c)) to its pathogenic form, PrP(sc), are still being elucidated. Using Surface Plasma Resonance (SPR), we provide evidence that a synthetic peptide (PrP(144-167)) corresponding to residues comprising the alpha helix 1-beta strand 2 domain of PrP(c) is able to interact and bind to immobilised recombinant human PrP (rHuPrP) in a dose-dependent manner. The interaction is pH dependent with an increase in binding observed as the pH is lowered, particularly between pH 6.5 and pH 5.5 suggesting a specific role for His(155) in the interaction, confirmed by covalent modification of this residue in the peptide with diethylpyrocarbonate (DEPC). Circular dichroism analysis of PrP(144-167) revealed no secondary structure motifs across the pH range investigated. Possible pH related structural changes of immobilised rHuPrP are also discussed with regard to the increased affinity for PrP(144-167). PMID:17761148
Hesp, J Richard; Raven, Neil D H; Sutton, J Mark
Interactions between the Conserved Hydrophobic Region of the Prion Protein and Dodecylphosphocholine Micelles*
The three-dimensional structure of PrP110�136, a peptide encompassing the conserved hydrophobic region of the human prion protein, has been determined at high resolution in dodecylphosphocholine micelles by NMR. The results support the conclusion that the CtmPrP, a transmembrane form of the prion protein, adopts a different conformation than the reported structures of the normal prion protein determined in solution. Paramagnetic relaxation enhancement studies with gadolinium-diethylenetriaminepentaacetic acid indicated that the conserved hydrophobic region peptide is not inserted symmetrically in the micelle, thus suggesting the presence of a guanidium-phosphate ion pair involving the side chain of the terminal arginine and the detergent headgroup. Titration of dodecylphosphocholine into a solution of PrP110�136 revealed the presence of a surface-bound species. In addition, paramagnetic probes located the surface-bound peptide somewhere below the micelle-water interface when using the inserted helix as a positional reference. This localization of the unknown population would allow a similar ion pair interaction.
Sauve, Simon; Buijs, Daniel; Gingras, Genevieve; Aubin, Yves
Insights into alternative prion protein topologies induced under high hydrostatic pressure
NASA Astrophysics Data System (ADS)
The critical step in the pathogenesis of transmissible spongiform encephalopathies (TSEs) appears to be a conformational transition of a normal prion protein (PrPC) into a misfolded isoform (PrPSc). To gain insight into the structural conversion of the prion protein we have exploited the use of high hydrostatic pressure combined with various spectroscopic techniques. In vitro transitions of the recombinant PrP to a scrapie-like form have never resulted in an infectious structure. It is our hypothesis that the acquisition of the disease-causing conformation depends on folding pathways which are difficult to attain. We attempt to favour, via specific reaction conditions at high pressure, alternative routes of misfolding leading to a stable infectious amyloidogenic conformer. Our results have demonstrated the potential of high pressure to reveal various prion structural changes, which are inaccessible by conventional methods. Especially, we have characterized a pressure-induced conformer in which the normal agr-helical structure is changed into a highly aggregated bgr-sheet conformation showing markedly increased resistance to proteolysis (key markers of potential infectious agents). Our work may have important implications, not only for ultimately proving the protein-only hypothesis and for understanding the basic mechanism of the disease, but also for developing preventative and therapeutic measures.
Torrent, Joan; Alvarez-Martinez, Maria Teresa; Heitz, Fr�d�ric; Liautard, Jean-Pierre; Balny, Claude; Lange, Reinhard
The role of the cellular prion protein in the immune system
Prion protein (PrP) plays a key role in the pathogenesis of prion diseases. However, the normal function of the protein remains unclear. The cellular isoform (PrPC) is expressed widely in the immune system, in haematopoietic stem cells and mature lymphoid and myeloid compartments in addition to cells of the central nervous system. It is up-regulated in T cell activation and may be expressed at higher levels by specialized classes of lymphocyte. Furthermore, antibody cross-linking of surface PrP modulates T cell activation and leads to rearrangements of lipid raft constituents and increased phosphorylation of signalling proteins. These findings appear to indicate an important but, as yet, ill-defined role in T cell function. Although PrP�/� mice have been reported to have only minor alterations in immune function, recent work has suggested that PrP is required for self-renewal of haematopoietic stem cells. Here, we consider the evidence for a distinctive role for PrPC in the immune system and what the effects of anti-prion therapeutics may be on immune function.
Isaacs, J D; Jackson, G S; Altmann, D M
Absolute Quantification of Prion Protein (90-231) Using Stable Isotope-Labeled Chymotryptic Peptide Standards in a LC-MRM AQUA Workflow
NASA Astrophysics Data System (ADS)
Substantial evidence indicates that the disease-associated conformer of the prion protein (PrPTSE) constitutes the etiologic agent in prion diseases. These diseases affect multiple mammalian species. PrPTSE has the ability to convert the conformation of the normal prion protein (PrPC) into a ?-sheet rich form resistant to proteinase K digestion. Common immunological techniques lack the sensitivity to detect PrPTSE at subfemtomole levels, whereas animal bioassays, cell culture, and in vitro conversion assays offer higher sensitivity but lack the high-throughput the immunological assays offer. Mass spectrometry is an attractive alternative to the above assays as it offers high-throughput, direct measurement of a protein's signature peptide, often with subfemtomole sensitivities. Although a liquid chromatography-multiple reaction monitoring (LC-MRM) method has been reported for PrPTSE, the chemical composition and lack of amino acid sequence conservation of the signature peptide may compromise its accuracy and make it difficult to apply to multiple species. Here, we demonstrate that an alternative protease (chymotrypsin) can produce signature peptides suitable for a LC-MRM absolute quantification (AQUA) experiment. The new method offers several advantages, including: (1) a chymotryptic signature peptide lacking chemically active residues (Cys, Met) that can confound assay accuracy; (2) low attomole limits of detection and quantitation (LOD and LOQ); and (3) a signature peptide retaining the same amino acid sequence across most mammals naturally susceptible to prion infection as well as important laboratory models. To the authors' knowledge, this is the first report on the use of a non-tryptic peptide in a LC-MRM AQUA workflow.
Sturm, Robert; Sheynkman, Gloria; Booth, Clarissa; Smith, Lloyd M.; Pedersen, Joel A.; Li, Lingjun
The formation of fibrillar amyloid is most often associated with protein conformational disorders such as prion diseases, Alzheimer disease and Huntington disease. Interestingly, however, an increasing number of studies suggest that amyloid structures can sometimes play a functional role in normal biology. Several proteins form self-propagating amyloids called prions in the budding yeast Saccharomyces cerevisiae. These unique elements operate by creating a reversible, epigenetic change in phenotype. While the function of the non-prion conformation of the Rnq1 protein is unclear, the prion form, [RNQ+], acts to facilitate the de novo formation of other prions to influence cellular phenotypes. The [RNQ+] prion itself does not adversely affect the growth of yeast, but the overexpression of Rnq1p can form toxic aggregated structures that are not necessarily prions. The [RNQ+] prion is also involved in dictating the aggregation and toxicity of polyglutamine proteins ectopically expressed in yeast. Thus, the [RNQ+] prion provides a tractable model that has the potential to reveal significant insight into the factors that dictate how amyloid structures are initiated and propagated in both physiological and pathological contexts.
Stein, Kevin C
Crystallization and preliminary X-ray diffraction analysis of prion protein bound to the Fab fragment of the POM1 antibody.
Prion diseases are neurodegenerative diseases that are characterized by the conversion of the cellular prion protein PrP(c) to the pathogenic isoform PrP(sc). Several antibodies are known to interact with the cellular prion protein and to inhibit this transition. An antibody Fab fragment, Fab POM1, was produced that recognizes a structural motif of the C-terminal domain of mouse prion protein. To study the mechanism by which Fab POM1 recognizes and binds the prion molecule, the complex between Fab POM1 and the C-terminal domain of mouse prion (residues 120-232) was prepared and crystallized. Crystals of this binary complex belonged to the monoclinic space group C2, with unit-cell parameters a = 83.68, b = 106.9, c = 76.25 �, ? = 95.6�. PMID:22102029
Baral, Pravas Kumar; Wieland, Barbara; Swayampakula, Mridula; Polymenidou, Magdalini; Aguzzi, Adriano; Kav, Nat N V; James, Michael N G
Failure of Prion Protein Oxidative Folding Guides the Formation of Toxic Transmembrane Forms*
The mechanism by which pathogenic mutations in the globular domain of the cellular prion protein (PrPC) increase the likelihood of misfolding and predispose to diseases is not yet known. Differences in the evidences provided by structural and metabolic studies of these mutants suggest that in vivo folding could be playing an essential role in their pathogenesis. To address this role, here we use the single or combined M206S and M213S artificial mutants causing labile folds and express them in cells. We find that these mutants are highly toxic, fold as transmembrane PrP, and lack the intramolecular disulfide bond. When the mutations are placed in a chain with impeded transmembrane PrP formation, toxicity is rescued. These results suggest that oxidative folding impairment, as on aging, can be fundamental for the genesis of intracellular neurotoxic intermediates key in prion neurodegenerations.
Lisa, Silvia; Domingo, Beatriz; Martinez, Javier; Gilch, Sabine; Llopis, Juan F.; Schatzl, Hermann M.; Gasset, Maria
Transcriptomic Analysis Brings New Insight into the Biological Role of the Prion Protein during Mouse Embryogenesis
The biological function of the Prion protein remains largely unknown but recent data revealed its implication in early zebrafish and mammalian embryogenesis. To gain further insight into its biological function, comparative transcriptomic analysis between FVB/N and FVB/N Prnp knockout mice was performed at early embryonic stages. RNAseq analysis revealed the differential expression of 73 and 263 genes at E6.5 and E7.5, respectively. The related metabolic pathways identified in this analysis partially overlap with those described in PrP1 and PrP2 knockdown zebrafish embryos and prion-infected mammalian brains and emphasize a potentially important role for the PrP family genes in early developmental processes.
Khalife, Manal; Young, Rachel; Passet, Bruno; Halliez, Sophie; Vilotte, Marthe; Jaffrezic, Florence; Marthey, Sylvain; Beringue, Vincent; Vaiman, Daniel; Le Provost, Fabienne; Laude, Hubert; Vilotte, Jean-Luc
Transcriptomic analysis brings new insight into the biological role of the prion protein during mouse embryogenesis.
The biological function of the Prion protein remains largely unknown but recent data revealed its implication in early zebrafish and mammalian embryogenesis. To gain further insight into its biological function, comparative transcriptomic analysis between FVB/N and FVB/N Prnp knockout mice was performed at early embryonic stages. RNAseq analysis revealed the differential expression of 73 and 263 genes at E6.5 and E7.5, respectively. The related metabolic pathways identified in this analysis partially overlap with those described in PrP1 and PrP2 knockdown zebrafish embryos and prion-infected mammalian brains and emphasize a potentially important role for the PrP family genes in early developmental processes. PMID:21858045
Khalif�, Manal; Young, Rachel; Passet, Bruno; Halliez, Sophie; Vilotte, Marthe; Jaffrezic, Florence; Marthey, Sylvain; B�ringue, Vincent; Vaiman, Daniel; Le Provost, Fabienne; Laude, Hubert; Vilotte, Jean-Luc
Ablation of prion protein immunoreactivity by heating in saturated calcium hydroxide
Background Prions, the infectious agents that cause transmissible spongiform encephalopathies (TSEs), are relatively resistant to destruction by physical, enzymatic, and chemical treatments. Hydrolysis in boiling saturated calcium hydroxide (limewater) utilizes inexpensive chemicals to digest protein components of offal. The purpose of this work was to determine if incubating brain material from scrapie-infected sheep in near-boiling saturated calcium hydroxide solution (Ca(OH)2) would abolish immunoreactivity of the infectious prion (PrPSc) as determined by western blot. Findings After incubating for as few as 10 minutes in saturated calcium hydroxide at 99�C, immunoreactivity of protease resistant bands by western blot analysis is completely lost. Conclusion Boiling in limewater may offer an alternative for disposal of carcasses and enable alternative uses for rendered products from potentially infected carcasses.
Greenlee, Justin J; Nicholson, Eric M; Hamir, Amir N; Noyes, Gary P; Holtzapple, Mark T; Kehrli, Marcus E
Ultrastructural studies on scrapie prion protein crystals obtained from reverse micellar solutions.
The structural transition from the cellular prion protein (PrPC) that is rich in alpha-helices to the pathological form (PrPSc) that has a high beta-sheet content seems to be the fundamental event underlying the prion diseases. Determination of the structure of PrPSc and the N-terminally truncated PrP 27-30 has been complicated by their insolubility. Here we report the solubilization of PrP 27-30 through a system of reverse micelles that yields monomeric and dimeric PrP. Although solubilization of PrP 27-30 was not accompanied by any recognizable change in secondary structure as measured by FTIR spectroscopy, it did result in a loss of prion infectivity. The formation of small two- and three-dimensional crystals upon exposure to uranyl salts argues that soluble PrP 27-30 possesses considerable tertiary structure. The crystals of PrP 27-30 grown from reverse micellar solutions suggest a novel crystallization mechanism that might be applicable for other membrane proteins. A variety of different crystal lattices diffracted up to 1.85 nm by electron microscopy. Despite the lack of measurable biological activity, the structure of PrP 27-30 in these crystals may provide insight into the structural transition that occurs during PrPSc formation.
Wille, H; Prusiner, S B
Palladium complexes affect the aggregation of human prion protein PrP106-126.
Many neurodegenerative disorders are induced by protein conformational change. Prion diseases are characterized by protein conformational conversion from a normal cellular form (PrP(C)) to an abnormal scrapie isoform (PrP(Sc)). PrP106-126 is an accepted model for studying the characteristics of PrP(Sc) because they share many biological and physiochemical properties. To understand how metal complexes affect the property of the prion peptide, the present work investigated interactions between Pd complexes and PrP106-126 based on our previous research using Pt and Au complexes to target the peptide. The selected compounds (Pd(phen)Cl(2), Pd(bipy)Cl(2), and Pd(en)Cl(2)) showed strong binding affinity to PrP106-126 and affected the conformation and aggregation of this active peptide in a different binding mode. Our results indicate that it may be the metal ligand-induced spatial effect rather the binding affinity that contributes to better inhibition on peptide aggregation. This finding would prove valuable in helping design and develop novel metallodrugs against prion diseases. PMID:21504185
Wang, Yanli; Feng, Li; Zhang, Bingbing; Wang, Xuesong; Huang, Cheng; Li, Yiming; Du, Weihong
Spontaneous Generation of Infectious Prion Disease in Transgenic Mice
We generated transgenic mice expressing bovine cellular prion protein (PrPC) with a leucine substitution at codon 113 (113L). This protein is homologous to human protein with mutation 102L, and its genetic link with Gerstmann�Str�ussler�Scheinker syndrome has been established. This mutation in bovine PrPC causes a fully penetrant, lethal, spongiform encephalopathy. This genetic disease was transmitted by intracerebral inoculation of brain homogenate from ill mice expressing mutant bovine PrP to mice expressing wild-type bovine PrP, which indicated de novo generation of infectious prions. Our findings demonstrate that a single amino acid change in the PrPC sequence can induce spontaneous generation of an infectious prion disease that differs from all others identified in hosts expressing the same PrPC sequence. These observations support the view that a variety of infectious prion strains might spontaneously emerge in hosts displaying random genetic PrPC mutations.
Castilla, Joaquin; Pintado, Belen; Gutierrez-Adan, Alfonso; Andreoletti, Olivier; Aguilar-Calvo, Patricia; Arroba, Ana-Isabel; Parra-Arrondo, Beatriz; Ferrer, Isidro; Manzanares, Jorge; Espinosa, Juan-Carlos
New insights into cellular prion protein (PrP c) functions: The � ying and yang� of a relevant protein
The conversion of cellular prion protein (PrPc), a GPI-anchored protein, into a protease-K-resistant and infective form (generally termed PrPsc) is mainly responsible for Transmissible Spongiform Encephalopathies (TSEs), characterized by neuronal degeneration and progressive loss of basic brain functions. Although PrPc is expressed by a wide range of tissues throughout the body, the complete repertoire of its functions has not been
Oriol Nicolas; Rosalina Gav�n; Jos� A. del R�o
Structure of the ?2-?2 loop and interspecies prion transmission
Prions are misfolded, aggregated conformers of the prion protein that can be transmitted between species. The precise determinants of interspecies transmission remain unclear, although structural similarity between the infectious prion and host prion protein is required for efficient conversion to the misfolded conformer. The ?2-?2 loop region of endogenous prion protein, PrPC, has been implicated in barriers to prion transmission. We recently discovered that conversion was efficient when incoming and host prion proteins had similar ?2-?2 loop structures; however, the roles of primary vs. secondary structural homology could not be distinguished. Here we uncouple the effect of primary and secondary structural homology of the ?2-?2 loop on prion conversion. We inoculated prions from animals having a disordered or an ordered ?2-?2 loop into mice having a disordered loop or an ordered loop due to a single residue substitution (D167S). We found that prion conversion was driven by a homologous primary structure and occurred independently of a homologous secondary structure. Similarly, cell-free conversion using PrPC from mice with disordered or ordered loops and prions from 5 species correlated with primary but not secondary structural homology of the loop. Thus, our findings support a model in which efficient interspecies prion conversion is determined by small stretches of the primary sequence rather than the secondary structure of PrP.�Bett, C., Fern�ndez-Borges, N., Kurt, T. D., Lucero, M., Nilsson, K. P. R., Castilla, J., Sigurdson, C. J. Structure of the ?2-?2 loop and interspecies prion transmission.
Bett, Cyrus; Fernandez-Borges, Natalia; Kurt, Timothy D.; Lucero, Melanie; Nilsson, K. Peter R.; Castilla, Joaquin; Sigurdson, Christina J.
Two Misfolding Routes for the Prion Protein around pH 4.5
Using molecular dynamics simulations, we show that the prion protein (PrP) exhibits a dual behavior, with two possible transition routes, upon protonation of H187 around pH 4.5, which mimics specific conditions encountered in endosomes. Our results suggest a picture in which the protonated imidazole ring of H187 experiences an electrostatic repulsion with the nearby guanidinium group of R136, to which the system responds by pushing either H187 or R136 sidechains away from their native cavities. The regions to which H187 and R136 are linked, namely the C-terminal part of H2 and the loop connecting S1 to H1, respectively, are affected in a different manner depending on which pathway is taken. Specific in vivo or in vitro conditions, such as the presence of molecular chaperones or a particular experimental setup, may favor one transition pathway over the other, which can result in very different monomers. This has some possible connections with the observation of various fibril morphologies and the outcome of prion strains. In addition, the finding that the interaction of H187 with R136 is a weak point in mammalian PrP is supported by the absence of the residue pair in non-mammalian species that are known to be resistant to prion diseases.
Garrec, Julian; Tavernelli, Ivano; Rothlisberger, Ursula
Uptake and neuritic transport of scrapie prion protein coincident with infection of neuronal cells.
Invasion of the nervous system and neuronal spread of infection are critical, but poorly understood, steps in the pathogenesis of transmissible spongiform encephalopathies or prion diseases. To characterize pathways for the uptake and intraneuronal trafficking of infectious, protease-resistant prion protein (PrP-res), fluorescent-labeled PrP-res was used to infect a neuronally derived murine cell line (SN56) and adult hamster cortical neurons in primary culture. Concurrent with the establishment of persistent scrapie infection, SN56 cells internalized PrP-res aggregates into vesicles positive for markers for late endosomes and/or lysosomes but not synaptic, early endocytic, or raft-derived vesicles. Internalized PrP-res was then transported along neurites to points of contact with other cells. Similar trafficking was observed with dextran, Alzheimer's Abeta1-42 fibrils and noninfectious recombinant PrP fibrils, suggesting that PrP-res is internalized by a relatively nonspecific pinocytosis or transcytosis mechanism. Hamster cortical neurons were also capable of internalizing and disseminating exogenous PrP-res. Similar trafficking of exogenous PrP-res by cortical neurons cultured from the brains of PrP knock-out mice showed that uptake and neuritic transport did not require the presence of endogenous cellular PrP. These experiments visualize and characterize the initial steps associated with prion infection and transport within neuronal cells. PMID:15917460
Magalh�es, Ana Cristina; Baron, Gerald S; Lee, Kil Sun; Steele-Mortimer, Olivia; Dorward, David; Prado, Marco A M; Caughey, Byron
Single-Chain Fv Antibody Fragments Retain Binding Properties of the Monoclonal Antibody Raised Against Peptide P1 of the Human Prion Protein
Prion diseases are incurable neurodegenerative diseases that affect both humans and animals. The infectious agent is a pathogenic\\u000a form of the prion protein that accumulates in brain as amyloids. Currently, there is neither cure nor reliable preclinical\\u000a diagnostics on the market available. The growing number of reports shows that passive immunisation is one of the most promising\\u000a strategies for prion
Nives �krlj; Vladka ?urin �erbec; Marko Dolinar
Stability of the ?-structure in prion protein: A molecular dynamics study based on polarized force field
NASA Astrophysics Data System (ADS)
Conformational changes of the antiparallel ?-sheet in normal cellular prion protein (PrPC) of rat, bovine, and human are investigated by molecular dynamics simulations in both neutral and acidic environment. Using a recently developed simulation method based on an on-the-fly polarized protein-specific charge (PPC) update scheme during the simulation process, we evaluate and compare the cross-species performances of the ?-sheet during the early stage transition from the PrPC to its mutant configuration. Through this study, we observe the growth of the ?-sheet structure in all species studied with the extent of elongation in ?-sheet being different across the three species.
Xu, Zhijun; Lazim, Raudah; Mei, Ye; Zhang, Dawei
Nanoimaging for prion related diseases
Misfolding and aggregation of prion proteins is linked to a number of neurodegenerative disorders such as Creutzfeldt-Jacob disease (CJD) and its variants: Kuru, Gerstmann-Straussler-Scheinker syndrome and fatal familial insomnia. In prion diseases, infectious particles are proteins that propagate by transmitting a misfolded state of a protein, leading to the formation of aggregates and ultimately to neurodegeneration. Prion phenomenon is not restricted to humans. There are a number of prion-related diseases in a variety of mammals, including bovine spongiform encephalopathy (BSE, also known as �mad cow disease�) in cattle. All known prion diseases, collectively called transmissible spongiform encephalopathies (TSEs), are untreatable and fatal. Prion proteins were also found in some fungi where they are responsible for heritable traits. Prion proteins in fungi are easily accessible and provide a powerful model for understanding the general principles of prion phenomenon and molecular mechanisms of mammalian prion diseases. Presently, several fundamental questions related to prions remain unanswered. For example, it is not clear how prions cause the disease. Other unknowns include the nature and structure of infectious agent and how prions replicate. Generally, the phenomenon of misfolding of the prion protein into infectious conformations that have the ability to propagate their properties via aggregation is of significant interest. Despite the crucial importance of misfolding and aggregation, very little is currently known about the molecular mechanisms of these processes. While there is an apparent critical need to study molecular mechanisms underlying misfolding and aggregation, the detailed characterization of these single molecule processes is hindered by the limitation of conventional methods. Although some issues remain unresolved, much progress has been recently made primarily due to the application of nanoimaging tools. The use of nanoimaging methods shows great promise for understanding the molecular mechanisms of prion phenomenon, possibly leading toward early diagnosis and effective treatment of these devastating diseases. This review article summarizes recent reports which advanced our understanding of the prion phenomenon through the use of nanoimaging methods.
Portillo, Alexander M; Deckert-Gaudig, Tanja; Deckert, Volker
A simple quantitative model of macromolecular crowding effects on protein folding: Application to the murine prion protein(121-231)
NASA Astrophysics Data System (ADS)
A model of protein folding kinetics is applied to study the effects of macromolecular crowding on protein folding rate and stability. Macromolecular crowding is found to promote a decrease of the entropic cost of folding of proteins that produces an increase of both the stability and the folding rate. The acceleration of the folding rate due to macromolecular crowding is shown to be a topology-dependent effect. The model is applied to the folding dynamics of the murine prion protein (121-231). The differential effect of macromolecular crowding as a function of protein topology suffices to make non-native configurations relatively more accessible.
Bergasa-Caceres, Fernando; Rabitz, Herschel A.
Bovine prion protein as a modulator of protein kinase CK2.
On the basis of far-Western blot and plasmon resonance (BIAcore) experiments, we show here that recombinant bovine prion protein (bPrP) (25-242) strongly interacts with the catalytic alpha/alpha' subunits of protein kinase CK2 (also termed 'casein kinase 2'). This association leads to increased phosphotransferase activity of CK2alpha, tested on calmodulin or specific peptides as substrate. We also show that bPrP counteracts the inhibition of calmodulin phosphorylation promoted by the regulatory beta subunits of CK2. A truncated form of bPrP encompassing the C-terminal domain (residues 105-242) interacts with CK2 but does not affect its catalytic activity. The opposite is found with the N-terminal fragment of bPrP (residues 25-116), although the stimulation of catalysis is less efficient than with full-size bPrP. These results disclose the potential of the PrP to modulate the activity of CK2, a pleiotropic protein kinase that is particularly abundant in the brain.
Meggio, F; Negro, A; Sarno, S; Ruzzene, M; Bertoli, A; Sorgato, M C; Pinna, L A
The [PSI+] Prion Exists as a Dynamic Cloud of Variants
[PSI+] is an amyloid-based prion of Sup35p, a subunit of the translation termination factor. Prion �strains� or �variants� are amyloids with different conformations of a single protein sequence, conferring different phenotypes, but each relatively faithfully propagated. Wild Saccharomyces cerevisiae isolates have SUP35 alleles that fall into three groups, called reference, ?19, and E9, with limited transmissibility of [PSI+] between cells expressing these different polymorphs. Here we show that prion transmission pattern between different Sup35 polymorphs is prion variant-dependent. Passage of one prion variant from one Sup35 polymorph to another need not change the prion variant. Surprisingly, simple mitotic growth of a [PSI+] strain results in a spectrum of variant transmission properties among the progeny clones. Even cells that have grown for >150 generations continue to vary in transmission properties, suggesting that simple variant segregation is insufficient to explain the results. Rather, there appears to be continuous generation of a cloud of prion variants, with one or another becoming stochastically dominant, only to be succeeded by a different mixture. We find that among the rare wild isolates containing [PSI+], all indistinguishably �weak� [PSI+], are several different variants based on their transmission efficiencies to other Sup35 alleles. Most show some limitation of transmission, indicating that the evolved wild Sup35 alleles are effective in limiting the spread of [PSI+]. Notably, a �strong [PSI+]� can have any of several different transmission efficiency patterns, showing that �strong� versus �weak� is insufficient to indicate prion variant uniformity.
Bateman, David A.; Wickner, Reed B.
Copper Redox Cycling in the Prion Protein Depends Critically on Binding Mode
The prion protein (PrP) takes up four to six equivalents of copper in its extended N-terminal domain, composed of the octarepeat (OR) segment (human sequence residues 60�91) and two mononuclear binding sites (at His96 and His111; also referred to as the non-OR region). The OR segment responds to specific copper concentrations by transitioning from a multi-His mode at low copper levels to a single-His, amide nitrogen mode at high levels (Chattopadhyay et al. J. Am. Chem. Soc., 127, 12647�12656, 2005). The specific function of PrP in healthy tissue is unclear, but numerous reports link copper uptake to a neuroprotective role that regulates cellular stress (Stevens et al. PLoS Pathogens, 5(4): e1000390, 2009). A current working hypothesis is that the high occupancy binding mode quenches copper�s inherent redox cycling, thus protecting against the production of reactive oxygen species from unregulated Fenton type reactions. Here, we directly test this hypothesis by performing detailed pH-dependent electrochemical measurements on both low and high occupancy copper binding modes. In contrast to the current belief, we find that the low occupancy mode completely quenches redox cycling, but high occupancy leads to the gentle production of hydrogen peroxide through a catalytic reduction of oxygen facilitated by the complex. These electrochemical findings are supported by independent kinetic measurements that probe for ascorbate usage and also peroxide production. Hydrogen peroxide production is also observed from a segment corresponding to the non-OR region. Collectively, these results overturn the current working hypothesis and suggest, instead, that the redox cycling of copper bound to PrP in the high occupancy mode is not quenched, but is regulated. The observed production of hydrogen peroxide suggests a mechanism that could explain PrP�s putative role in cellular signaling.
Liu, Lin; Jiang, Dianlu; McDonald, Alex; Hao, Yuanqiang; Millhauser, Glenn L.; Zhou, Feimeng
Prion protein and Shadoo are involved in overlapping embryonic pathways and trophoblastic development.
The potential requirement of either the Prion or Shadoo protein for early mouse embryogenesis was recently suggested. However, the current data did not allow to precise the developmental process that was affected in the absence of both proteins and that led to the observed early lethal phenotype. In the present study, using various Prnp transgenic mouse lines and lentiviral vectors expressing shRNAs that target the Shadoo-encoding mRNA, we further demonstrate the specific requirement of at least one of these two PrP-related proteins at early developmental stages. Histological analysis reveals developmental defect of the ectoplacental cone and important hemorrhage surrounding the Prnp-knockout-Sprn-knockdown E7.5 embryos. By restricting the RNA interference to the trophoblastic cell lineages, the observed lethal phenotype could be attributed to the sole role of these proteins in this trophectoderm-derived compartment. RNAseq analysis performed on early embryos of various Prnp and Sprn genotypes indicated that the simultaneous down-regulation of these two proteins affects cell-adhesion and inflammatory pathways as well as the expression of ectoplacental-specific genes. Overall, our data provide biological clues in favor of a crucial and complementary embryonic role of the prion protein family in Eutherians and emphasizes the need to further evaluate its implication in normal and pathological human placenta biology. PMID:22860039
Passet, Bruno; Young, Rachel; Makhzami, Samira; Vilotte, Marthe; Jaffrezic, Florence; Halliez, Sophie; Bouet, St�phan; Marthey, Sylvain; Khalif�, Manal; Kanellopoulos-Langevin, Colette; B�ringue, Vincent; Le Provost, Fabienne; Laude, Hubert; Vilotte, Jean-Luc
Prion sequence polymorphisms and chronic wasting disease resistance in Illinois white-tailed deer (Odocoileus virginianus)
Nucleic acid sequences of the prion gene (PRNP) were examined and genotypes compiled for 76 white-tailed deer from northern Illinois, which previously tested positive for chronic wasting disease (CWD), and 120 negative animals selected to control for geographic location and age. Nine nucleotide polymorphisms, seven silent and two coding, were found in the sampled population. All observed polymorphisms except two of very low frequency were observed in both negative and positive animals, although five polymorphic loci had significantly different distributions of alleles between infected and non-infected individuals. Nucleotide base changes 60C/T, 285A/C, 286G/A and 555C/T were observed with higher than expected frequencies in CWD negative animals suggesting disease resistance, while 153C/T was observed more than expected in positive animals, suggesting susceptibility. The two coding polymorphisms, 285A/C (Q95H) and 286G/A (G96S), have been described in white-tailed deer populations sampled in Colorado and Wisconsin. Frequency distributions of coding polymorphisms in Wisconsin and Illinois deer populations were different, an unexpected result considering the sampled areas are less than 150 km apart. The total number of polymorphisms per animal, silent or coding, was negatively correlated to disease status. The potential importance of silent polymorphisms (60C/T, 153C/T, 555C/T), either individually or cumulatively, in CWD disease status has not been previously reported.
Kelly, Amy C; Mateus-Pinilla, Nohra E; Diffendorfer, Jay; Jewell, Emily; Ruiz, Marilyn O; Killefer, John; Shelton, Paul; Beissel, Tom
The complexity and implications of yeast prion domains
Prions are infectious proteins with altered conformations converted from otherwise normal host proteins. While there is only one known mammalian prion protein, PrP, a handful of prion proteins have been identified in the yeast Saccharomyces cerevisiae. Yeast prion proteins usually have a defined region called prion domain (PrD) essential for prion properties, which are typically rich in glutamine (Q) and asparagine (N). Despite sharing several common features, individual yeast PrDs are generally intricate and divergent in their compositional characteristics, which potentially implicates their prion phenotypes, such as prion-mediated transcriptional regulations.
Conservation of a portion of the S. cerevisiae Ure2p prion domain that interacts with the full-length protein
The [URE3] prion of Saccharomyces cerevisiae is a self-propagating inactive amyloid form of the Ure2 protein. Ure2p residues 1�65 constitute the prion domain, and the remaining C-terminal portion regulates nitrogen catabolism. We have examined the URE2 genes of wild-type isolates of S. cerevisiae and those of several pathogenic yeasts and a filamentous fungus. We find that the normal function of the S. cerevisiae Ure2p in nitrogen regulation is fully complemented by the Ure2p of Candida albicans, Candida glabrata, Candida kefyr, Candida maltosa, Saccharomyces bayanus, and Saccharomyces paradoxus, all of which have high homology in the C-terminal nitrogen regulation domain. However, there is considerable divergence of their N-terminal domains from that of Ure2p of S. cerevisiae. [URE3Sc] showed efficient transmission into S. cerevisiae ure2? cells if expressing a Ure2p of species within Saccharomyces. However, [URE3Sc] did not seed self-propagating inactivation of the Ure2p's from the other yeasts. When overexpressed as a fusion with green fluorescent protein, residues 5�47 of the S. cerevisiae prion domain are necessary for curing the [URE3] prion. Residues 11�39 are necessary for an inactivating interaction with the full-length Ure2p. A nearly identical region is highly conserved among many of the yeasts examined in this study, despite the wide divergence of sequences found in other parts of the N-terminal domains.
Edskes, Herman K.; Wickner, Reed B.
Characterization of prion protein-enriched domains, isolated from rat cerebellar granule cells in culture.
The biological functions of prion protein (PrP(C)) and its possible interaction with other specific molecular membrane partners remain largely unknown. The aim of this study is to gain information on the molecular environment of PrP(C) by analyzing the lipid and protein composition of a PrP(C)-enriched membrane subfraction, called prion domain, PrD. This domain was obtained by immunoprecipitation of detergent-resistant microdomains (DRM) of rat cerebellar granule cells under conditions designed to preserve lipid-mediated membrane organization. The electrophoretic pattern of PrD, after staining with Coomassie blue, showed the enrichment of some protein bands in comparison with DRM. microLiquid chromatography-electrospray ionization-mass spectrometry (microLC-ESI-MS)/MS analysis showed that Thy-1 and different types of myosin were strongly enriched in PrD and, in a lesser extent, also OBCAM, LSAMP and tubulin, present altogether in a single band. Experiments using the chemical cross-linker BS(3) suggested the existence of an interaction between PrP(C) and neural cell adhesion molecule (NCAM). Concerning lipids, the comparison between PrD and DRM showed a similar phospholipid/sphingolipid ratio, a phospholipid/cholesterol ratio doubled, and a strong decrease of plasmenilethanolamine (19.7 +/- 3.5% vs. 38.3 +/- 1.2%). In conclusion, the peculiar lipid composition and in particular the presence of proteins involved in synaptic plasticity, cell adhesion, cytoskeleton regulation and signalling, suggest an important physiological role in neurons of Prion Domain. PMID:19493159
Farina, Francesca; Botto, Laura; Chinello, Clizia; Cunati, Diana; Magni, Fulvio; Masserini, Massimo; Palestini, Paola
Interaction between a recombinant prion protein and organo-mineral complexes as evidenced by CPMAS 13C-NMR
NASA Astrophysics Data System (ADS)
Prion proteins (PrP) are the main responsible for Transmissible Spongiform Encephalopathies (TSE). The TSE etiological agent is a misfolded form of the normal cellular prion protein. The amyloidal aggregates accumulated in the brain of infected animals and mainly composed of PrPSc exhibit resistance to protease attack and many conventional inactivating procedures. The prion protein diseases cause an environmental issue because the environment and in particular the soil compartment can be contaminated and then become a potential reservoir and diffuser of TSEs infectivity as a consequence of (i) accidental dispersion from storage plants of meat and bone meal, (ii) incorporation of contaminated material in fertilizers, (iii) possible natural contamination of pasture soils by grazing herds, and (v) burial of carcasses. The environmental problem can be even more relevant because very low amounts of PrPSc are able to propagate the disease. Several studies evidenced that infectious prion protein remains active in soils for years. Contaminated soils result, thus, a possible critical route of TSE transmission in wild animals. Soil can also protect prion protein toward degradation processes due to the presence of humic substances and inorganic components such as clays. Mineral and organic colloids and the more common association between clay minerals and humic substances can contribute to the adsorption/entrapment of molecules and macromolecules. The polymerization of organic monomeric humic precursors occurring in soil in the presence of oxidative enzymes or manganese and iron oxides, is considered one of the most important processes contributing to the formation of humic substances. The process is very fast and produces a population of polymeric products of different molecular structures, sizes, shapes and complexity. Other molecules and possibly biomacromolecules such as proteins may be involved. The aim of the present work was to study by CPMAS 13C-NMR the interactions between a non pathogenic ovine recombinant prion protein and a model soil system represented by a manganese oxide in the form of birnessite (?-MnO2), coated with a polymerized catechol. To better understand the effect of the polymerization process, PrP was added to the birnessite-cathecol system either before or after the polymerization processes. The NMR spectra of the prion protein interacting directly with birnessite revealed disappearance of the signals due to the paramagnetic nature of manganese oxide or abiotic degradation. Conversely, the signal pattern of the protein re-appeared as it is mixed to the soil-like system either during or after the catechol polymerization process. Results suggested that the possible interactions of the prion protein on soil systems can be mediated by natural organic matter. However, deeper studies on more complex real soil systems are needed to definitely confirm such hypothesis.
Russo, F.; Scotti, R.; Gianfreda, L.; Conte, P.; Rao, M. A.
Prions of fungi: inherited structures and biological roles
The term 'prion' means an infectious protein that does not need an accompanying nucleic acid. There are six fungal prions, including four self-propagating amyloids and two enzymes that are necessary to activate their inactive precursors. Here we explore the scope of the prion phenomenon, the biological and evolutionary roles of prions, the structural basis of the amyloid prions and the
Herman K. Edskes; Frank Shewmaker; Toru Nakayashiki; Reed B. Wickner
Copper-induced structural propensities of the amyloidogenic region of human prion protein.
Transmissible spongiform encephalopathies are associated with the misfolding of the cellular Prion Protein (PrP(C)) to an abnormal protein isoform, called scrapie prion protein (PrP(Sc)). The structural rearrangement of the fragment of N-terminal domain of the protein spanning residues 91-127 is critical for the observed structural transition. The amyloidogenic domain of the protein encloses two copper-binding sites corresponding to His-96 and His-111 residues that act as anchors for metal ion binding. Previous studies have shown that Cu(II) sequestration by both sites may modulate the peptide's tendency to aggregation as it inflicts the hairpin-like structure that stabilizes the transition states leading to ?-sheet formation. On the other hand, since both His sites differ in their ability to Cu(II) sequestration, with His-111 as a preferred binding site, we found it interesting to test the role of Cu(II) coordination to this single site on the structural properties of amyloidogenic domain. The obtained results reveal that copper binding to His-111 site imposes precise backbone bending and weakens the natural tendency of apo peptide to ?-sheet formation. PMID:24737041
Migliorini, Caterina; Sinicropi, Adalgisa; Kozlowski, Henryk; Luczkowski, Marek; Valensin, Daniela
PrP Expression Level and Sensitivity to Prion Infection.
Mice overexpressing the prion protein (PrP) sequence from various host species are widely used for measuring infectious titers in prion disease. However, the impact that the transgene expression level might have on the susceptibility to infection raises some concerns about the final biological relevance of these models. Here we report that endpoint titration of a sheep scrapie isolate in sheep and in mice overexpressing the ovine PrP results in similar estimates of the infectious titer. PMID:24574409
Douet, Jean-Yves; Lacroux, Caroline; Corbi�re, Fabien; Litaise, Claire; Simmons, Hugh; Lugan, S�verine; Costes, Pierrette; Cassard, Herv�; Weisbecker, Jean-Louis; Schelcher, Fran�ois; Andreoletti, Olivier
Molecular determinants of the physicochemical properties of a critical prion protein region comprising residues 106-126.
Prion diseases are marked by the cerebral accumulation of conformationally modified forms of the cellular prion protein (PrP(C)), known as PrP(res). The region comprising the residues 106-126 of human PrP seems to have a key role in this conformational conversion, because a synthetic peptide homologous with this sequence (PrP106-126) adopts different secondary structures in different environments. To investigate the molecular determinants of the physicochemical characteristics of PrP106-126, we synthesized a series of analogues including PrP106-126 H(D), PrP106-126 A and PrP106-126 K, with l-His-->d-His, His-->Ala and His-->Lys substitutions respectively at position 111, PrP106-126 NH(2) with amidation of the C-terminus, PrP106-126 V with an Ala-->Val substition at position 117, and PrP106-126 VNH(2) with an Ala-->Val substitution at position 117 and amidation of the C-terminus. The analysis of the secondary structure and aggregation properties of PrP106-126 and its analogues showed the following. (1) His(111) is central to the conformational changes of PrP peptides. (2) Amidation of the C-terminal Gly(126) yields a predominantly random coil structure, abolishes the molecular polymorphism and decreases the propensity of PrP106-126 to generate amyloid fibrils. (3) PrP106-126 V, carrying an Ala-->Val substitution at position 117, does not demonstrate a fibrillogenic ability superior to that of PrP106-126. However, the presence of Val at position 117 increases the aggregation properties of the amidated peptide. (4) Amyloid fibrils are not required for neurotoxicity because the effects of PrP106-126 NH(2) on primary neuronal cultures were similar to those of the wild-type sequence. Conversely, astroglial proliferation is related to the presence of amyloid fibrils, suggesting that astrogliosis in prion encephalopathies without amyloid deposits is a mediated effect rather than a direct effect of disease-specific PrP isoforms.
Salmona, M; Malesani, P; De Gioia, L; Gorla, S; Bruschi, M; Molinari, A; Della Vedova, F; Pedrotti, B; Marrari, M A; Awan, T; Bugiani, O; Forloni, G; Tagliavini, F
Fate of prions in soil: adsorption kinetics of recombinant unglycosylated ovine prion protein onto mica in laminar flow conditions and subsequent desorption.
Prions can be disseminated in soils. Their interaction with soil minerals is a key factor for the assessment of risks associated with the transport of their infectivity. We did not examine here the infectivity itself but the adsorption kinetics of an ovine recombinant prion protein (ovine PrPrec), as a noninfectious model protein, on muscovite mica, a phyllosilicate with surface properties analogous to soil clays, in conditions of laminar flow through a channel. The protein was labeled with (125)I, and its adsorption examined between pH 4.0 and 9.0. At wall shear rate 100 s(-1), we found the process to be controlled mainly by transport at the beginning of the adsorption process. Additional experiments at 1000 s(-1) (pH 5 and 6) determined that the diffusion coefficient was in accordance with the hydrodynamic radius measured by size exclusion chromatography. The pseudo-plateau of the interfacial concentration with time was compatible with more than a monolayer and suggests the presence of aggregates. Desorption was not observed in the presence of buffer between pH 4 and 9 and sheep plasma, while the addition of alkaline detergent or 10(-1) M NaOH allowed an almost complete removal from the interface. The ensemble of results suggests that the largely irreversible adsorption of the ovine PrPrec onto mica is mainly due to electrostatic attraction between the protein and the highly negatively charged mica surface. Possible consequences for the mode of dissemination of prion proteins in soils are indicated. PMID:16283775
Vasina, Elena N; D�jardin, Philippe; Rezaei, Human; Grosclaude, Jeanne; Quiquampoix, Herv�
Anti-prion activity generated by a novel vaccine formulation.
Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) of domestic and wild cervids in North America. To address possible prevention regimens for CWD, we have used a mouse model system and the Rocky Mountain Laboratory (RML) mouse-adapted scrapie prion strain to screen efficacy of potential vaccine candidates. Three peptides derived from the primary amino acid sequence of the prion protein were conjugated to blue carrier protein (BCP) and formulated in an adjuvant containing M. avium subsp. avium. CL57/BL6 mice were vaccinated and boosted with 50 microg of the carrier protein-peptide conjugate formulation; all vaccines produced a humoral immune response as measured by ELISA. Disease challenge with the RML scrapie prion strain revealed anti-prion activity was generated by the vaccine formulations as measured by a delay in clinical disease onset and prolonged survivorship. PMID:18023980
Pilon, John; Loiacono, Christina; Okeson, Danelle; Lund, Sharon; Vercauteren, Kurt; Rhyan, Jack; Miller, Lowell
Degradation of the disease-associated prion protein by a serine protease from lichens
The disease-associated prion protein (PrPTSE), the probable etiological agent of the transmissible spongiform encephalopathies (TSEs), is resistant to degradation and can persist in the environment. Lichens, mutualistic symbioses containing fungi, algae, bacteria and occasionally cyanobacteria, are ubiquitous in the environment and have evolved unique biological activities allowing their survival in challenging ecological niches. We investigated PrPTSE inactivation by lichens and found acetone extracts of three lichen species (Parmelia sulcata, Cladonia rangiferina and Lobaria pulmonaria) have the ability to degrade prion protein (PrP) from TSE-infected hamsters, mice and deer. Immunoblots measuring PrP levels and protein misfolding cyclic amplification indicated at least two logs of reductions in PrPTSE. Degradative activity was not found in closely related lichen species or in algae or a cyanobacterium that inhabit lichens. Degradation was blocked by Pefabloc SC, a serine protease inhibitor, but not inhibitors of other proteases or enzymes. Additionally, we found that PrP levels in PrPTSE-enriched preps or infected brain homogenates are also reduced following exposure to freshly-collected P. sulcata or an aqueous extract of the lichen. Our findings indicate that these lichen extracts efficiently degrade PrPTSE and suggest that some lichens could have potential to inactivate TSE infectivity on the landscape or be a source for agents to degrade prions. Further work to clone and characterize the protease, assess its effect on TSE infectivity and determine which organism or organisms present in lichens produce or influence the protease activity is warranted.
Johnson, C. J.; Bennett, J. P.; Biro, S. M.; Duque-Velasquez, J. C.; Rodriguez, C. M.; Bessen, R. A.; Rocke, T. E.
Degradation of the disease-associated prion protein by a serine protease from lichens.
The disease-associated prion protein (PrPTSE), the probable etiological agent of the transmissible spongiform encephalopathies (TSEs), is resistant to degradation and can persist in the environment. Lichens, mutualistic symbioses containing fungi, algae, bacteria and occasionally cyanobacteria, are ubiquitous in the environment and have evolved unique biological activities allowing their survival in challenging ecological niches. We investigated PrPTSE inactivation by lichens and found acetone extracts of three lichen species (Parmelia sulcata, Cladonia rangiferina and Lobaria pulmonaria) have the ability to degrade prion protein (PrP) from TSE-infected hamsters, mice and deer. Immunoblots measuring PrP levels and protein misfolding cyclic amplification indicated at least two logs of reductions in PrPTSE. Degradative activity was not found in closely related lichen species or in algae or a cyanobacterium that inhabit lichens. Degradation was blocked by Pefabloc SC, a serine protease inhibitor, but not inhibitors of other proteases or enzymes. Additionally, we found that PrP levels in PrPTSE-enriched preps or infected brain homogenates are also reduced following exposure to freshly-collected P. sulcata or an aqueous extract of the lichen. Our findings indicate that these lichen extracts efficiently degrade PrPTSE and suggest that some lichens could have potential to inactivate TSE infectivity on the landscape or be a source for agents to degrade prions. Further work to clone and characterize the protease, assess its effect on TSE infectivity and determine which organism or organisms present in lichens produce or influence the protease activity is warranted.
Johnson, C. J.; Bennett, J. P.; Biro, S. M.; Duque-Velasquez, J. C.; Rodriguez, C. M.; Bessen, R. A.; Rocke, T. E.
Degradation of the disease-associated prion protein by a serine protease from lichens
The disease-associated prion protein (PrP(TSE)), the probable etiological agent of the transmissible spongiform encephalopathies (TSEs), is resistant to degradation and can persist in the environment. Lichens, mutualistic symbioses containing fungi, algae, bacteria and occasionally cyanobacteria, are ubiquitous in the environment and have evolved unique biological activities allowing their survival in challenging ecological niches. We investigated PrP(TSE) inactivation by lichens and found acetone extracts of three lichen species (Parmelia sulcata, Cladonia rangiferina and Lobaria pulmonaria) have the ability to degrade prion protein (PrP) from TSE-infected hamsters, mice and deer. Immunoblots measuring PrP levels and protein misfolding cyclic amplification indicated at least two logs of reductions in PrP(TSE). Degradative activity was not found in closely related lichen species or in algae or a cyanobacterium that inhabit lichens. Degradation was blocked by Pefabloc SC, a serine protease inhibitor, but not inhibitors of other proteases or enzymes. Additionally, we found that PrP levels in PrP(TSE)-enriched preps or infected brain homogenates are also reduced following exposure to freshly-collected P. sulcata or an aqueous extract of the lichen. Our findings indicate that these lichen extracts efficiently degrade PrP(TSE) and suggest that some lichens could have potential to inactivate TSE infectivity on the landscape or be a source for agents to degrade prions. Further work to clone and characterize the protease, assess its effect on TSE infectivity and determine which organism or organisms present in lichens produce or influence the protease activity is warranted.
Johnson, C.J.; Bennett, J.P.; Biro, S.M.; Duque-Velasquez, J.C.; Rodriguez, C.M.; Bessen, R.A.; Rocke, T.E.
Degradation of the Disease-Associated Prion Protein by a Serine Protease from Lichens
The disease-associated prion protein (PrPTSE), the probable etiological agent of the transmissible spongiform encephalopathies (TSEs), is resistant to degradation and can persist in the environment. Lichens, mutualistic symbioses containing fungi, algae, bacteria and occasionally cyanobacteria, are ubiquitous in the environment and have evolved unique biological activities allowing their survival in challenging ecological niches. We investigated PrPTSE inactivation by lichens and found acetone extracts of three lichen species (Parmelia sulcata, Cladonia rangiferina and Lobaria pulmonaria) have the ability to degrade prion protein (PrP) from TSE-infected hamsters, mice and deer. Immunoblots measuring PrP levels and protein misfolding cyclic amplification indicated at least two logs of reductions in PrPTSE. Degradative activity was not found in closely related lichen species or in algae or a cyanobacterium that inhabit lichens. Degradation was blocked by Pefabloc SC, a serine protease inhibitor, but not inhibitors of other proteases or enzymes. Additionally, we found that PrP levels in PrPTSE-enriched preps or infected brain homogenates are also reduced following exposure to freshly-collected P. sulcata or an aqueous extract of the lichen. Our findings indicate that these lichen extracts efficiently degrade PrPTSE and suggest that some lichens could have potential to inactivate TSE infectivity on the landscape or be a source for agents to degrade prions. Further work to clone and characterize the protease, assess its effect on TSE infectivity and determine which organism or organisms present in lichens produce or influence the protease activity is warranted.
Johnson, Christopher J.; Bennett, James P.; Biro, Steven M.; Duque-Velasquez, Juan Camilo; Rodriguez, Cynthia M.; Bessen, Richard A.; Rocke, Tonie E.
Loss of amino-terminal acetylation suppresses a prion phenotype by modulating global protein folding.
Amino-terminal acetylation is among the most ubiquitous of protein modifications in eukaryotes. Although loss of N-terminal acetylation is associated with many abnormalities, the molecular basis of these effects is known for only a few cases, where acetylation of single factors has been linked to binding avidity or metabolic stability. In contrast, the impact of N-terminal acetylation for the majority of the proteome, and its combinatorial contributions to phenotypes, are unknown. Here, by studying the yeast prion [PSI(+)], an amyloid of the Sup35 protein, we show that loss of N-terminal acetylation promotes general protein misfolding, a redeployment of chaperones to these substrates, and a corresponding stress response. These proteostasis changes, combined with the decreased stability of unacetylated Sup35 amyloid, reduce the size of prion aggregates and reverse their phenotypic consequences. Thus, loss of N-terminal acetylation, and its previously unanticipated role in protein biogenesis, globally resculpts the proteome to create a unique phenotype. PMID:25023910
Holmes, William M; Mannakee, Brian K; Gutenkunst, Ryan N; Serio, Tricia R
Amyloid-? induced signaling by cellular prion protein and Fyn kinase in Alzheimer disease
Alzheimer disease (AD) is the most prevalent cause of dementia. Amyloid-? (A?) oligomers are potent synaptotoxins thought to mediate AD-related phenotypes. Cellular prion protein (PrPC) has been identified as a high-affinity receptor for A? oligomers. Herein, we review the functional consequences of A? oligomer binding to PrPC on the neuronal surface. We highlight recent evidence that Fyn kinase mediates signal transduction downstream of the PrPC-A? oligomer complex. These studies suggest that PrPC has a central role in AD pathogenesis and may provide a target for therapeutic intervention in AD.
Um, Ji Won; Strittmatter, Stephen M.
Distinct Proteinase K-Resistant Prion Protein Fragment in Goats with No Signs of Disease in a Classical Scrapie Outbreak?�
Considerable efforts have been directed toward the identification of small-ruminant prion diseases, i.e., classical and atypical scrapie as well as bovine spongiform encephalopathy (BSE). Here we report the in-depth molecular analysis of the proteinase K-resistant prion protein core fragment (PrPres) in a highly scrapie-affected goat flock in Greece. The PrPres profile by Western immunoblotting in most animals was that of classical scrapie in sheep. However, in a series of clinically healthy goats we identified a unique C- and N-terminally truncated PrPres fragment, which is akin but not identical to that observed for atypical scrapie. These findings reveal novel aspects of the nature and diversity of the molecular PrPres phenotypes in goats and suggest that these animals display a previously unrecognized prion protein disorder.
Bouzalas, Ilias G.; Lortscher, Florian; Dovas, Chrysostomos I.; Oevermann, Anna; Langeveld, Jan P. M.; Papanastassopoulou, Maria; Papadopoulos, Orestis; Zurbriggen, Andreas; Seuberlich, Torsten
Mass spectrometric detection of attomole amounts of the prion protein by nanoLC\\/MS\\/MS
Sensitive quantitation of prions in biological samples is an extremely important and challenging analytical problem. Prions\\u000a are the cause of several fatal neurodegenerative diseases known as transmissible spongiform encephalopathies (TSEs). At this\\u000a time, there are no methods to diagnose TSEs in live animals or to assure a prion-free blood supply for humans. Prions have\\u000a been shown to be present in
Bruce Onisko; Irina Dynin; Jes�s R. Requena; Christopher J. Silva; Melissa Erickson; John Mark Cartera
Activation of phosphatidylinositol 3-kinase by cellular prion protein and its role in cell survival.
The cellular prion protein (PrP(C)) is thought to be involved in protection against cell death, however the exact cellular mechanisms involved are still controversial. Herein we present data that strongly indicate a functional link between PrP(C) expression and phosphatidylinositol 3-kinase (PI 3-kinase) activation, a protein kinase that plays a pivotal role in cell survival. Both mouse neuroblastoma N2a cells and immortalized murine hippocampal neuronal cell lines expressing wild-type PrP(C) had significantly higher PI 3-kinase activity levels than their respective controls. Moreover, PI 3-kinase activity was found to be elevated in brain lysates from wild-type mice, as compared to prion protein-knockout mice. Recruitment of PI 3-kinase by PrP(C) was shown to contribute to cellular survival toward oxidative stress by using 3-morpholinosydnonimine (SIN-1) and serum deprivation. Moreover, both PI 3-kinase activation and cytoprotection by PrP(C) appeared to rely on copper binding to the N-terminal octapeptide of PrP(C). Thus, we propose a model in which the interaction of copper(II) with the N-terminal domain of PrP(C) enables transduction of a signal to PI 3-kinase; the latter, in turn, mediates downstream regulation of cell survival. PMID:15896301
Vassallo, Neville; Herms, Jochen; Behrens, Christina; Krebs, Bjarne; Saeki, Keiichi; Onodera, Takashi; Windl, Otto; Kretzschmar, Hans A
Mapping the early steps in the pH-induced conformational conversion of the prion protein
Under certain conditions, the prion protein (PrP) undergoes a conformational change from the normal cellular isoform, PrPC, to PrPSc, an infectious isoform capable of causing neurodegenerative diseases in many mammals. Conversion can be triggered by low pH, and in vivo this appears to take place in an endocytic pathway and/or caveolae-like domains. It has thus far been impossible to characterize the conformational change at high resolution by experimental methods. Therefore, to investigate the effect of acidic pH on PrP conformation, we have performed 10-ns molecular dynamics simulations of PrPC in water at neutral and low pH. The core of the protein is well maintained at neutral pH. At low pH, however, the protein is more dynamic, and the sheet-like structure increases both by lengthening of the native ?-sheet and by addition of a portion of the N terminus to widen the sheet by another two strands. The side chain of Met-129, a polymorphic codon in humans associated with variant Creutzfeldt�Jakob disease, pulls the N terminus into the sheet. Neutralization of Asp-178 at low pH removes interactions that inhibit conversion, which is consistent with the Asp-178�Asn mutation causing human prion diseases.
Alonso, Darwin O. V.; DeArmond, Stephen J.; Cohen, Fred E.; Daggett, Valerie
Single-particle tracking of quantum dot-conjugated prion proteins inside yeast cells
Research highlights: {yields} We develop a method to track a quantum dot-conjugated protein in yeast cells. {yields} We incorporate the conjugated quantum dot proteins into yeast spheroplasts. {yields} We track the motions by conventional or 3D tracking microscopy. -- Abstract: Yeast is a model eukaryote with a variety of biological resources. Here we developed a method to track a quantum dot (QD)-conjugated protein in the budding yeast Saccharomyces cerevisiae. We chemically conjugated QDs with the yeast prion Sup35, incorporated them into yeast spheroplasts, and tracked the motions by conventional two-dimensional or three-dimensional tracking microscopy. The method paves the way toward the individual tracking of proteins of interest inside living yeast cells.
Tsuji, Toshikazu; Kawai-Noma, Shigeko [Department of Biomolecular Engineering, Graduate School of Biosciences and Biotechnology, Tokyo Institute of Technology, B56, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501 (Japan)] [Department of Biomolecular Engineering, Graduate School of Biosciences and Biotechnology, Tokyo Institute of Technology, B56, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501 (Japan); Pack, Chan-Gi [Cellular Informatics Laboratory, RIKEN Advanced Science Institute, Wako-shi, Saitama 351-0198 (Japan)] [Cellular Informatics Laboratory, RIKEN Advanced Science Institute, Wako-shi, Saitama 351-0198 (Japan); Terajima, Hideki [Department of Biomolecular Engineering, Graduate School of Biosciences and Biotechnology, Tokyo Institute of Technology, B56, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501 (Japan)] [Department of Biomolecular Engineering, Graduate School of Biosciences and Biotechnology, Tokyo Institute of Technology, B56, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501 (Japan); Yajima, Junichiro; Nishizaka, Takayuki [Department of Physics, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588 (Japan)] [Department of Physics, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588 (Japan); Kinjo, Masataka [Laboratory of Molecular Cell Dynamics, Graduate School of Life Sciences, Hokkaido University, Sapporo 001-0021 (Japan)] [Laboratory of Molecular Cell Dynamics, Graduate School of Life Sciences, Hokkaido University, Sapporo 001-0021 (Japan); Taguchi, Hideki, E-mail: email@example.com [Department of Biomolecular Engineering, Graduate School of Biosciences and Biotechnology, Tokyo Institute of Technology, B56, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501 (Japan)] [Department of Biomolecular Engineering, Graduate School of Biosciences and Biotechnology, Tokyo Institute of Technology, B56, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501 (Japan)
Structural intermediates in the putative pathway from the cellular prion protein to the pathogenic form.
The conversion of the alpha-helical, protease sensitive and noninfectious form of the prion protein (PrP(C)) into an insoluble, protease resistant, predominantly beta-sheeted and infectious form (PrP(Sc)) is the fundamental event in prion formation. In the present work, two soluble and stable intermediate structural states are newly identified for recombinant Syrian hamster PrP(90-231) (recPrP), a dimeric alpha-helical state and a tetra- or oligomeric, beta-sheet rich state. In 0.2% SDS at room temperature, recPrP is soluble and exhibits alpha-helical and random coil secondary structure as determined by circular dichroism. Reduction of the SDS concentration to 0.06% leads first to a small increase in alpha-helical content, whereas further dilution to 0.02% results in the aquisition of beta-sheet structure. The reversible transition curve is sigmoidal within a narrow range of SDS concentrations (0.04 to 0.02%). Size exclusion chromatography and chemical crosslinking revealed that the alpha-helical form is dimeric, while the beta-sheet rich form is tetra- or oligomeric. Both the alpha-helical and beta-sheet rich intermediates are soluble and stable. Thus, they should be accessible to further structural and mechanistic studies. At 0.01% SDS, the oligomeric intermediates aggregated into large, insoluble structures as observed by fluorescence correlation spectroscopy. Our results are discussed with respect to the mechanism of PrP(Sc) formation and the propagation of prions. PMID:11405232
Jansen, K; Sch�fer, O; Birkmann, E; Post, K; Serban, H; Prusiner, S B; Riesner, D
Paradoxical Role of Prion Protein Aggregates in Redox-Iron Induced Toxicity
Background Imbalance of iron homeostasis has been reported in sporadic Creutzfeldt-Jakob-disease (sCJD) affected human and scrapie infected animal brains, but the contribution of this phenotype to disease associated neurotoxicity is unclear. Methodology/Principal Findings Using cell models of familial prion disorders, we demonstrate that exposure of cells expressing normal prion protein (PrPC) or mutant PrP forms to a source of redox-iron induces aggregation of PrPC and specific mutant PrP forms. Initially this response is cytoprotective, but becomes increasingly toxic with time due to accumulation of PrP-ferritin aggregates. Mutant PrP forms that do not aggregate are not cytoprotective, and cells show signs of acute toxicity. Intracellular PrP-ferritin aggregates induce the expression of LC3-II, indicating stimulation of autophagy in these cells. Similar observations are noted in sCJD and scrapie infected hamster brains, lending credence to these results. Furthermore, phagocytosis of PrP-ferritin aggregates by astrocytes is cytoprotective, while culture in astrocyte conditioned medium (CM) shows no measurable effect. Exposure to H2O2, on the other hand, does not cause aggregation of PrP, and cells show acute toxicity that is alleviated by CM. Conclusions/Significance These observations suggest that aggregation of PrP in response to redox-iron is cytoprotective. However, subsequent co-aggregation of PrP with ferritin induces intracellular toxicity unless the aggregates are degraded by autophagosomes or phagocytosed by adjacent scavenger cells. H2O2, on the other hand, does not cause aggregation of PrP, and induces toxicity through extra-cellular free radicals. Together with previous observations demonstrating imbalance of iron homeostasis in prion disease affected brains, these observations provide insight into the mechanism of neurotoxicity by redox-iron, and the role of PrP in this process.
Das, Dola; Luo, Xiu; Singh, Ajay; Gu, Yaping; Ghosh, Soumya; Mukhopadhyay, Chinmay K.; Chen, Shu G.; Sy, Man-Sun; Kong, Qingzhong; Singh, Neena
Expression of prion protein increases cellular copper binding and antioxidant enzyme activities but not copper delivery.
The N-terminal region of the prion protein PrP(C) contains a series of octapeptide repeats. This region has been implicated in the binding of divalent metal ions, particularly copper. PrP(C) has been suggested to be involved in copper transport and metabolism and in cell defense mechanisms against oxidative insult, possibly through the regulation of the intracellular CuZn superoxide dismutase activity (CuZn-SOD) or a SOD-like activity of PrP(C) itself. However, up to now the link between PrP(C) expression and copper metabolism or SOD activity has still to be formally established; particularly because conflicting results have been obtained in vivo. In this study, we report a link between PrP(C), copper binding, and resistance to oxidative stress. Radioactive copper ((64)Cu) was used at a physiological concentration to demonstrate that binding of copper to the outer plasma cell membrane is related to the level of PrP(C) expression in a cell line expressing a doxycycline-inducible murine PrP(C) gene. Cellular PIPLC pretreatment indicated that PrP(C) was not involved in copper delivery at physiological concentrations. We also demonstrated that murine PrP(C) expression increases several antioxidant enzyme activities and glutathione levels. Prion protein may be a stress sensor sensitive to copper and able to initiate, following copper binding, a signal transduction process acting on the antioxidant systems to improve cell defenses. PMID:12500977
Rachidi, Walid; Vilette, Didier; Guiraud, Pascale; Arlotto, Marie; Riondel, Jacqueline; Laude, Hubert; Lehmann, Sylvain; Favier, Alain
The N-Terminal, Polybasic Region Is Critical for Prion Protein Neuroprotective Activity
Several lines of evidence suggest that the normal form of the prion protein, PrPC, exerts a neuroprotective activity against cellular stress or toxicity. One of the clearest examples of such activity is the ability of wild-type PrPC to suppress the spontaneous neurodegenerative phenotype of transgenic mice expressing a deleted form of PrP (?32�134, called F35). To define domains of PrP involved in its neuroprotective activity, we have analyzed the ability of several deletion mutants of PrP (?23�31, ?23�111, and ?23�134) to rescue the phenotype of Tg(F35) mice. Surprisingly, all of these mutants displayed greatly diminished rescue activity, although ?23�31 PrP partially suppressed neuronal loss when expressed at very high levels. Our results pinpoint the N-terminal, polybasic domain as a critical determinant of PrPC neuroprotective activity, and suggest that identification of molecules interacting with this region will provide important clues regarding the normal function of the protein. Small molecule ligands targeting this region may also represent useful therapeutic agents for treatment of prion diseases.
Unterberger, Ursula; Biasini, Emiliano; Harris, David A.
Nanomechanical Properties of Human Prion Protein Amyloid as Probed by Force Spectroscopy
Amyloids are associated with a number of protein misfolding disorders, including prion diseases. In this study, we used single-molecule force spectroscopy to characterize the nanomechanical properties and molecular structure of amyloid fibrils formed by human prion protein PrP90-231. Force-extension curves obtained by specific attachment of a gold-covered atomic force microscope tip to engineered Cys residues could be described by the worm-like chain model for entropic elasticity of a polymer chain, with the size of the N-terminal segment that could be stretched entropically depending on the tip attachment site. The data presented here provide direct information about the forces required to extract an individual monomer from the core of the PrP90-231 amyloid, and indicate that the ?-sheet core of this amyloid starts at residue ?164�169. The latter finding has important implications for the ongoing debate regarding the structure of PrP amyloid.
Ganchev, Dragomir N.; Cobb, Nathan J.; Surewicz, Krystyna; Surewicz, Witold K.
Cellular Prion Protein Regulates Its Own ?-Cleavage through ADAM8 in Skeletal Muscle*
The ubiquitously expressed cellular prion protein (PrPC) is subjected to the physiological ?-cleavage at a region critical for both PrP toxicity and the conversion of PrPC to its pathogenic prion form (PrPSc), generating the C1 and N1 fragments. The C1 fragment can activate caspase 3 while the N1 fragment is neuroprotective. Recent articles indicate that ADAM10, ADAM17, and ADAM9 may not play a prominent role in the ?-cleavage of PrPC as previously thought, raising questions on the identity of the responsible protease(s). Here we show that, ADAM8 can directly cleave PrP to generate C1 in vitro and PrP C1/full-length ratio is greatly decreased in the skeletal muscles of ADAM8 knock-out mice; in addition, the PrP C1/full-length ratio is linearly correlated with ADAM8 protein level in myoblast cell line C2C12 and in skeletal muscle tissues of transgenic mice. These results indicate that ADAM8 is the primary protease responsible for the ?-cleavage of PrPC in muscle cells. Moreover, we found that overexpression of PrPC led to up-regulation of ADAM8, suggesting that PrPC may regulate its own ?-cleavage through modulating ADAM8 activity.
Liang, Jingjing; Wang, Wei; Sorensen, Debra; Medina, Sarah; Ilchenko, Sergei; Kiselar, Janna; Surewicz, Witold K.; Booth, Stephanie A.; Kong, Qingzhong
Expression of the prion-like protein Shadoo in the developing mouse embryo.
The prion-like protein Shadoo has been suggested to compensate for the lack of PrP in Prnp-knockout mice, explaining their lack of extreme phenotype. In adult mice, both PrP and Shadoo have shown overlapping expression patterns and shared functions. Their expression in the mouse embryo has also been suggested to be complementary, as invalidation of both genes results in embryonic lethality. The developmental expression profile of PrP has been described from post-implantation stages up until birth. However the spatial expression pattern of Shadoo in the developing mouse embryo is not known. We previously described the expression profile of the prion-like protein Shadoo in adult mice using Sprn reporter mice (Sprn-GFP and Sprn-LacZ). Here we used these mice to describe the developmental expression of Shadoo between 10.5 and 14.5 dpc. The observed pattern in specific embryonic cell lineages and in extra-embryonic tissues is consistent with the previously reported phenotype resulting from its knockdown. PMID:22093825
Young, Rachel; Bouet, St�phan; Polyte, Jacqueline; Le Guillou, Sandrine; Passet, Bruno; Vilotte, Marthe; Castille, Johan; Beringue, Vincent; Le Provost, Fabienne; Laude, Hubert; Vilotte, Jean-Luc
Conformational detection of prion protein with biarsenical labeling and FlAsH fluorescence
Prion diseases are associated with the misfolding of the host-encoded cellular prion protein (PrP{sup C}) into a disease associated form (PrP{sup Sc}). Recombinant PrP can be refolded into either an {alpha}-helical rich conformation ({alpha}-PrP) resembling PrP{sup C} or a {beta}-sheet rich, protease resistant form similar to PrP{sup Sc}. Here, we generated tetracysteine tagged recombinant PrP, folded this into {alpha}- or {beta}-PrP and determined the levels of FlAsH fluorescence. Insertion of the tetracysteine tag at three different sites within the 91-111 epitope readily distinguished {beta}-PrP from {alpha}-PrP upon FlAsH labeling. Labelling of tetracysteine tagged PrP in the {alpha}-helical form showed minimal fluorescence, whereas labeling of tagged PrP in the {beta}-sheet form showed high fluorescence indicating that this region is exposed upon conversion. This highlights a region of PrP that can be implicated in the development of diagnostics and is a novel, protease free mechanism for distinguishing PrP{sup Sc} from PrP{sup C}. This technique may also be applied to any protein that undergoes conformational change and/or misfolding such as those involved in other neurodegenerative disorders including Alzheimer's, Huntington's and Parkinson's diseases.
Coleman, Bradley M. [Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010 (Australia); Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010 (Australia); Nisbet, Rebecca M.; Han, Sen [Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010 (Australia); Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010 (Australia); Department of Pathology and Mental Health Research Institute, University of Melbourne, Parkville, Victoria 3010 (Australia); Cappai, Roberto [Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010 (Australia); Department of Pathology and Mental Health Research Institute, University of Melbourne, Parkville, Victoria 3010 (Australia); Hatters, Danny M. [Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010 (Australia); Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010 (Australia); Department of Pathology and Mental Health Research Institute, University of Melbourne, Parkville, Victoria 3010 (Australia); Hill, Andrew F. [Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010 (Australia); Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010 (Australia); Department of Pathology and Mental Health Research Institute, University of Melbourne, Parkville, Victoria 3010 (Australia)], E-mail: email@example.com
Understanding Factors Influencing The Propagation of Prions.
National Technical Information Service (NTIS)
Prions are altered conformations of a protein that have gained the ability to convert the normal form of the protein into the prion form. They are an infectious, misfolded and aggregated form of a protein. In mammals, prions are associated with neurodegen...
S. W. Liebman
The Rich Electrochemistry and Redox Reactions of the Copper Sites in the Cellular Prion Protein
This paper reviews recent electrochemical studies of the copper complexes of prion protein (PrP) and its related peptides, and correlates their redox behavior to chemical and biologically relevant reactions. Particular emphasis is placed on the difference in redox properties between copper in the octarepeat (OR) and the non-OR domains of PrP, as well as differences between the high and low copper occupancy states in the OR domain. Several discrepancies in literature concerning these differences are discussed and reconciled. The PrP copper complexes, in comparison to copper complexes of other amyloidogenic proteins/peptides, display a more diverse and richer redox chemistry. The specific protocols and caveats that need to be considered in studying the electrochemistry and redox reactions of copper complexes of PrP, PrP-derived peptides, and other related amyloidogenic proteins are summarized.
Zhou, Feimeng; Millhauser, Glenn L.
A novel expression system for production of soluble prion proteins in E. coli
Expression of eukaryotic proteins in Escherichia coli is challenging, especially when they contain disulfide bonds. Since the discovery of the prion protein (PrP) and its role in transmissible spongiform encephalopathies, the need to obtain large quantities of the recombinant protein for research purposes has been essential. Currently, production of recombinant PrP is achieved by refolding protocols. Here, we show that the co-expression of two different PrP with the human Quiescin Sulfhydryl OXidase (QSOX), a human chaperone with thiol/disulfide oxidase activity, in the cytoplasm of E. coli produces soluble recombinant PrP. The structural integrity of the soluble PrP has been confirmed by nuclear magnetic resonance spectroscopy, demonstrating that properly folded PrP can be easily expressed in bacteria. Furthermore, the soluble recombinant PrP produced with this method can be used for functional and structural studies.
A novel expression system for production of soluble prion proteins in E. coli.
Expression of eukaryotic proteins in Escherichia coli is challenging, especially when they contain disulfide bonds. Since the discovery of the prion protein (PrP) and its role in transmissible spongiform encephalopathies, the need to obtain large quantities of the recombinant protein for research purposes has been essential. Currently, production of recombinant PrP is achieved by refolding protocols. Here, we show that the co-expression of two different PrP with the human Quiescin Sulfhydryl OXidase (QSOX), a human chaperone with thiol/disulfide oxidase activity, in the cytoplasm of E. coli produces soluble recombinant PrP. The structural integrity of the soluble PrP has been confirmed by nuclear magnetic resonance spectroscopy, demonstrating that properly folded PrP can be easily expressed in bacteria. Furthermore, the soluble recombinant PrP produced with this method can be used for functional and structural studies. PMID:22233534
Abskharon, Romany N N; Ramboarina, Stephanie; El Hassan, Hassan; Gad, Wael; Apostol, Marcin I; Giachin, Gabriele; Legname, Giuseppe; Steyaert, Jan; Messens, Joris; Soror, Sameh H; Wohlkonig, Alexandre
Associations between lamb survival and prion protein genotype: analysis of data for ten sheep breeds in Great Britain
BACKGROUND: Selective breeding programmes, based on prion protein (PrP) genotype, have been introduced throughout the European Union to reduce the risk of sheep transmissible spongiform encephalopathies (TSEs). These programmes could have negative consequences on other important traits, such as fitness and production traits, if the PrP gene has pleiotropic effects or is in linkage disequilibrium with genes affecting these traits.
Simon Gubbins; Charlotte J Cook; Kieran Hyder; Kay Boulton; Carol Davis; Eurion Thomas; Will Haresign; Stephen C Bishop; Beatriz Villanueva; Rachel D Eglin
Neuronal low-density lipoprotein receptor-related protein 1 binds and endocytoses prion fibrils via receptor cluster 4
For infectious prion protein (designated PrPSc) to act as a template to convert normal cellular protein (PrPC) to its distinctive pathogenic conformation, the two forms of prion protein (PrP) must interact closely. The neuronal receptor that rapidly endocytoses PrPC is the low-density lipoprotein receptor-related protein 1 (LRP1). We show here that on sensory neurons LRP1 is also the receptor that binds and rapidly endocytoses smaller oligomeric forms of infectious prion fibrils, and recombinant PrP fibrils. Although LRP1 binds two molecules of most ligands independently to its receptor clusters 2 and 4, PrPC and PrPSc fibrils bind only to receptor cluster 4. PrPSc fibrils out-compete PrPC for internalization. When endocytosed, PrPSc fibrils are routed to lysosomes, rather than recycled to the cell surface with PrPC. Thus, although LRP1 binds both forms of PrP, it traffics them to separate fates within sensory neurons. The binding of both to ligand cluster 4 should enable genetic modification of PrP binding without disrupting other roles of LRP1 essential to neuronal viability and function, thereby enabling in vivo analysis of the role of this interaction in controlling both prion and LRP1 biology.
Jen, Angela; Parkyn, Celia J.; Mootoosamy, Roy C.; Ford, Melanie J.; Warley, Alice; Liu, Qiang; Bu, Guojun; Baskakov, Ilia V.; Moestrup, S?ren; McGuinness, Lindsay; Emptage, Nigel; Morris, Roger J.
Iron in neurodegenerative disorders of protein misfolding: a case of prion disorders and Parkinson's disease.
Abstract Significance: Intracellular and extracellular aggregation of a specific protein or protein fragments is the principal pathological event in several neurodegenerative conditions. We describe two such conditions: sporadic Creutzfeldt-Jakob disease (sCJD), a rare but potentially infectious and invariably fatal human prion disorder, and Parkinson's disease (PD), a common neurodegenerative condition second only to Alzheimer's disease in prevalence. In sCJD, a cell surface glycoprotein known as the prion protein (PrP(C)) undergoes a conformational change to PrP-scrapie, a pathogenic and infectious isoform that accumulates in the brain parenchyma as insoluble aggregates. In PD, ?-synuclein, a cytosolic protein, forms insoluble aggregates that accumulate in neurons of the substantia nigra and cause neurotoxicity. Recent Advances: Although distinct processes are involved in the pathogenesis of sCJD and PD, both share brain iron dyshomeostasis as a common associated feature that is reflected in the cerebrospinal fluid in a disease-specific manner. Critical Issues: Since PrP(C) and ?-synuclein play a significant role in maintaining cellular iron homeostasis, it is important to understand whether the aggregation of these proteins and iron dyshomeostasis are causally related. Here, we discuss recent information on the normal function of PrP(C) and ?-synuclein in cellular iron metabolism and the cellular and biochemical processes that contribute to iron imbalance in sCJD and PD. Future Directions: Improved understanding of the relationship between brain iron imbalance and protein aggregation is likely to help in the development of therapeutic strategies that can restore brain iron homeostasis and mitigate neurotoxicity. Antioxid. Redox Signal. 21, 471-484. PMID:24512387
Singh, Neena; Haldar, Swati; Tripathi, Ajai K; McElwee, Matthew K; Horback, Katharine; Beserra, Amber
Expression of Prion Protein in Mouse Erythroid Progenitors and Differentiating Murine Erythroleukemia Cells
Prion diseases have been observed to deregulate the transcription of erythroid genes, and prion protein knockout mice have demonstrated a diminished response to experimental anemia. To investigate the role of the cellular prion protein (PrPC) in erythropoiesis, we studied the protein's expression on mouse erythroid precursors in vivo and utilized an in vitro model of the erythroid differentiation of murine erythroleukemia cells (MEL) to evaluate the effect of silencing PrPC through RNA interference. The expression of PrPC and selected differentiation markers was analyzed by quantitative multicolor flow cytometry, western blot analysis and quantitative RT-PCR. The silencing of PrPC expression in MEL cells was achieved by expression of shRNAmir from an integrated retroviral vector genome. The initial upregulation of PrPC expression in differentiating erythroid precursors was detected both in vivo and in vitro, suggesting PrPC's importance to the early stages of differentiation. The upregulation was highest on early erythroblasts (16200�3700 PrPC / cell) and was followed by the gradual decrease of PrPC level with the precursor's maturation reaching 470�230 PrPC / cell on most mature CD71?Ter119+ small precursors. Interestingly, the downregulation of PrPC protein with maturation of MEL cells was not accompanied by the decrease of PrP mRNA. The stable expression of anti-Prnp shRNAmir in MEL cells led to the efficient (>80%) silencing of PrPC levels. Cell growth, viability, hemoglobin production and the transcription of selected differentiation markers were not affected by the downregulation of PrPC. In conclusion, the regulation of PrPC expression in differentiating MEL cells mimics the pattern detected on mouse erythroid precursors in vivo. Decrease of PrPC protein expression during MEL cell maturation is not regulated on transcriptional level. The efficient silencing of PrPC levels, despite not affecting MEL cell differentiation, enables created MEL lines to be used for studies of PrPC cellular function.
Panigaj, Martin; Glier, Hana; Wildova, Marcela; Holada, Karel
The Role of the Octarepeat Region in Neuroprotective Function of the Cellular Prion Protein
Structural alterations of the cellular prion protein (PrPC) seem to be the core of the pathogenesis of prion diseases. However, the physiological function of PrPC remains an enigma. Cell culture experiments have indicated that PrPC and in particular its N-terminal octarepeat region together with the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways have a fundamental involvement in neuroprotection and oxidative stress reactions. We used wild-type mice, PrP knockout (Prnp?/?) animals and transgenic mice that lack the octarepeat region (C4/?) and subjected them to controlled ischemia. We identified an increased cleavage and synthesis of PrPC in ischemic brain areas of wild-type mice compared with sham controls. The infarct size in Prnp?/? animals was increased threefold when compared with wild-type mice. The infarct size in C4/? animals was identical to Prnp?/? mice, that is, around three times larger than in wild-type mice. We showed that the PrP in C4/? mice does not functionally rescue the Prnp?/? phenotype; furthermore it is unable to undergo ? cleavage, although an increased amount of C1 fragments was found in ischemic brain areas compared with sham controls. We demonstrated that the N-terminal octarepeat region has a lead function in PrPC physiology and neuroprotection against oxidative stress in vivo.
Mitteregger, Gerda; Vosko, Milan; Krebs, Bjarne; Xiang, Wei; Kohlmannsperger, Veronika; Nolting, Svenja; Hamann, Gerhard F; Kretzschmar, Hans A
Cloning and expression analysis of a prion protein encoding gene in guppy ( Poecilia reticulata)
NASA Astrophysics Data System (ADS)
The full length cDNA of a prion protein (PrP) encoding gene of guppy ( Poecilia reticulata) and the corresponding genomic DNA were cloned. The cDNA was 2245 bp in length and contained an open reading frame (ORF) of 1545 bp encoding a protein of 515 amino acids, which held all typical structural characteristics of the functional PrP. The cloned genomic DNA fragment corresponding to the cDNA was 3720 bp in length, consisting of 2 introns and 2 exons. The 5' untranslated region of cDNA originated from the 2 exons, while the ORF originated from the second exon. Although the gene was transcribed in diverse tissues including brain, eye, liver, intestine, muscle and tail, its transcript was most abundant in the brain. In addition, the transcription of the gene was enhanced by 5 salinity, implying that it was associated with the response of guppy to saline stress.
Wu, Suihan; Wei, Qiwei; Yang, Guanpin; Wang, Dengqiang; Zou, Guiwei; Chen, Daqing
Polymorphic distribution of the ovine prion protein ( PrP) gene in scrapie-infected sheep flocks in which embryo transfer was used to circumvent the transmissions of scrapie
The genetic sequence of the ovine prion protein (PrP) gene between codons 102 and 175 with emphasis on ovine PrP gene codons 136 and 171 was determined, and the polymorphic distribution of the ovine PrP gene in the scrapie-exposed Suffolk embryo donors and offspring from these donors that were transferred to scrapie-free recipient ewes was investigated in this study. The
Shiquan Wang; Noelle E. Cockett; Janice M. Miller; Tracy L. Shay; Alma Maciulis; Diane L. Sutton; Warren C. Foote; Gilbert R. Holyoak; Ronald C. Evans; Thomas D. Bunch; Jonathan E. Beever; Jay W. Call; William D. Taylor; Michael R. Marshall
Is the presence of abnormal prion protein in the renal glomeruli of feline species presenting with FSE authentic?
In a recent paper written by Hilbe et al (BMC vet res, 2009), the nature and specificity of the prion protein deposition in the kidney of feline species affected with feline spongiform encephalopathy (FSE) were clearly considered doubtful. This article was brought to our attention because we published several years ago an immunodetection of abnormal prion protein in the kidney of a cheetah affected with FSE. At this time we were convinced of its specificity but without having all the possibilities to demonstrate it. As previously published by another group, the presence of abnormal prion protein in some renal glomeruli in domestic cats affected with FSE is indeed generally considered as doubtful mainly because of low intensity detected in this organ and because control kidneys from safe animals present also a weak prion immunolabelling. Here we come back on these studies and thought it would be helpful to relay our last data to the readers of BMC Vet res for future reference on this subject. Here we come back on our material as it is possible to study and demonstrate the specificity of prion immunodetection using the PET-Blot method (Paraffin Embedded Tissue - Blot). It is admitted that this method allows detecting the Proteinase K (PK) resistant form of the abnormal prion protein (PrPres) without any confusion with unspecific immunoreaction. We re-analysed the kidney tissue versus adrenal gland and brain samples from the same cheetah affected with TSE using this PET-Blot method. The PET-Blot analysis revealed specific PrPres detection within the brain, adrenal gland and some glomeruli of the kidney, with a complete identicalness compared to our previous detection using immunohistochemistry. In conclusion, these new data enable us to confirm with assurance the presence of specific abnormal prion protein in the adrenal gland and in the kidney of the cheetah affected with FSE. It also emphasizes the usefulness for the re-examination of any available tissue blocks with the PET-Blot method as a sensitive complementary tool in case of doubtful PrP IHC results.
Is the presence of abnormal prion protein in the renal glomeruli of feline species presenting with FSE authentic?
In a recent paper written by Hilbe et al (BMC vet res, 2009), the nature and specificity of the prion protein deposition in the kidney of feline species affected with feline spongiform encephalopathy (FSE) were clearly considered doubtful. This article was brought to our attention because we published several years ago an immunodetection of abnormal prion protein in the kidney of a cheetah affected with FSE. At this time we were convinced of its specificity but without having all the possibilities to demonstrate it. As previously published by another group, the presence of abnormal prion protein in some renal glomeruli in domestic cats affected with FSE is indeed generally considered as doubtful mainly because of low intensity detected in this organ and because control kidneys from safe animals present also a weak prion immunolabelling. Here we come back on these studies and thought it would be helpful to relay our last data to the readers of BMC Vet res for future reference on this subject.Here we come back on our material as it is possible to study and demonstrate the specificity of prion immunodetection using the PET-Blot method (Paraffin Embedded Tissue--Blot). It is admitted that this method allows detecting the Proteinase K (PK) resistant form of the abnormal prion protein (PrPres) without any confusion with unspecific immunoreaction. We re-analysed the kidney tissue versus adrenal gland and brain samples from the same cheetah affected with TSE using this PET-Blot method. The PET-Blot analysis revealed specific PrPres detection within the brain, adrenal gland and some glomeruli of the kidney, with a complete identicalness compared to our previous detection using immunohistochemistry. In conclusion, these new data enable us to confirm with assurance the presence of specific abnormal prion protein in the adrenal gland and in the kidney of the cheetah affected with FSE. It also emphasizes the usefulness for the re-examination of any available tissue blocks with the PET-Blot method as a sensitive complementary tool in case of doubtful PrP IHC results. PMID:20684771
Lezmi, St�phane; Baron, Thierry G M; Bencsik, Anna A
Shadoo (Sprn) and prion disease incubation time in mice.
Prion diseases are transmissible neurodegenerative disorders of mammalian species and include scrapie, bovine spongiform encephalopathy (BSE), and variant Creutzfeldt-Jakob disease (vCJD). The prion protein (PrP) plays a key role in the disease, with coding polymorphism in both human and mouse influencing disease susceptibility and incubation time, respectively. Other genes are also thought to be important and a plausible candidate is Sprn, which encodes the PrP-like protein Shadoo (Sho). Sho is expressed in the adult central nervous system and exhibits neuroprotective activity reminiscent of PrP in an in vitro assay. To investigate the role of Sprn in prion disease incubation time we sequenced the open reading frame (ORF) in a diverse panel of mice and saw little variation except in strains derived from wild-trapped mice. Sequencing the untranslated regions revealed polymorphisms that allowed us to carry out an association study of incubation period in the Northport heterogeneous stock of mice inoculated with Chandler/RML prions. We also examined the expression level of Sprn mRNA in the brains of normal and prion-infected mice and saw no correlation with either genotype or incubation time. We therefore conclude that Sprn does not play a major role in prion disease incubation time in these strains of mice. PMID:19513788
Lloyd, Sarah E; Grizenkova, Julia; Pota, Hirva; Collinge, John
An N-terminal Polybasic Domain and Cell Surface Localization Are Required for Mutant Prion Protein Toxicity*
There is evidence that alterations in the normal physiological activity of PrPC contribute to prion-induced neurotoxicity. This mechanism has been difficult to investigate, however, because the normal function of PrPC has remained obscure, and there are no assays available to measure it. We recently reported that cells expressing PrP deleted for residues 105�125 exhibit spontaneous ionic currents and hypersensitivity to certain classes of cationic drugs. Here, we utilize cell culture assays based on these two phenomena to test how changes in PrP sequence and/or cellular localization affect the functional activity of the protein. We report that the toxic activity of ?105�125 PrP requires localization to the plasma membrane and depends on the presence of a polybasic amino acid segment at the N terminus of PrP. Several different deletions spanning the central region as well as three disease-associated point mutations also confer toxic activity on PrP. The sequence domains identified in our study are also critical for PrPSc formation, suggesting that common structural features may govern both the functional activity of PrPC and its conversion to PrPSc.
Solomon, Isaac H.; Khatri, Natasha; Biasini, Emiliano; Massignan, Tania; Huettner, James E.; Harris, David A.
Possible role of region 152-156 in the structural duality of a peptide fragment from sheep prion protein
The conformational conversion of the nonpathogenic �cellular� prion isoform into a pathogenic �scrapie� protease-resistant isoform is a fundamental event in the onset of transmissible spongiform encephalopathies (TSE). During this pathogenic conversion, helix H1 and its two flanking loops of the normal prion protein are thought to undergo a conformational transition into a ?-like structure. A peptide spanning helix H1 and ?-strand S2 (residues 142�166 in human numbering) was studied by circular dichroism and nuclear magnetic resonance spectroscopies. This peptide in aqueous solution, in contrast to many prion fragments studied earlier (1) is highly soluble and (2) does not aggregate until the millimolar concentration range, and (3) exhibits an intrinsic propensity to a ?-hairpin-like conformation at neutral pH. We found that this peptide can also fold into a helix H1 conformation when dissolved in a TFE/PB mixture. The structures of the peptide calculated by MD showed solvent-dependent internal stabilizing forces of the structures and evidenced a higher mobility of the residues following the end of helix H1. These data suggest that the molecular rearrangement of this peptide in region 152�156, particularly in position 155, could be associated with the pathogenic conversion of the prion protein.
Megy, Simon; Bertho, Gildas; Kozin, Sergey A.; Debey, Pascale; Hui Bon Hoa, Gaston; Girault, Jean-Pierre
Current concepts in human prion protein (Prp) misfolding, Prnp gene polymorphisms and their contribution to Creutzfeldt-Jakob Disease (CJD).
Transmissible spongiform encephalopathies are a group of neural degenerative diseases that may be infectious, sporadic, or hereditary and are associated with an abnormally folded prion protein. Unfortunately at the current time it is not at all clear what the normal structure of the prion protein actually is or how it is toxic to cells. Extensive research on prion diseases has led to a dramatic increase in understanding of the pathogenesis of prion disorders, which will hopefully lead to the development of effective treatments. The inability to detect the disease in blood using current technology has made screening difficult. While fortunately there has been a decline in the number of clinical cases of transmissible variant CJD, evidence indicates that very long incubation periods can occur in humans so there may be a long slow, gradual epidemic. In particular, clinical cases in genotypes other than those homozygous for methionine at codon 129 of PRNP have not yet occurred, but such cases might be expected to have longer incubation periods and show differences in pathology to those seen to date. Transgenic animal studies have shown that a large proportion of infected animals develop sub-clinical disease. Moreover, results from a large prevalence study in humans show that several cases test positive but do not develop clinical disease. It is possible therefore that further cases of secondary transmission could occur by iatrogenic spread, which could result in vCJD persisting in the UK at low levels for many years, highlighting the importance of continued vigilance. PMID:17616941
Michalczyk, K; Ziman, M
Distinct Transmissibility Features of TSE Sources Derived from Ruminant Prion Diseases by the Oral Route in a Transgenic Mouse Model (TgOvPrP4) Overexpressing the Ovine Prion Protein
Transmissible spongiform encephalopathies (TSEs) are a group of fatal neurodegenerative diseases associated with a misfolded form of host-encoded prion protein (PrP). Some of them, such as classical bovine spongiform encephalopathy in cattle (BSE), transmissible mink encephalopathy (TME), kuru and variant Creutzfeldt�Jakob disease in humans, are acquired by the oral route exposure to infected tissues. We investigated the possible transmission by the oral route of a panel of strains derived from ruminant prion diseases in a transgenic mouse model (TgOvPrP4) overexpressing the ovine prion protein (A136R154Q171) under the control of the neuron-specific enolase promoter. Sources derived from Nor98, CH1641 or 87V scrapie sources, as well as sources derived from L-type BSE or cattle-passaged TME, failed to transmit by the oral route, whereas those derived from classical BSE and classical scrapie were successfully transmitted. Apart from a possible effect of passage history of the TSE agent in the inocula, this implied the occurrence of subtle molecular changes in the protease-resistant prion protein (PrPres) following oral transmission that can raises concerns about our ability to correctly identify sheep that might be orally infected by the BSE agent in the field. Our results provide proof of principle that transgenic mouse models can be used to examine the transmissibility of TSE agents by the oral route, providing novel insights regarding the pathogenesis of prion diseases.
Arsac, Jean-Noel; Baron, Thierry
Prion protein as a mediator of neurocardiosympathetic interactions.
A proteomic approach to study cardiovascular disease includes the examination of proteins associated with risk factors such as left ventricular hypertrophy (LVH). PrP(C) is a host-coded membrane-bound glycoprotein found in most cell types, including myocardium, and whose physiological function is uncertain. We have taken a selective proteomic approach and performed mechanistic studies to determine whether PrP(C) levels are related to left ventricular (LV) structure or function. Echocardiograms were performed at baseline in 65 mice comprising three strains of the same C57Bl/6J � 129SV genetic background but expressing different levels of PrP(C) (wild-type mice (WT), PrP(-/-) , and PrP(C) over-expressing transgenic mice (tga20)). There were no significant differences in LV mass or LV ejection fraction between the three groups. Either normal saline (n = 60) or isoproterenol (n = 55) was then administered intraperitoneally (50 mg/kg/day) for 5 days/wk for two consecutive weeks to induce LVH. Body weight decreased significantly in the PrP(-/-) group (18%). On multivariate analysis, higher LV mass index posttreatment was independently associated with the tga20 group (versus PrP(-/-) versus WT, p = 0.002) after adjusting for treatment (isoproterenol versus saline), and weight change (r(2) = 0.13 for model, p = 0.016). Therefore, PrP(C) appears unrelated to LV mass and function in the basal state. Isoproterenol causes transient enhancement of PrP(C) expression in WT mice and a more pronounced increase in tga20 mice at 2 h posttreatment. Overexpression of PrP(C) in the tga20 group may be associated with higher LV mass after a 2 wk regimen of isoproterenol. PMID:23161471
Rubenstein, Richard; Chiu, Allen; Salciccioli, Louis; Kamran, Haroon; Lazar, Jason
Age-Dependent Impairment of Eyeblink Conditioning in Prion Protein-Deficient Mice
Mice lacking the prion protein (PrPC) gene (Prnp), Ngsk Prnp0/0 mice, show late-onset cerebellar Purkinje cell (PC) degeneration because of ectopic overexpression of PrPC-like protein (PrPLP/Dpl). Because PrPC is highly expressed in cerebellar neurons (including PCs and granule cells), it may be involved in cerebellar synaptic function and cerebellar cognitive function. However, no studies have been conducted to investigate the possible involvement of PrPC and/or PrPLP/Dpl in cerebellum-dependent discrete motor learning. Therefore, the present cross-sectional study was designed to examine cerebellum-dependent delay eyeblink conditioning in Ngsk Prnp0/0 mice in adulthood (16, 40, and 60 weeks of age). The aims of the present study were two-fold: (1) to examine the role of PrPC and/or PrPLP/Dpl in cerebellum-dependent motor learning and (2) to confirm the age-related deterioration of eyeblink conditioning in Ngsk Prnp0/0 mice as an animal model of progressive cerebellar degeneration. Ngsk Prnp0/0 mice aged 16 weeks exhibited intact acquisition of conditioned eyeblink responses (CRs), although the CR timing was altered. The same result was observed in another line of PrPc-deficient mice, ZrchI PrnP0/0 mice. However, at 40 weeks of age, CR incidence impairment was observed in Ngsk Prnp0/0 mice. Furthermore, Ngsk Prnp0/0 mice aged 60 weeks showed more significantly impaired CR acquisition than Ngsk Prnp0/0 mice aged 40 weeks, indicating the temporal correlation between cerebellar PC degeneration and motor learning deficits. Our findings indicate the importance of the cerebellar cortex in delay eyeblink conditioning and suggest an important physiological role of prion protein in cerebellar motor learning.
Kishimoto, Yasushi; Hirono, Moritoshi; Atarashi, Ryuichiro; Sakaguchi, Suehiro; Yoshioka, Tohru; Katamine, Shigeru; Kirino, Yutaka
GFP-tagged prion protein is correctly localized and functionally active in the brains of transgenic mice
Prion diseases result from conversion of PrPC, a neuronal membrane glycoprotein of unknown function, into PrPSc, an abnormal conformer that is thought to be infectious. To facilitate analysis of PrP distribution in the brain, we have generated transgenic mice in which a PrP promoter drives expression of PrP-EGFP, a fusion protein consisting of enhanced green fluorescent protein inserted adjacent to
Sami Barmada; Pedro Piccardo; Keiji Yamaguchi; Bernardino Ghetti; David A Harris
Evaluation of non-immunoaffinity methods for isolation of cellular prion protein from bovine brain.
Transmissible spongiform encephalopathies (TSEs) are progressive neurodegenerative diseases that affect the central nervous system of many animals, including humans. Research suggests that TSEs are caused by conversion of the cellular prion protein (PrP(C)), which is encoded in many tissues, especially brain, to the pathological form (PrP(Sc)). This conversion affects PrP(Sc) structure, conferring different biochemical properties, such as the increased resistance to proteinase K, that have been widely used for its purification. By contrast, PrP(C) is less resistant and its isolation is more challenging. Here, we propose a purification strategy to efficiently recover PrP(C) from healthy bovine brain using conventional non-immunoaffinity methods. The applicability of extraction using detergents, size exclusion chromatography, diafiltration with molecular weight cutoff (MWCO) filters, and immobilized metal affinity chromatography (IMAC) using Western blot (WB) analysis to detect the presence of PrP(C) is discussed in detail. PMID:24463017
Borges-Alvarez, M; Benavente, F; M�rquez, M; Barbosa, J; Sanz-Nebot, V
Cellular prion protein participates in amyloid-? transcytosis across the blood-brain barrier
The blood�brain barrier (BBB) facilitates amyloid-? (A?) exchange between the blood and the brain. Here, we found that the cellular prion protein (PrPc), a putative receptor implicated in mediating A? neurotoxicity in Alzheimer's disease (AD), participates in A? transcytosis across the BBB. Using an in vitro BBB model, [125I]-A?1?40 transcytosis was reduced by genetic knockout of PrPc or after addition of a competing PrPc-specific antibody. Furthermore, we provide evidence that PrPc is expressed in endothelial cells and, that monomeric A?1?40 binds to PrPc. These observations provide new mechanistic insights into the role of PrPc in AD.
Pflanzner, Thorsten; Petsch, Benjamin; Andre-Dohmen, Bettina; Muller-Schiffmann, Andreas; Tschickardt, Sabrina; Weggen, Sascha; Stitz, Lothar; Korth, Carsten; Pietrzik, Claus U
Sulphated glycosaminoglycans prevent the neurotoxicity of a human prion protein fragment.
Although a number of features distinguish the disease isoform of the prion protein (PrPSc) from its normal cellular counterpart (PrPC) in the transmissible spongiform encephalopathies (TSEs), the neuropathogenesis of these diseases remains an enigma. The amyloid fibrils formed by fragments of human PrP have, however, been shown to be directly neurotoxic in vitro. We show here that sulphated polysaccharides (heparin, keratan and chondroitin) inhibit the neurotoxicity of these amyloid fibrils and this appears to be mediated via inhibition of the polymerization of the PrP peptide into fibrils. This provides a rationale for the therapeutic effects of sulphated polysaccharides and suggests a rapid in vitro functional screen for TSE therapeutics.
Perez, M; Wandosell, F; Colaco, C; Avila, J
Expression and Knockdown of Cellular Prion Protein (PrPC) in Differentiating Mouse Embryonic Stem Cells
The mammalian cellular prion protein (PrPC) is a highly conserved glycoprotein that may undergo conversion into a conformationally altered isoform (scrapie prion protein or PrPSc), widely believed to be the pathogenic agent of transmissible spongiform encephalopathies (TSEs). Although much is known about pathogenic PrP conversion and its role in TSEs, the normal function of PrPC is poorly understood. Given the abundant expression of PrPC in the developing mammalian CNS and the spatial association with differentiated stages of neurogenesis, recently it has been proposed that PrPC participates in neural cell differentiation. In the present study, we investigated the role of PrPC in neural development during early embryogenesis. In bovine fetuses, PrPC was differentially expressed in the neuroepithelium, showing higher levels at the intermediate and marginal layers where more differentiated states of neurogenesis were located. We utilized differentiating mouse embryonic stem (ES) cells to test whether PrPC contributed to the process of neural differentiation during early embryogenesis. PrPC showed increasing levels of expression starting on Day 9 until Day 18 of ES cell differentiation. PrPC expression was negatively correlated with pluripotency marker Oct-4 confirming that ES cells had indeed differentiated. Induction of ES cells in the presence of retinoic acid (RA) resulted in up-regulation of PrPC at Day 20 and nestin at Day 12. PrPC expression was knocked down in PrP-targeted siRNA ES cells between Days 12 and 20. PrPC knockdown in ES cells resulted in nestin reduction at Days 16 and 20. Analysis in early bovine fetuses suggests the participation of PrPC in neural cell differentiation during early embryogenesis. The positive association between PrPC and nestin expression provide evidence for the contribution of PrPC to ES cell differentiation into neural progenitor cells.
Peralta, Oscar A.; Huckle, William R.; Eyestone, Willard H.
Developmental expression of the cellular prion protein (PrPC) in bovine embryos
The mammalian cellular prion protein (PrPC) is a highly conserved glycoprotein that may undergo conversion into a conformationally altered isoform (scrapie prion protein or PrPSc), widely believed to be the pathogenic agent of transmissible spongiform encephalopathies (TSEs). Although much is known about PrPSc conversion and its role in TSEs, the normal function of PrPC has not been elucidated. In adult mammals, PrPC is most abundant in the central nervous tissue, with intermediate levels in the intestine and heart, and lower levels in the pancreas and liver. PrPC is expressed during neurogenesis throughout development, and it has recently been proposed that PrPC participates in neural cell differentiation during embryogenesis. In order to establish the developmental timing and to address the cell-specific expression of PrPC during mammalian development, we examined PrPC expression in bovine gametes and embryos through gestation Day 39. Our data revealed differential levels of Prnp mRNA at Days 4 and 18 in pre-attachment embryos. PrPC was detected in the developing central and peripheral nervous systems in Day 27, 32, and 39 embryos. PrPC was particularly expressed in differentiated neural cells located in the marginal regions of the central nervous system, but was absent from mitotically active, periventricular areas. Moreover, a PrPC cell-specific pattern of expression was detected in non-nervous tissues, including liver and mesonephros, during these stages. The potential participation of PrPC in neural cell differentiation is supported by its specific expression in differentiated states of neurogenesis.
Peralta, Oscar A.; Huckle, William R.; Eyestone, Willard H.
Prion protein M129V polymorphism affects retrieval-related brain activity.
The prion protein Met129Val polymorphism has recently been related to human long-term memory with carriers of either the 129MM or the 129MV genotype recalling 17% more words than 129(VV) carriers at 24h following learning. Here, we sampled genotype differences in retrieval-related brain activity at 30min and 24h following learning. Furthermore, genotype groups were compared regarding grey matter concentrations and cognitive profiles. We used event-related functional magnetic resonance imaging (fMRI) during a word recognition task on 12 Met/Met carriers, 12 Val/Met carriers, and 12 Val/Val carriers. These groups were matched for retrieval performance, gender, age, education, and other memory-related genetic polymorphisms. Although retrieval performance was matched, Val carriers exhibited enhanced retrieval-related brain activity at 30min and 24h following learning. At both time lags, correlations between retrieval-related brain activity and retrieval success were negative for Val homozygotes (the more activity, the worse retrieval success), while correlations showed no significance or were positive for Met homozygotes and heterozygotes. These results suggest a less economic use of retrieval-related neural resources in Val relative to Met carriers. Furthermore, Val carriers exhibited higher neocortical grey matter concentrations compared to Met carriers. When controlling for grey matter concentration, genotype effects in retrieval-related brain activity remained significant. Val and Met carriers yielded comparable brain activations for correct rejections of non-studied words and for working memory, which speaks to the specificity of the genotype effect. Findings suggest that the prion protein Met129Val polymorphism affects neural plasticity following learning at a time-scale of minutes to hours. PMID:18423780
Buchmann, Andreas; Mondadori, Christian R A; H�nggi, J�rgen; Aerni, Amanda; Vrticka, Pascal; Luechinger, Roger; Boesiger, Peter; Hock, Christoph; Nitsch, Roger M; de Quervain, Dominique J-F; Papassotiropoulos, Andreas; Henke, Katharina
Conversion of alpha-helices into beta-sheets features in the formation of the scrapie prion proteins.
Prions are composed largely, if not entirely, of prion protein (PrPSc in the case of scrapie). Although the formation of PrPSc from the cellular prion protein (PrPC) is a post-translational process, no candidate chemical modification was identified, suggesting that a conformational change features in PrPSc synthesis. To assess this possibility, we purified both PrPC and PrPSc by using nondenaturing procedures and determined the secondary structure of each. Fourier-transform infrared (FTIR) spectroscopy demonstrated that PrPC has a high alpha-helix content (42%) and no beta-sheet (3%), findings that were confirmed by circular dichroism measurements. In contrast, the beta-sheet content of PrPSc was 43% and the alpha-helix 30% as measured by FTIR. As determined in earlier studies, N-terminally truncated PrPSc derived by limited proteolysis, designated PrP 27-30, has an even higher beta-sheet content (54%) and a lower alpha-helix content (21%). Neither PrPC nor PrPSc formed aggregates detectable by electron microscopy, while PrP 27-30 polymerized into rod-shaped amyloids. While the foregoing findings argue that the conversion of alpha-helices into beta-sheets underlies the formation of PrPSc, we cannot eliminate the possibility that an undetected chemical modification of a small fraction of PrPSc initiates this process. Since PrPSc seems to be the only component of the "infectious" prion particle, it is likely that this conformational transition is a fundamental event in the propagation of prions. Images Fig. 1 Fig. 4
Pan, K M; Baldwin, M; Nguyen, J; Gasset, M; Serban, A; Groth, D; Mehlhorn, I; Huang, Z; Fletterick, R J; Cohen, F E
Roles of the cellular prion protein in the regulation of cell-cell junctions and barrier function
The cellular prion protein was historically characterized owing to its misfolding in prion disease. Although its physiological role remains incompletely understood, PrPC has emerged as an evolutionary conserved, multifaceted protein involved in a wide-range of biological processes. PrPC is a GPI-anchored protein targeted to the plasma membrane, in raft microdomains, where its interaction with a repertoire of binding partners, which differ depending on cell models, mediates its functions. Among identified PrPC partners are cell adhesion molecules. This review will focus on the multiple implications of PrPC in cell adhesion processes, mainly the regulation of cell-cell junctions in epithelial and endothelial cells and the consequences on barrier properties. We will show how recent findings argue for a role of PrPC in the recruitment of signaling molecules, which in turn control the targeting or the stability of adhesion complexes at the plasma membrane.
Petit, Constance S.V.; Besnier, Laura; Morel, Etienne; Rousset, Monique; Thenet, Sophie
Impact of the tail and mutations G131V and M129V on prion protein flexibility.
Within the "protein-only" hypothesis, a detailed mechanism for the conversion of a alpha-helix to beta-sheet structure is unclear. We have investigated the effects of the tail 90-123 and the point mutations G131V and M129V on prion protein conformational plasticity at neutral pH. Molecular dynamics simulations show that the dynamics of the core 124-226 is essentially independent of the tail and that the point mutation G131V does not affect PrP thermodynamic stability. Both mutations, however, enhance the flexibility of residues that participate in the two-step process for prion propagation. They also extend the short beta-sheet in the normal protein into a larger sheet at neutral pH. This finding suggests a critical role of the tail for triggering the topological change. PMID:12660994
Santini, S�bastien; Claude, Jean-Baptiste; Audic, St�phane; Derreumaux, Philippe
RNA-binding proteins with prion-like domains in ALS and FTLD-U.
Amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig's disease) is a debilitating, and universally fatal, neurodegenerative disease that devastates upper and lower motor neurons. The causes of ALS are poorly understood. A central role for RNA-binding proteins and RNA metabolism in ALS has recently emerged. The RNA-binding proteins, TDP-43 and FUS, are principal components of cytoplasmic inclusions found in motor neurons of ALS patients and mutations in TDP-43 and FUS are linked to familial and sporadic ALS. Pathology and genetics also connect TDP-43 and FUS with frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). It was unknown whether mechanisms of FUS aggregation and toxicity were similar or different to those of TDP-43. To address this issue, we have employed yeast models and pure protein biochemistry to define mechanisms underlying TDP-43 and FUS aggregation and toxicity, and to identify genetic modifiers relevant for human disease. We have identified prion-like domains in FUS and TDP-43 and provide evidence that these domains are required for aggregation. Our studies have defined key similarities as well as important differences between the two proteins. Collectively, however, our findings lead us to suggest that FUS and TDP-43, though similar RNA-binding proteins, likely aggregate and confer disease phenotypes via distinct mechanisms. PMID:21847013
Gitler, Aaron D; Shorter, James
Convergent Replication of Mouse Synthetic Prion Strains
Prion diseases are neurodegenerative disorders characterized by the aberrant folding of endogenous proteins into self-propagating pathogenic conformers. Prion disease can be initiated in animal models by inoculation with amyloid fibrils formed from bacterially derived recombinant prion protein. The synthetic prions that accumulated in infected organisms are structurally distinct from the amyloid preparations used to initiate their formation and change conformationally on repeated passage. To investigate the nature of synthetic prion transformation, we infected mice with a conformationally diverse set of amyloids and serially passaged the resulting prion strains. At each passage, we monitored changes in the biochemical and biological properties of the adapting strain. The physicochemical properties of each synthetic prion strain gradually changed on serial propagation until attaining a common adapted state with shared physicochemical characteristics. These results indicate that synthetic prions can assume multiple intermediate conformations before converging into one conformation optimized for in�vivo propagation.
Ghaemmaghami, Sina; Colby, David W.; Nguyen, Hoang-Oanh B.; Hayashi, Shigenari; Oehler, Abby; DeArmond, Stephen J.; Prusiner, Stanley B.
In vitro conversion and seeded fibrillization of posttranslationally modified prion protein.
The conversion of the cellular isoform of the prion protein (PrP(C)) into the pathologic isoform (PrP(Sc)) is the key event in prion diseases. To study the conversion process, an in vitro system based on varying the concentration of low amounts of sodium dodecyl sulfate (SDS) has been employed. In the present study, the conversion of full-length PrP(C) isolated from Chinese hamster ovary cells (CHO-PrP(C)) was examined. CHO-PrP(C) harbors native, posttranslational modifications, including the GPI anchor and two N-linked glyco-sylation sites. The properties of CHO-PrP(C) were compared with those of full-length and N-terminally truncated recombinant PrP. As shown earlier with recombinant PrP (recPrP90-231), transition from a soluble ?-helical state as known for native PrP(C) into an aggregated, ?-sheet-rich PrP(Sc)-like state could be induced by dilution of SDS. The aggregated state is partially proteinase K (PK)-resistant, exhibiting a cleavage site similar to that found with PrP(Sc). Compared to recPrP (90-231), fibril formation with CHO-PrP(C) requires lower SDS concentrations (0.0075%), and can be drastically accelerated by seeding with PrP(Sc) purified from brain homogenates of terminally sick hamsters. Our results show that recPrP 90-231 and CHO-PrPC behave qualitatively similar but quantitatively different. The in vivo situation can be simulated closer with CHO-PrP(C) because the specific PK cleave site could be shown and the seed-assisted fibrillization was much more efficient. PMID:21476870
St�hr, Jan; Elfrink, Kerstin; Weinmann, Nicole; Wille, Holger; Willbold, Dieter; Birkmann, Eva; Riesner, Detlev
Biochemical typing of pathological prion protein in aging cattle with BSE
Background The broad enforcement of active surveillance for bovine spongiform encephalopathy (BSE) in 2000 led to the discovery of previously unnoticed, atypical BSE phenotypes in aged cattle that differed from classical BSE (C-type) in biochemical properties of the pathological prion protein. Depending on the molecular mass and the degree of glycosylation of its proteinase K resistant core fragment (PrPres), mainly determined in samples derived from the medulla oblongata, these atypical cases are currently classified into low (L)-type or high (H)-type BSE. In the present study we address the question to what extent such atypical BSE cases are part of the BSE epidemic in Switzerland. Results To this end we analyzed the biochemical PrPres type by Western blot in a total of 33 BSE cases in cattle with a minimum age of eight years, targeting up to ten different brain regions. Our work confirmed H-type BSE in a zebu but classified all other cases as C-type BSE; indicating a very low incidence of H- and L-type BSE in Switzerland. It was documented for the first time that the biochemical PrPres type was consistent across different brain regions of aging animals with C-type and H-type BSE, i.e. independent of the neuroanatomical structure investigated. Conclusion Taken together this study provides further characteristics of the BSE epidemic in Switzerland and generates new baseline data for the definition of C- and H-type BSE phenotypes, thereby underpinning the notion that they indeed represent distinct prion disease entities.
Tester, Seraina; Juillerat, Valerie; Doherr, Marcus G; Haase, Bianca; Polak, Miroslaw; Ehrensperger, Felix; Leeb, Tosso; Zurbriggen, Andreas; Seuberlich, Torsten
Molecular Barriers to Zoonotic Transmission of Prions
The risks posed to human health by individual animal prion diseases cannot be determined a priori and are difficult to address empirically. The fundamental event in prion disease pathogenesis is thought to be the seeded conversion of normal prion protein to its pathologic isoform. We used a rapid molecular conversion assay (protein misfolding cyclic amplification) to test whether brain homogenates from specimens of classical bovine spongiform encephalopathy (BSE), atypical BSE (H-type BSE and L-type BSE), classical scrapie, atypical scrapie, and chronic wasting disease can convert normal human prion protein to the abnormal disease-associated form. None of the tested prion isolates from diseased animals were as efficient as classical BSE in converting human prion protein. However, in the case of chronic wasting disease, there was no absolute barrier to conversion of the human prion protein.
Barria, Marcelo A.; Balachandran, Aru; Morita, Masanori; Kitamoto, Tetsuyuki; Barron, Rona; Manson, Jean; Knight, Richard; Ironside, James W.
Copper is required for prion protein-associated superoxide dismutase-I activity in Pichia pastoris.
The prion protein (PrP) is the key protein implicated in transmissible spongiform encephalopathies. It is a metalloprotein that binds manganese and copper. The latter is involved in the physiological function of the protein. We have previously found that PrP expression in Pichia pastoris affects intracellular metal ion concentrations and that formation of protease-resistant PrP is induced by additional copper and/or manganese. In this study, we show that heterologously expressed PrP is post-translationally modified and transported to the cell wall. We found by combining three different test systems that PrP itself had gained superoxide dismutase-like activity in P. pastoris. However, this activity could not be inhibited by KCN and depended on additional copper in the medium. Thus, this study defines the conditions under which PrP exhibits superoxide dismutase-like activity by showing that copper must be present for the protein to participate in scavenging and detoxification of reactive oxygen species. PMID:17263729
Treiber, Carina; Pipkorn, R�diger; Weise, Christoph; Holland, Gudrun; Multhaup, Gerd
Prion strain discrimination using luminescent conjugated polymers
The occurrence of multiple strains of prions may reflect conformational variability of PrPSc, a disease-associated, aggregated variant of the cellular prion protein, PrPC. Here we used luminescent conjugated polymers (LCPs), which emit conformation-dependent fluorescence spectra, for characterizing prion strains. LCP reactivity and emission spectra of brain sections discriminated among four immunohistochemically indistinguishable, serially mouse-passaged prion strains derived from sheep scrapie,
Christina J Sigurdson; K Peter R Nilsson; Simone Hornemann; Giuseppe Manco; Magdalini Polymenidou; Petra Schwarz; Mario Leclerc; Per Hammarstr�m; Kurt W�thrich; Adriano Aguzzi
Combinatorial Approach of Gene Silencing and Expression Profiling in Deciphering the Roles of Prion and Auxiliary Molecules in Aberrant Prion Replication.
National Technical Information Service (NTIS)
Infectious, self-propagating protein aggregates (prions) and deposition of amyloid fibrils are universal components of prion-based diseases. Prion null mice are fertile, neurologically normal and show resistant to scrapie infection than their wild-type co...
R. Kumar
Strain Specific Resistance to Murine Scrapie Associated with a Naturally Occurring Human Prion Protein Polymorphism at Residue 171
Transmissible spongiform encephalopathies (TSE) or prion diseases are neurodegenerative disorders associated with conversion of normal host prion protein (PrP) to a misfolded, protease-resistant form (PrPres).�Genetic variations of prion protein in humans and animals can alter susceptibility to both familial and infectious prion diseases. The N171S PrP polymorphism is found mainly in humans of African descent, but its low incidence has precluded study of its possible influence on prion disease. Similar to previous experiments of others, for laboratory studies we created a transgenic model expressing the mouse PrP homolog, PrP-170S, of human PrP-171S. Since PrP polymorphisms can vary in their effects on different TSE diseases, we tested these mice with four different strains of mouse-adapted scrapie. Whereas 22L and ME7 scrapie strains induced typical clinical disease, neuropathology and accumulation of PrPres in all transgenic mice at 99-128 average days post-inoculation, strains RML and 79A produced clinical disease and PrPres formation in only a small subset of mice at very late times. When mice expressing both PrP-170S and PrP-170N were inoculated with RML scrapie, dominant-negative inhibition of disease did not occur, possibly because interaction of strain RML with PrP-170S was minimal. Surprisingly, in vitro PrP conversion using protein misfolding cyclic amplification (PMCA), did not reproduce the in vivo findings, suggesting that the resistance noted in live mice might be due to factors or conditions not present in vitro. These findings suggest that in vivo conversion of PrP-170S by RML and 79A scrapie strains was slow and inefficient. PrP-170S mice may be an example of the conformational selection model where the structure of some prion strains does not favor interactions with PrP molecules expressing certain polymorphisms.
Striebel, James F.; Race, Brent; Meade-White, Kimberly D.; LaCasse, Rachel; Chesebro, Bruce
Different allelic effects of the codons 136 and 171 of the prion protein gene in sheep with natural scrapie
Scrapie is a transmissible degenerative disease of the central nervous system occurring naturally in sheep. It belongs to the group ofprion diseases also affecting man in which an abnormal isoform of the host-encoded prion protein (PrP) accumulating in the brain is responsible for neuronal death. Three main polymorphisms have been described in the sheep PrP gene, at positions 136, 154
C. Clouscard; P. Beaudry; J. M. Elsen; D. Milan; M. Dussaucy; C. Bounneau; F. Schelcher; J. Chatelain; J. M. Launay; J. L. Laplanche
Prion Protein-Deficient Cells Show Altered Response to Oxidative Stress Due to Decreased SOD1 Activity
The cellular function of the prion protein (PrPC), a cell surface glycoprotein expressed in neurones and astrocytes, has not been elucidated. Cell culture experiments reveal that cerebellar cells lacking PrPCare more sensitive to oxidative stress and undergo cell death more readily than wild-type cells. This effect is reversible by treatment with vitamin E.In vivostudies show that the activity of Cu\\/Zn
David R. Brown; Walter J. Schulz-Schaeffer; Bernhard Schmidt; Hans A. Kretzschmar
Cellular prion protein regulates the motor behaviour performance and anxiety-induced responses in genetically modified mice
The cellular prion protein (PrPC) is a sialoglycoprotein involved in neuroplasticity processes and synaptic transmission. This study investigated behavioural responses (balance in the rota-rod test at 24rpm, motility in the open-field test, anxiety in the elevated plus-maze test) in Zurich developed wild-type adult mice (WT, controls of normal PrPC expression), in knockout (KO) mice (Prnp0\\/0, with no PrPC expression), and
Bruno Lob�o-Soares; Roger Walz; Carlos Gilberto Carlotti; Am�rico Ceiki Sakamoto; Fabr�cio Calvo; Ana Luiza Bernardes Terzian; Juliana Almeida da Silva; Lauro Wichert-Ana; Norberto Cysne Coimbra; Marino Muxfeldt Bianchin
Introducing a Rigid Loop Structure from Deer into Mouse Prion Protein Increases Its Propensity for Misfolding In Vitro
Prion diseases are fatal neurodegenerative disorders characterized by misfolding of the cellular prion protein (PrPc) into the disease-associated isoform (PrPSc) that has increased ?-sheet content and partial resistance to proteolytic digestion. Prion diseases from different mammalian species have varying propensities for transmission upon exposure of an uninfected host to the infectious agent. Chronic Wasting Disease (CWD) is a highly transmissible prion disease that affects free ranging and farmed populations of cervids including deer, elk and moose, as well as other mammals in experimental settings. The molecular mechanisms allowing CWD to maintain comparatively high transmission rates have not been determined. Previous work has identified a unique structural feature in cervid PrP, a rigid loop between ?-sheet 2 and ?-helix 2 on the surface of the protein. This study was designed to test the hypothesis that the rigid loop has a direct influence on the misfolding process. The rigid loop was introduced into murine PrP as the result of two amino acid substitutions: S170N and N174T. Wild-type and rigid loop murine PrP were expressed in E. coli and purified. Misfolding propensity was compared for the two proteins using biochemical techniques and cell free misfolding and conversion systems. Murine PrP with a rigid loop misfolded in cell free systems with greater propensity than wild type murine PrP. In a lipid-based conversion assay, rigid loop PrP converted to a PK resistant, aggregated isoform at lower concentrations than wild-type PrP. Using both proteins as substrates in real time quaking-induced conversion, rigid loop PrP adopted a misfolded isoform more readily than wild type PrP. Taken together, these findings may help explain the high transmission rates observed for CWD within cervids.
Kyle, Leah M.; John, Theodore R.; Schatzl, Hermann M.; Lewis, Randolph V.
Effects of post-translational modifications on prion protein aggregation and the propagation of scrapie-like characteristics in vitro
Prion diseases, or transmissible spongiform encephalopathies (TSEs) are typically characterised by CNS accumulation of PrPSc, an aberrant conformer of a normal cellular protein PrPC. It is thought PrPSc is itself infectious and the causative agent of such diseases. To date, no chemical modifications of PrPSc, or a sub-population thereof, have been reported. In this study we have investigated whether chemical
Denise V. Dear; Duncan S. Young; Jurate Kazlauskaite; Filip Meersman; David Oxley; Judith Webster; Teresa J. T. Pinheiro; Andrew C. Gill; Igor Bronstein; Christopher R. Lowe
Aggravation of ischemic brain injury by prion protein deficiency: Role of ERK1\\/-2 and STAT1
The cellular isoform of prion protein, PrPc, may confer neuroprotection in the brain, according to recent studies. To elucidate the role of PrPc in stroke pathology, we subjected PrPc-knockout (Prnp0\\/0), wild-type and PrPc-transgenic (tga20) mice to 30 min of intraluminal middle cerebral artery occlusion, followed by 3, 24 or 72 h reperfusion, and examined how PrPc levels influence brain injury
Annett Spudich; Rico Frigg; Ertugrul Kilic; �lkan Kilic; Bruno Oesch; Alex Raeber; Claudio L. Bassetti; Dirk M. Hermann
Pathologic Prion Protein Infects Cells by Lipid-Raft Dependent Macropinocytosis
Transmissible spongiform encephalopathies, including variant-Creutzfeldt-Jakob disease (vCJD) in humans and bovine spongiform encephalopathies in cattle, are fatal neurodegenerative disorders characterized by protein misfolding of the host cellular prion protein (PrPC) to the infectious scrapie form (PrPSc). However, the mechanism that exogenous PrPSc infects cells and where pathologic conversion of PrPC to the PrPSc form occurs remains uncertain. Here we report that similar to the mechanism of HIV-1 TAT-mediated peptide transduction, processed mature, full length PrP contains a conserved N-terminal cationic domain that stimulates cellular uptake by lipid raft-dependent, macropinocytosis. Inhibition of macropinocytosis by three independent means prevented cellular uptake of recombinant PrP; however, it did not affect recombinant PrP cell surface association. In addition, fusion of the cationic N-terminal PrP domain to a Cre recombinase reporter protein was sufficient to promote both cellular uptake and escape from the macropinosomes into the cytoplasm. Inhibition of macropinocytosis was sufficient to prevent conversion of PrPC to the pathologic PrPSc form in N2a cells exposed to strain RML PrPSc infected brain homogenates, suggesting that a critical determinant of PrPC conversion occurs following macropinocytotic internalization and not through mere membrane association. Taken together, these observations provide a cellular mechanism that exogenous pathological PrPSc infects cells by lipid raft dependent, macropinocytosis.
Wadia, Jehangir S.; Schaller, Monica; Williamson, R. Anthony; Dowdy, Steven F.
Electron paramagnetic resonance evidence for binding of Cu(2+) to the C-terminal domain of the murine prion protein.
Transmissible spongiform encephalopathies in mammals are believed to be caused by scrapie form of prion protein (PrP(Sc)), an abnormal, oligomeric isoform of the monomeric cellular prion protein (PrP(C)). One of the proposed functions of PrP(C) in vivo is a Cu(II) binding activity. Previous studies revealed that Cu(2+) binds to the unstructured N-terminal PrP(C) segment (residues 23-120) through conserved histidine residues. Here we analyzed the Cu(II) binding properties of full-length murine PrP(C) (mPrP), of its isolated C-terminal domain mPrP(121-231) and of the N-terminal fragment mPrP(58-91) in the range of pH 3-8 with electron paramagnetic resonance spectroscopy. We find that the C-terminal domain, both in its isolated form and in the context of the full-length protein, is capable of interacting with Cu(2+). Three Cu(II) coordination types are observed for the C-terminal domain. The N-terminal segment mPrP(58-91) binds Cu(2+) only at pH values above 5.0, whereas both mPrP(121-231) and mPrP(23-231) already show identical Cu(II) coordination in the pH range 3-5. As the Cu(2+)-binding N-terminal segment 58-91 is not required for prion propagation, our results open the possibility that Cu(2+) ions bound to the C-terminal domain are involved in the replication of prions, and provide the basis for further analytical studies on the specificity of Cu(II) binding by PrP.
Cereghetti, G M; Schweiger, A; Glockshuber, R; Van Doorslaer, S
Phenotypic characterization of cells participating in transport of prion protein aggregates across the intestinal mucosa of sheep
The oral route is considered to be the main entry site of several transmissible spongiform encephalopathies or prion diseases of animals and man. Following natural and experimental oral exposure to scrapie, sheep first accumulate disease associated prion protein (PrPd) in Peyer�s patch (PP) lymphoid follicles. In this study, recombinant ovine prion protein (rPrP) was inoculated into gut loops of young lambs and the transportation across the intestinal wall studied. In particular, the immunohistochemical phenotypes of cells bearing the inoculated prion protein were investigated. The rPrP was shown to be transported across the villi of the gut, into the lacteals and submucosal lymphatics, mimicking the transport route of PrPd from scrapie brain inoculum observed in a previous intestinal loop experiment. The cells bearing the inoculated rPrP were mainly mononuclear cells, and multicolor immunofluorescence procedures were used to show that the rPrP bearing cells were professional antigen presenting cells expressing Major histocompatibility complex II (MHCII). In addition, the rPrP bearing cells labeled with CD205, CD11b and the macrophage marker CD68, and not with the dendritic cell markers CD11c and CD209. Others have reported that cells expressing CD205 and CD11b in the absence of CD11c have been shown to induce T cell tolerance or regulatory T cells. Based on this association, it was speculated that the rPrP and by extension PrPd and scrapie infective material may exploit the physiological process of macromolecular uptake across the gut, and that this route of entry may have implications for immune surveillance.
Piercey Akesson, Caroline; Press, Charles McL.; Tranulis, Michael A.; Jeffrey, Martin; Aleksandersen, Mona; Landsverk, Thor; Espenes, Arild
NSDL National Science Digital Library
This problem space introduces basic skills in protein structure exploration, utilizing prions -- relatively small proteins that display dramatically alternate conformations for similar primary structures. We will learn to search databases for protein structures, explore the Cn3D software, and propose questions that may be answered with these tools.
Stephen Everse (University of Vermont College of Medicine;Biochemistry)
Redox behaviors of the neurotoxic portion in human prion protein, HuPrP(106-126)
NASA Astrophysics Data System (ADS)
A peptide fragment of human prion protein, HuPrP(106-126), has been reported to mimic the pathological features underlying prion diseases. Although the actual neurotoxic mechanism of HuPrP(106-126) has not been elucidated, several hypotheses has been proposed based on the role for copper. In this study, to understand the toxic function of HuPrP(106-126) from a viewpoint of electrochemical competence, we investigated redox properties of copper ion complexes with four different binding motifs of a model of HuPrP(106-126) based on density functional theory calculations. We found that the HuPrP(106-126)-derived models exhibited diverse redox activities that depended on copper-binding conformations.
Yamamoto, Norifumi; Kuwata, Kazuo
Double replacement gene targeting for the production of a series of mouse strains with different prion protein gene alterations
We have developed a double replacement gene targeting strategy which enables the production of a series of mouse strains bearing different subtle alterations to endogenous genes. This is a two-step process in which a region of the gene of interest is first replaced with a selectable marker to produce an inactivated allele, which is then re-targeted with a second vector to reconstruct the inactivated allele, concomitantly introducing an engineered mutation. Five independent embryonic stem cell lines have been produced bearing different targeted alterations to the prion protein gene, including one which raises the level of expression. We have constructed mice bearing the codon 101 proline to leucine substitution linked to the human familial prion disease, Gerstmann-Straussler-Scheinker syndrome. We anticipate that this procedure will have applications to the study of human inherited diseases and the development of therapies. 43 refs., 6 figs., 1 tab.
Moore, R.C.; Redhead, N.J.; Selfridge, J. [Univ. of Edinburgh (United Kingdom)] [and others] [Univ. of Edinburgh (United Kingdom); and others
MRS in Early and Presymptomatic Carriers of a Novel Octapeptide Repeat Insertion in the Prion Protein Gene
To evaluate the proton MR spectroscopy (1H MRS) changes in carriers of a novel octapeptide repeat insertion in the Prion Protein Gene (PRNP) and family history of frontotemporal dementia with ataxia. Four at-risk mutation carriers and 13 controls were compared using single voxel, short TE, 1H MRS from the posterior cingulate gyrus. The mutation carriers had an increased choline/creatine, p=0.003 and increased myoinositol/creatine ratio, p=0.003. 1H MRS identified differences in markers of glial activity and choline metabolism in pre- and early symptomatic carriers of a novel PRNP gene octapeptide insertion. These findings expand the possible diagnostic utility of 1H MRS in familial prion disorders.
McDade, Eric M; Boeve, Bradley F.; Fields, Julie A; Kumar, Neeraj; Rademakers, Rosa; Baker, Matt C.; Knopman, David S.; Petersen, Ronald C.; Jack, Clifford R.; Kantarci, Kejal
Crystallization and preliminary X-ray diffraction analysis of a specific VHH domain against mouse prion protein
Prion disorders are infectious diseases that are characterized by the conversion of the cellular prion protein PrPC into the pathogenic isoform PrPSc. Specific antibodies that interact with the cellular prion protein have been shown to inhibit this transition. Recombinant VHHs (variable domain of dromedary heavy-chain antibodies) or nanobodies are single-domain antibodies, making them the smallest antigen-binding fragments. A specific nanobody (Nb_PrP_01) was raised against mouse PrPC. A crystallization condition for this recombinant nanobody was identified using high-throughput screening. The crystals were optimized using streak-seeding and the hanging-drop method. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a�=�30.04, b = 37.15, c = 83.00?�, and diffracted to 1.23?� resolution using synchrotron radiation. The crystal structure of this specific nanobody against PrPC together with the known PrPC structure may help in understanding the PrPC/PrPSc transition mechanism.
Abskharon, Romany N. N.; Soror, Sameh H.; Pardon, Els; El Hassan, Hassan; Legname, Giuseppe; Steyaert, Jan; Wohlkonig, Alexandre
Clinical features of genetic Creutzfeldt-Jakob disease with V180I mutation in the prion protein gene
Objectives Genetic Creutzfeldt-Jakob disease (CJD) due to V180I mutation in the prion protein gene (PRNP) is of great interest because of the differences from sporadic CJD and other genetic prion diseases in terms of clinical features, as well as pathological and biochemical findings. However, few systematic observations about the clinical features in patients with this unique mutation have been published. Therefore, the goal of this study was to relate this mutation to other forms of CJD from a clinical perspective. Design We analysed clinical symptoms, prion protein genetics, biomarkers in cerebrospinal fluid (CSF) and MRI of patients. Participants 186 Japanese patients with the V180I mutation in PRNP. Results Our results indicate that the V180I mutation caused CJD at an older age, with a slower progression and a lower possibility of developing myoclonus, cerebellar, pyramidal signs and visual disturbance compared with classical sporadic CJD with methionine homozygosity at codon 129 of PRNP. Cognitive impairment was the major symptom. Diffuse hyperintensity of the cerebral cortex in diffusion-weighted MRI might be helpful for diagnosis. Owing to the low positivity of PrPSc in the CSF, genetic analysis was often required for a differential diagnosis from slowly progressive dementia. Conclusions We conclude that the V180I mutation in PRNP produces a late-developing and slow-developing, less severe form of CJD, whose lesions are uniquely distributed compared with sporadic and other genetic forms of CJD.
Qina, Temu; Sanjo, Nobuo; Hizume, Masaki; Higuma, Maya; Tomita, Makoto; Atarashi, Ryuichiro; Satoh, Katsuya; Nozaki, Ichiro; Hamaguchi, Tsuyoshi; Nakamura, Yosikazu; Kobayashi, Atsushi; Kitamoto, Tetsuyuki; Murayama, Shigeo; Murai, Hiroyuki; Yamada, Masahito; Mizusawa, Hidehiro
The Prion Protein and Its Paralogue Doppel Affect Calcium Signaling in Chinese Hamster Ovary Cells
The function of the prion protein (PrPc), implicated in transmissible spongiform encephalopathies (TSEs), is largely unknown. We examined the possible influence of PrPc on Ca2+ homeostasis, by analyzing local Ca2+ fluctuations in cells transfected with PrPc and Ca2+-sensitive aequorin chimeras targeted to defined subcellular compartments. In agonist-stimulated cells, the presence of PrPc sharply increases the Ca2+ concentration of subplasma membrane Ca2+ domains, a feature that may explain the impairment of Ca2+-dependent neuronal excitability observed in TSEs. PrPc also limits Ca2+ release from the endoplasmic reticulum and Ca2+ uptake by mitochondria, thus rendering unlikely the triggering of cell death pathways. Instead, cells expressing Doppel, a PrPc paralogue, display opposite effects, which, however, are abolished by the coexpression of PrPc. These findings are consistent with the functional interplay and antagonistic role attributed to the proteins, whereby PrPc protects, and Doppel sensitizes, cells toward stress conditions.
Brini, Marisa; Miuzzo, Manuela; Pierobon, Nicola; Negro, Alessandro; Sorgato, Maria Catia
Neurodegeneration in humans caused by prions.
Prion diseases include kuru, Creutzfeldt-Jakob disease, Gerstmann-Str�ussler-Scheinker disease, and fatal familial insomnia of humans as well as scrapie and bovine spongiform encephalopathy of animals. For many years, the prion diseases were thought to be caused by viruses despite evidence to the contrary. The unique characteristic common to all of these disorders, whether sporadic, dominantly inherited, or acquired by infection, is that they involve aberrant metabolism of the prion protein. In many cases, the cellular prion protein is converted into the scrapie variant by a process after translation that involves a conformational change. Often the human prion diseases are transmissible experimentally to animals, and all of the inherited prion diseases segregate with prion protein gene mutations. Images
Prusiner, S B
Real-time kinetics of discontinuous and highly conformational metal-ion binding sites of prion protein.
The prion protein (PrP) is a metalloprotein with an unstructured region covering residues 60-91 that bind two to six Cu(II) ions cooperatively. Cu can bind to PrP regions C-terminally to the octarepeat region involving residues His111 and/or His96. In addition to Cu(II), PrP binds Zn(II), Mn(II) and Ni(II) with binding constants several orders of magnitudes lower than those determined for Cu. We used for the first time surface plasmon resonance (SPR) analysis to dissect metal binding to specific sites of PrP domains and to determine binding kinetics in real time. A biosensor assay was established to measure the binding of PrP-derived synthetic peptides and recombinant PrP to nitrilotriacetic acid chelated divalent metal ions. We have identified two separate binding regions for binding of Cu to PrP by SPR, one in the octarepeat region and the second provided by His96 and His111, of which His96 is more essential for Cu coordination. The octarepeat region at the N-terminus of PrP increases the affinity for Cu of the full-length protein by a factor of 2, indicating a cooperative effect. Since none of the synthetic peptides covering the octarepeat region bound to Mn and recombinant PrP lacking this sequence were able to bind Mn, we propose a conformational binding site for Mn involving residues 91-230. A novel low-affinity binding site for Co(II) was discovered between PrP residues 104 and 114, with residue His111 being the key amino acid for coordinating Co(II). His111 is essential for Co(II) binding, whereas His96 is more important than His111 for binding of Cu(II). PMID:17345106
Treiber, Carina; Thompsett, Andrew R; Pipkorn, R�diger; Brown, David R; Multhaup, Gerd
Molecular Dynamics Studies on the Structural Stability of Wild-type Dog Prion Protein
Prion diseases such as Creutzfeldt-Jakob disease, variant Creutzfeldt-Jakob diseases, Gerstmann-Str�ussler-Scheinker syndrome, Fatal Familial Insomnia, Kuru in humans, scrapie in sheep, bovine spongiform encephalopathy (or �mad-cow� disease) and chronic wasting disease in cattle are invariably fatal and highly infectious neurodegenerative diseases affecting humans and animals. However, by now there have not been some effective therapeutic approaches to treat all these prion
Jiapu Zhang; David D. W. Liu
Early Host Responses to Prion Infection and Development of an In Vitro Bioassay.
National Technical Information Service (NTIS)
The goal of this project is to identify pathways and networks of genes and proteins perturbed by prion replication. The unusual nature of prion disease prompted a systems approach to identify networks specifically perturbed by prion infections and to dete...
G. A. Carlson L. E. Hood
Early Host Responses to Prion Infection and Development of an In Vitro Bioassay.
National Technical Information Service (NTIS)
The goal of this project is to identify pathways and networks of genes and proteins perturbed by prion replication. The unusual nature of prion disease prompted a systems approach to identify networks specifically perturbed by prion infections and to dete...
G. A. Carison
Prions: Generation and Spread Versus Neurotoxicity
Neurodegenerative diseases are characterized by the aggregation of misfolded proteins in the brain. Among these disorders are the prion diseases, which are transmissible, and in which the misfolded proteins (�prions�) are also the infectious agent. Increasingly, it appears that misfolded proteins in Alzheimer and Parkinson diseases and the tauopathies also propagate in a �prion-like� manner. However, the association between prion formation, spread, and neurotoxicity is not clear. Recently, we showed that in prion disease, protein misfolding leads to neurodegeneration through dysregulation of generic proteostatic mechanisms, specifically, the unfolded protein response. Genetic and pharmacological manipulation of the unfolded protein response was neuroprotective despite continuing prion replication, hence dissociating this from neurotoxicity. The data have clear implications for treatment across the spectrum of these disorders, targeting pathogenic processes downstream of protein misfolding.
Halliday, Mark; Radford, Helois; Mallucci, Giovanna R.
Adsorption of prion and tissue proteins to surgical stainless steel surfaces and the efficacy of decontamination following dry and wet storage conditions.
Iatrogenic transmission of the infectious prion protein (PrP(Sc)) is a potential threat due to its resistance to many chemical and enzymatic decontamination protocols and its strong adhesive properties to stainless steel. The conditions in which surgical instruments are handled during and after surgery may affect the level of tissue protein, prion attachment and the efficacy of subsequent decontamination regimes. This study investigated the adhesion of tissue protein and prion-associated amyloid to surgical stainless steel with respect to time and various storage conditions, and the subsequent outcome on the efficacy of enzymatic cleaning chemistries. Surfaces were contaminated with ME7-infected brain homogenate and left to dry between 0 and 120�min at room temperature or 24�h, in dry or moist conditions. Residual contamination before and after cleaning was visualised using sensitive fluorescent staining and episcopic differential interference contrast/epifluorescence microscopy. Longer drying times increased both protein and prion amyloid adsorption and affected the efficacy of the cleaning chemistries tested. A moist environment post-contamination significantly reduced the attachment of both protein and prion amyloid to the surgical stainless steel surface. Maintaining moist conditions could potentially improve the subsequent decontamination of reusable surgical instruments, also reducing process time and cost. PMID:21658801
Secker, T J; Herv�, R; Keevil, C W
Prion Protein Complexed to N2a Cellular RNAs through Its N-terminal Domain Forms Aggregates and Is Toxic to Murine Neuroblastoma Cells*S?
Conversion of the cellular prion protein (PrPC) into its altered conformation, PrPSc, is believed to be the major cause of prion diseases. Although PrP is the only identified agent for these diseases, there is increasing evidence that other molecules can modulate the conversion. We have found that interaction of PrP with double-stranded DNA leads to a protein with higher ?-sheet content and characteristics similar to those of PrPSc. RNA molecules can also interact with PrP and potentially modulate PrPC to PrPSc conversion or even bind differentially to both PrP isoforms. Here, we investigated the interaction of recombinant murine PrP with synthetic RNA sequences and with total RNA extracted from cultured neuroblastoma cells (N2aRNA). We found that PrP interacts with N2aRNA with nanomolar affinity, aggregates upon this interaction, and forms species partially resistant to proteolysis. RNA does not bind to N-terminal deletion mutants of PrP, indicating that the N-terminal region is important for this process. Cell viability assays showed that only the N2aRNA extract induces PrP-RNA aggregates that can alter the homeostasis of cultured cells. Small RNAs bound to PrP give rise to nontoxic small oligomers. Nuclear magnetic resonance measurements of the PrP-RNA complex revealed structural changes in PrP, but most of its native fold is maintained. These results indicate that there is selectivity in the species generated by interaction with different molecules of RNA. The catalytic effect of RNA on the PrPC?PrPSc conversion depends on the RNA sequence, and small RNA molecules may exert a protective effect.
Gomes, Mariana P. B.; Millen, Thiago A.; Ferreira, Priscila S.; e Silva, Narcisa L. Cunha; Vieira, Tuane C. R. G.; Almeida, Marcius S.; Silva, Jerson L.; Cordeiro, Yraima
Strain-specific kinetics of prion protein formation in vitro and in vivo.
The molecular basis of prion strain diversity is proposed to be encoded by distinct conformations of the abnormal scrapie isoform of the prion protein (PrP(Sc)). PrP(Sc) formation for the hyper (HY) and drowsy (DY) strains of the transmissible mink encephalopathy (TME) agent was investigated using the cell-free PrP conversion reaction to determine the role of distinct PrP(Sc) conformations in the rate of in vitro conversion of cellular PrP into protease-resistant PrP. PrP conversion increased at an exponential rate for both TME strains until peak levels were reached at 72-96 h of reaction time. The amount and rate of PrP conversion for HY TME was greater than those for DY TME between 48 h and the peak level of PrP conversion. Between 96 and 120 h, there was a negative rate of PrP conversion; and between 120 and 168 h, the net rate of HY and DY PrP conversion approached zero. These findings suggest that PrP conversion can occur in three distinct stages: an elongation phase, a depolymerization phase, and a steady-state phase. Strain-specific properties between the TME strains were identified only during the elongation phase. The steady-state phase could be disrupted by the addition of PrP(Sc) to, or by sonication of, the cell-free PrP conversion reaction. These treatments resulted in an increase in the amount of PrP conversion that was equal to or greater than that found during the peak level of PrP conversion for both TME strains, indicating that the steady-state phase was in dynamic equilibrium. In a related study, the rate of accumulation of HY and DY PrP(Sc) in hamster brain exhibited a strain-specific pattern that had similarities to the strain-specific PrP conversion reaction during the elongation phase. These results suggest that strain-specific conformations of PrP(Sc) have the ability to influence the rate of additional PrP(Sc) formation from cellular PrP both in vitro and in vivo. PMID:14573620
Mulcahy, Ellyn R; Bessen, Richard A
Analysis of prion protein aggregates in blood and brain from pre-clinical and clinical BSE cases.
Prion diseases are infectious neurodegenerative diseases affecting humans and animals. The food-borne bovine spongiform encephalopathy (BSE) had serious impact on both economy and public health, respectively. To follow the pathogenesis of BSE, oral challenge studies were previously conducted, among others on the Isle of Riems, Germany (Balkema-Buschmann et al., 2011b). In the present work brain and plasma samples from this pathogenesis study were subjected to surface fluorescence distribution analysis (sFIDA). sFIDA is a diagnostic tool that exploits the aggregated state of the disease-related prion protein (PrP) as a biomarker for prion disorders. With the exception of one animal, all tested brain samples from clinical cattle exhibited a high titer of PrP particles. Moreover we could detect PrP aggregates already 16 and 24 months after infection. In contrast to our previous demonstration of PrP particles in blood plasma from scrapie sheep, however, no aggregates could be identified in plasma from pre-clinical and clinical cattle. This is in accordance with other studies suggesting a restriction of the BSE infection to the central nervous system. PMID:23845735
Bannach, O; Reinartz, E; Henke, F; Dre�en, F; Oelschlegel, A; Kaatz, M; Groschup, M H; Willbold, D; Riesner, D; Birkmann, E
Mutation and polymorphism of the prion protein gene in Libyan Jews with Creutzfeldt-Jakob disease (CJD)
The inherited prion diseases are neurodegenerative disorders which are not only genetic but also transmissible. More than a dozen mutations in the prion protein gene that result in nonconservative amino acid substitutions segregate with the inherited prion diseases including familial Creutzfeldt-Jakob disease (CJD). In Israel, the incidence of CJD is about 1 case/10[sup 4] Libyan Jews. A Lys[sub 200] substitution segregates with CJD and is reported here to be genetically linked to CJD with a lod score of >4.8. Some healthy elderly Lys[sub 200] carriers > age 65 years were identified, suggesting the possibility of incomplete penetrance. In contrast, no linkage was found between the development of familial CJD and a polymorphism encoding either Met[sub 129] or Val[sub 129]. All Libyan Jewish CJD patients with the Lys[sub 200] mutation encode a Met[sub 129] on the mutant allele. Homozygosity for Met[sub 129] did not correlate with age at disease onset or the duration of illness. The frequency of the Met[sub 129] allele was higher in the affected pedigrees than in a control population of Libyan Jews. The frequency of the Met[sub 129] and Val[sub 129] alleles in the control Libyan population was similar to that found in the general Caucasian population. The identification of three Libyan Jews homozygous for the Lys[sub 200] mutation suggests frequent intrafamilial marriages, a custom documented by genealogical investigations. 26 refs., 3 figs., 6 tabs.
Gabizon, R.; Rosenmann, H.; Meiner, Z.; Kahana, I. (Hadassah Univ., Jerusalem (Israel)); Kahana, E. (Barzilai Medical Center, Ashkelon (Israel)); Shugart, Y.; Ott, J. (Columbia Univ., New York, NY (United States)); Prusiner, S.B. (Univ. of California, San Francisco, CA (United States))
Diverse effects on the native ?-sheet of the human prion protein due to disease-associated mutations.
Prion diseases are fatal neurodegenerative disorders that involve the conversion of the normal cellular form of the prion protein (PrP(C)) to a misfolded pathogenic form (PrP(Sc)). There are many genetic mutations of PrP associated with human prion diseases. Three of these point mutations are located at the first strand of the native ?-sheet in human PrP: G131V, S132I, and A133V. To understand the underlying structural and dynamic effects of these disease-causing mutations on the human PrP, we performed molecular dynamics of wild-type and mutated human PrP. The results indicate that the mutations induced different effects but they were all related to misfolding of the native ?-sheet: G131V caused the elongation of the native ?-sheet, A133V disrupted the native ?-sheet, and S132I converted the native ?-sheet to an ?-sheet. The observed changes were due to the reorientation of side chain-side chain interactions upon introducing the mutations. In addition, all mutations impaired a structurally conserved water site at the native ?-sheet. Our work suggests various misfolding pathways for human PrP in response to mutation. PMID:20949975
Chen, Wei; van der Kamp, Marc W; Daggett, Valerie
Prions: pathogenesis and reverse genetics.
Spongiform encephalopathies are a group of infectious neurodegenerative diseases. The infectious agent that causes transmissible spongiform encephalopathies was termed prion by Stanley Prusiner. The prion hypothesis states that the partially protease-resistant and detergent-insoluble prion protein (PrPsc) is identical with the infectious agent, and lacks any detectable nucleic acids. Since the latter discovery, transgenic mice have contributed many important insights into the field of prion biology. The prion protein (PrPc) is encoded by the Prnp gene, and disruption of Prnp leads to resistance to infection by prions. Introduction of mutant PrPc genes into PrPc-deficient mice was used to investigate structure-activity relationships of the PrPc gene with regard to scrapie susceptibility. Ectopic expression of PrPc in PrPc knockout mice proved a useful tool for the identification of host cells competent for prion replication. Finally, the availability of PrPc knockout and transgenic mice overexpressing PrPc allowed selective reconstitution experiments aimed at expressing PrPc in neurografts or in specific populations of hemato- and lymphopoietic cells. The latter studies helped in elucidating some of the mechanisms of prion spread and disease pathogenesis. PMID:11193143
Aguzzi, A; Klein, M A; Montrasio, F; Pekarik, V; Brandner, S; Furukawa, H; K�ser, P; R�ckl, C; Glatzel, M
Overcoming barriers and thresholds - signaling of oligomeric A? through the prion protein to Fyn
Evidence has been mounting for an involvement of the prion protein (PrP) in a molecular pathway assumed to play a critical role in the etiology of Alzheimer disease. A currently popular model sees oligomeric amyloid ? (oA?) peptides bind directly to PrP to emanate a signal that causes activation of the cytoplasmic tyrosine kinase Fyn, an essential player in a cascade of events that ultimately leads to NMDA receptor-mediated excitotoxicity and hyper-phosphorylation of tau. The model does not reveal, however, how extracellular binding of oA? to PrP is communicated across the plasma membrane barrier to affect activation of Fyn. A scenario whereby PrP may adapt a transmembrane topology to affect Fyn activation in the absence of additional partners is currently not supported by evidence. A survey of known candidate PrP interactors leads to a small number of molecules that are known to acquire a transmembrane topology and understood to contribute to Fyn activation. Because multiple signaling pathways converge onto Fyn, a realistic model needs to take into account a reality of Fyn acting as a hub that integrates signals from multiple inhibitory and activating effectors. To clarify the role of PrP in oA?-dependent excitotoxicity, future studies may need to incorporate experimental designs that can probe the contributions of Fyn modulator pathways and rely on analogous readouts, rather than threshold effects, known to underlie excitotoxic signaling.
Evidence for degradation of abnormal prion protein in tissues from sheep with scrapie during composting
This study investigated whether the abnormal prion protein (PrPSc) in tissues from sheep with scrapie would be destroyed by composting. Tissues from sheep naturally infected with scrapie were placed within fiberglass mesh bags and buried in compost piles for 108 d in experiment 1 or 148 d in experiment 2. The temperature in the compost piles rose quickly; it was above 60�C for about 2 wk and then slowly declined to the ambient temperature. Before composting, PrPSc was detected in all the tissues by Western blotting. In experiment 1, PrPSc was not detected after composting in the tissue remnants or the surrounding sawdust. In experiment 2, 1 of 5 specimens tested negative after composting, whereas PrPSc was detected in the other 4 bags, though in reduced amounts compared with those before composting. Tissue weights were reduced during composting. Analysis of the tissue remnants for microbial 16S ribosomal DNA demonstrated that there were more diverse microbes involved in experiment 1 than in experiment 2 and that the guanine and cytosine content of the microbial 16S DNA was higher in the specimens of experiment 1 than in those of experiment 2, which suggests greater dominance of thermophilic microbes in experiment 1. These results indicate that composting may have value as a means for degrading PrPSc in carcasses and other wastes.
Huang, Hongsheng; Spencer, J. Lloyd; Soutyrine, Andrei; Guan, Jeiwen; Rendulich, Jasmine; Balachandran, Aru
Membrane Toxicity of Abnormal Prion Protein in Adrenal Chromaffin Cells of Scrapie Infected Sheep
Transmissible spongiform encephalopathies (TSEs) or prion diseases are associated with accumulations of disease specific PrP (PrPd) in the central nervous system (CNS) and often the lymphoreticular system (LRS). Accumulations have additionally been recorded in other tissues including the peripheral nervous system and adrenal gland. Here we investigate the effect of sheep scrapie on the morphology and the accumulation of PrPd in the adrenal medulla of scrapie affected sheep using light and electron microscopy. Using immunogold electron microscopy, non-fibrillar forms of PrPd were shown to accumulate mainly in association with chromaffin cells, occasional nerve endings and macrophages. PrPd accumulation was associated with distinctive membrane changes of chromaffin cells including increased electron density, abnormal linearity and invaginations. Internalisation of PrPd from the chromaffin cell plasma membrane occurred in association with granule recycling following hormone exocytosis. PrPd accumulation and internalisation from membranes is similarly associated with perturbations of membrane structure and trafficking in CNS neurons and tingible body macrophages of the LRS. These data suggest that a major toxic effect of PrPd is at the level of plasma membranes. However, the precise nature of PrPd-membrane toxicity is tissue and cell specific suggesting that the normal protein may act as a multi-functional scaffolding molecule. We further suggest that the co-localisation of PrPd with exocytic granules of the hormone trafficking system may provide an additional source of infectivity in blood.
McGovern, Gillian; Jeffrey, Martin
Cellular prion protein (PrP(C)) modulates ethanol-induced behavioral adaptive changes in mice.
Chronic consumption of drugs with addictive potential induces profound synaptic changes in the dopaminergic mesocorticolimbic pathway that underlie the long-term behavioral alterations seen in addicted subjects. Thus, exploring modulation systems of dopaminergic function may reveal novel targets to interfere with drug addiction. We recently showed that cellular prion protein (PrP(C)) affects the homeostasis of the dopaminergic system by interfering with dopamine synthesis, content, receptor density and signaling pathways in different brain areas. Here we report that the genetic deletion of PrP(C) modulates ethanol (EtOH)-induced behavioral alterations including the maintenance of drug seeking, voluntary consumption and the development of EtOH tolerance, all pivotal steps in drug addiction. Notably, these behavioral changes were accompanied by a significant depletion of dopamine levels in the prefrontal cortex and reduced dopamine D1 receptors in PrP(C) knockout mice. Furthermore, the pharmacological blockade of dopamine D1 receptors, but not D2 receptors, attenuated the abnormal EtOH consumption in PrP(C) knockout mice. Altogether, these findings provide new evidence that the PrP(C)/dopamine interaction plays a pivotal role in EtOH addictive properties in mice. PMID:24975422
Rial, Daniel; Pandolfo, Pablo; Bitencourt, Rafael M; Pamplona, Fabr�cio A; Moreira, Karin M; Hipolide, D�bora; Dombrowski, Patr�cia A; Da Cunha, Claudio; Walz, Roger; Cunha, Rodrigo A; Takahashi, Reinaldo N; Prediger, Rui D
Copper attachment to prion protein at a non-octarepeat site
NASA Astrophysics Data System (ADS)
Prion protein (PrP) plays a causative role in a group of neurodegenerative diseases, which include "mad cow disease" or its human form variant Creutzfeld-Jacob disease. Normal function of PrP remains unknown, but it is now well established that PrP can efficiently bind copper ions and this ability has been linked to its function. The primary binding sites are located in the so-called octarepeat region located between residues 60-91. While these are by now well characterized, the sites located outside these region remain mostly undetermined. In this work,