text: DSpace Community: http://hdl.handle.net/2381/69 2015-04-02T03:11:21Z Characterization of novel markers of senescence and their prognostic potential in cancer. http://hdl.handle.net/2381/31943 Title: Characterization of novel markers of senescence and their prognostic potential in cancer. Authors: Althubiti, M.; Lezina, L.; Carrera, S.; Jukes-Jones, R.; Giblett, S. M.; Antonov, A.; Barlev, N.; Saldanha, G. S.; Pritchard, C. A.; Cain, K.; Macip, S. Abstract: Cellular senescence is a terminal differentiation state that has been proposed to have a role in both tumour suppression and ageing. This view is supported by the fact that accumulation of senescent cells can be observed in response to oncogenic stress as well as a result of normal organismal ageing. Thus, identifying senescent cells in in vivo and in vitro has an important diagnostic and therapeutic potential. The molecular pathways involved in triggering and/or maintaining the senescent phenotype are not fully understood. As a consequence, the markers currently utilized to detect senescent cells are limited and lack specificity. In order to address this issue, we screened for plasma membrane-associated proteins that are preferentially expressed in senescent cells. We identified 107 proteins that could be potential markers of senescence and validated 10 of them (DEP1, NTAL, EBP50, STX4, VAMP3, ARMX3, B2MG, LANCL1, VPS26A and PLD3). We demonstrated that a combination of these proteins can be used to specifically recognize senescent cells in culture and in tissue samples and we developed a straightforward fluorescence-activated cell sorting-based detection approach using two of them (DEP1 and B2MG). Of note, we found that expression of several of these markers correlated with increased survival in different tumours, especially in breast cancer. Thus, our results could facilitate the study of senescence, define potential new effectors and modulators of this cellular mechanism and provide potential diagnostic and prognostic tools to be used clinically. Description: PMCID: PMC4260747 2015-03-30T11:07:17Z DJ-1 interactions with α-synuclein attenuate aggregation and cellular toxicity in models of Parkinson's disease. http://hdl.handle.net/2381/31942 Title: DJ-1 interactions with α-synuclein attenuate aggregation and cellular toxicity in models of Parkinson's disease. Authors: Zondler, L.; Miller-Fleming, L.; Repici, M.; Gonçalves, S.; Tenreiro, S.; Rosado-Ramos, R.; Betzer, C.; Straatman, K. R.; Jensen, P. H.; Giorgini, F.; Outeiro, T. F. Abstract: Parkinson's disease (PD) is a devastating neurodegenerative disorder characterized by the loss of neurons in the substantia nigra pars compacta and the presence of Lewy bodies in surviving neurons. These intracellular protein inclusions are primarily composed of misfolded α-synuclein (aSyn), which has also been genetically linked to familial and sporadic forms of PD. DJ-1 is a small ubiquitously expressed protein implicated in several pathways associated with PD pathogenesis. Although mutations in the gene encoding DJ-1 lead to familial early-onset PD, the exact mechanisms responsible for its role in PD pathogenesis are still elusive. Previous work has found that DJ-1--which has protein chaperone-like activity--modulates aSyn aggregation. Here, we investigated possible physical interactions between aSyn and DJ-1 and any consequent functional and pathological relevance. We found that DJ-1 interacts directly with aSyn monomers and oligomers in vitro, and that this also occurs in living cells. Notably, several PD-causing mutations in DJ-1 constrain this interaction. In addition, we found that overexpression of DJ-1 reduces aSyn dimerization, whereas mutant forms of DJ-1 impair this process. Finally, we found that human DJ-1 as well as yeast orthologs of DJ-1 reversed aSyn-dependent cellular toxicity in Saccharomyces cerevisiae. Taken together, these data suggest that direct interactions between DJ-1 and aSyn constitute the basis for a neuroprotective mechanism and that familial mutations in DJ-1 may contribute to PD by disrupting these interactions. Description: PMCID: PMC4123098 2015-03-30T11:00:02Z DNA gyrase can cleave short DNA fragments in the presence of quinolone drugs http://hdl.handle.net/2381/31858 Title: DNA gyrase can cleave short DNA fragments in the presence of quinolone drugs Authors: Cove, Matthew E.; Tingey, Andrew P.; Maxwell, Anthony Abstract: We have analysed the DNA cleavage reaction of DNA gyrase using oligonucleotides annealed to a singlestranded M13 derivative containing a preferred gyrase cleavage site. We find that gyrase can cleave duplexes down to ∼20 bp in size in the presence of the quinolone drugs ciprofloxacin and oxolinic acid. Ciprofloxacin shows a variation in its site specificity with an apparent preference for G bases adjacent to the cleavage sites, whereas oxolinic acid stimulates cleavage predominantly at the previously determined site. With either drug, cleavage will not occur within 6 bases from the end of a DNA duplex or a nick. We suggest that cleavage site specificity with short DNA duplexes is determined by drug-DNA interactions whereas with longer fragments the positioning effect of the DNA wrap around gyrase prescribes the site of cleavage. 2015-03-11T15:14:16Z Probing the role of the ATP-operated clamp in the strand-passage reaction of DNA gyrase http://hdl.handle.net/2381/31857 Title: Probing the role of the ATP-operated clamp in the strand-passage reaction of DNA gyrase Authors: Tingey, Andrew P.; Maxwell, Anthony Abstract: The high-resolution structure of the 43 kDa N-terminal fragment of the DNA gyrase B protein shows a large cavity within the protein dimer. The approximate size of this cavity is 20 Å , suggesting it could accommodate a DNA helix. Computer-modelling studies of this cavity suggest that it contains a constriction, reducing the width to ∼13 Å, principally caused by the side chain of Arg286. We have used site-directed mutagenesis to alter this residue to Gln. Gyrase bearing this mutation shows virtually no supercoiling activity and near normal relaxation and DNA cleavage activities. The mutated protein has ATPase activity which cannot be stimulated by DNA. These data support the proposed role of the 43 kDa domain as an ATP-operated clamp which binds DNA during the supercoiling cycle. The lack of DNA-dependent ATPase of the mutant may indicate that binding of DNA within the clamp is a prerequisite for stimulation of the ATPase activity 2015-03-11T14:44:24Z Microtubule association of EML proteins and the EML4-ALK variant 3 oncoprotein require an N-terminal trimerization domain. http://hdl.handle.net/2381/31855 Title: Microtubule association of EML proteins and the EML4-ALK variant 3 oncoprotein require an N-terminal trimerization domain. Authors: Richards, Mark W.; O'Regan, Laura; Roth, D.; Montgomery, Jessica M.; Straube, A.; Fry, Andrew M.; Bayliss, R. Abstract: Proteins of the echinoderm microtubule associated protein-like (EML) family contribute to formation of the mitotic spindle and interphase microtubule (MT) network. EML1-4 consist of WD40 repeats and an N-terminal region containing a putative coiled-coil. Recurrent gene rearrangements in non-small cell lung cancer (NSCLC) fuse EML4 to anaplastic lymphoma kinase (ALK) causing expression of several oncogenic fusion variants. The fusions have constitutive ALK activity due to self-association through the EML4 coiled-coil. We have determined crystal structures of the coiled-coils from EML2 and EML4, which describe the structural basis of both EML self-association and oncogenic EML4-ALK activation. The structures reveal a trimeric oligomerization state directed by a conserved pattern of hydrophobic residues and salt bridges. We show that the trimerization domain (TD) of EML1 is necessary and sufficient for self-association. The TD is also essential for MT binding, however this property requires an adjacent basic region. These observations prompted us to investigate MT association of EML4-ALK and EML1-ABL1 fusions in which variable portions of the EML component are present. Uniquely, EML4-ALK variant 3, which includes the TD and basic region of EML4 but none of the WD40 repeats, was localized to MTs, both when expressed recombinantly and in a patient-derived NSCLC cell line (H2228). This raises the question of whether the mislocalization of ALK activity to MTs might influence downstream signalling and malignant properties of cells. Furthermore, the structure of EML4 TD may enable the development of protein-protein interaction inhibitors targeting the trimerization interface, providing a possible avenue towards therapeutic intervention in EML4-ALK NSCLC. Description: We thank the beamline support staff at DLS I02 and I04 for assistance with data collection and the Centre for Core Biotechnology Services (University of Leicester) for imaging support. 2015-03-11T12:18:43Z TACC3-ch-TOG track the growing tips of microtubules independently of clathrin and Aurora-A phosphorylation. http://hdl.handle.net/2381/31740 Title: TACC3-ch-TOG track the growing tips of microtubules independently of clathrin and Aurora-A phosphorylation. Authors: Gutiérrez-Caballero, C.; Burgess, Selena G.; Bayliss, Richard; Royle, Stephen J. Abstract: The interaction between TACC3 (transforming acidic coiled coil protein 3) and the microtubule polymerase ch-TOG (colonic, hepatic tumor overexpressed gene) is evolutionarily conserved. Loading of TACC3-ch-TOG onto mitotic spindle microtubules requires the phosphorylation of TACC3 by Aurora-A kinase and the subsequent interaction of TACC3 with clathrin to form a microtubule-binding surface. Recent work indicates that TACC3 can track the plus-ends of microtubules and modulate microtubule dynamics in non-dividing cells via its interaction with ch-TOG. Whether there is a pool of TACC3-ch-TOG that is independent of clathrin in human cells, and what is the function of this pool, are open questions. Here, we describe the molecular interaction between TACC3 and ch-TOG that permits TACC3 recruitment to the plus-ends of microtubules. This TACC3-ch-TOG pool is independent of EB1, EB3, Aurora-A phosphorylation and binding to clathrin. We also describe the distinct combinatorial subcellular pools of TACC3, ch-TOG and clathrin. TACC3 is often described as a centrosomal protein, but we show that there is no significant population of TACC3 at centrosomes. The delineation of distinct protein pools reveals a simplified view of how these proteins are organized and controlled by post-translational modification. 2015-02-24T11:55:33Z The structure of C290A:C393A Aurora A provides structural insights into kinase regulation http://hdl.handle.net/2381/31737 Title: The structure of C290A:C393A Aurora A provides structural insights into kinase regulation Authors: Burgess, Selena G.; Bayliss, Richard Abstract: Aurora A is a Ser/Thr protein kinase that functions in cell-cycle regulation and is implicated in cancer development. During mitosis, Aurora A is activated by autophosphorylation on its activation loop at Thr288. The Aurora A catalytic domain (amino acids 122-403) expressed in Escherichia coli autophosphorylates on two activation-loop threonine residues (Thr288 and Thr287), whereas a C290A,C393A double point mutant of the Aurora A catalytic domain autophosphorylates only on Thr288. The structure of the complex of this mutant with ADP and magnesium was determined to 2.1 Å resolution using molecular replacement. This is an improvement on the existing 2.75 Å resolution structure of the equivalent wild-type complex. The structure confirms that single phosphorylation of the activation loop on Thr288 is insufficient to stabilize a `fully active' conformation of the activation loop in the absence of binding to TPX2. Description: PDB reference: 4ceg http://www.rcsb.org/pdb/explore.do?structureId=4ceg 2015-02-24T11:34:32Z Mitotic phosphorylation of SUN1 loosens its connection with the nuclear lamina while the LINC complex remains intact. http://hdl.handle.net/2381/31583 Title: Mitotic phosphorylation of SUN1 loosens its connection with the nuclear lamina while the LINC complex remains intact. Authors: Patel, Jennifer T.; Bottrill, Andrew; Prosser, Suzanna L.; Jayaraman, Sangeetha; Straatman, Kees; Fry, Andrew M.; Shackleton, Sue Abstract: At the onset mitosis in higher eukaryotes, the nuclear envelope (NE) undergoes dramatic deconstruction to allow separation of duplicated chromosomes. Studies have shown that during this process of nuclear envelope breakdown (NEBD), the extensive protein networks of the nuclear lamina are disassembled through phosphorylation of lamins and several inner nuclear membrane (INM) proteins. The LINC complex, composed of SUN and nesprin proteins, is involved in multiple interactions at the NE and plays vital roles in nuclear and cellular mechanics by connecting the nucleus to the cytoskeleton. Here, we show that SUN1, located in the INM, undergoes mitosis-specific phosphorylation on at least 3 sites within its nucleoplasmic N-terminus. We further identify Cdk1 as the kinase responsible for serine 48 and 333 phosphorylation, while serine 138 is phosphorylated by Plk1. In mitotic cells, SUN1 loses its interaction with N-terminal domain binding partners lamin A/C, emerin, and short nesprin-2 isoforms. Furthermore, a triple phosphomimetic SUN1 mutant displays increased solubility and reduced retention at the NE. In contrast, the central LINC complex interaction between the SUN1 C-terminus and the KASH domain of nesprin-2 is maintained during mitosis. Together, these data support a model whereby mitotic phosphorylation of SUN1 disrupts interactions with nucleoplasmic binding partners, promoting disassembly of the nuclear lamina and, potentially, its chromatin interactions. At the same time, our data add to an emerging picture that the core LINC complex plays an active role in NEBD. Description: PMCID: PMC4164488 2015-02-04T13:54:31Z Muscular dystrophy-associated SUN1 and SUN2 variants disrupt nuclear-cytoskeletal connections and myonuclear organization. http://hdl.handle.net/2381/31582 Title: Muscular dystrophy-associated SUN1 and SUN2 variants disrupt nuclear-cytoskeletal connections and myonuclear organization. Authors: Meinke, P.; Mattioli, E.; Haque, Farhana; Antoku, S.; Columbaro, M.; Straatman, Kees R.; Worman, H. J.; Gundersen, G. G.; Lattanzi, G.; Wehnert, M.; Shackleton, Sue Abstract: Proteins of the nuclear envelope (NE) are associated with a range of inherited disorders, most commonly involving muscular dystrophy and cardiomyopathy, as exemplified by Emery-Dreifuss muscular dystrophy (EDMD). EDMD is both genetically and phenotypically variable, and some evidence of modifier genes has been reported. Six genes have so far been linked to EDMD, four encoding proteins associated with the LINC complex that connects the nucleus to the cytoskeleton. However, 50% of patients have no identifiable mutations in these genes. Using a candidate approach, we have identified putative disease-causing variants in the SUN1 and SUN2 genes, also encoding LINC complex components, in patients with EDMD and related myopathies. Our data also suggest that SUN1 and SUN2 can act as disease modifier genes in individuals with co-segregating mutations in other EDMD genes. Five SUN1/SUN2 variants examined impaired rearward nuclear repositioning in fibroblasts, confirming defective LINC complex function in nuclear-cytoskeletal coupling. Furthermore, myotubes from a patient carrying compound heterozygous SUN1 mutations displayed gross defects in myonuclear organization. This was accompanied by loss of recruitment of centrosomal marker, pericentrin, to the NE and impaired microtubule nucleation at the NE, events that are required for correct myonuclear arrangement. These defects were recapitulated in C2C12 myotubes expressing exogenous SUN1 variants, demonstrating a direct link between SUN1 mutation and impairment of nuclear-microtubule coupling and myonuclear positioning. Our findings strongly support an important role for SUN1 and SUN2 in muscle disease pathogenesis and support the hypothesis that defects in the LINC complex contribute to disease pathology through disruption of nuclear-microtubule association, resulting in defective myonuclear positioning. Description: PMCID: PMC4161305 2015-02-04T13:41:32Z Precision medicines for B-cell leukaemias and lymphomas; progress and potential pitfalls. http://hdl.handle.net/2381/31557 Title: Precision medicines for B-cell leukaemias and lymphomas; progress and potential pitfalls. Authors: Dyer, Martin J. S.; Vogler, Meike; Samuel, Jesvin; Jayne, Sandrine; Wagner, Simon; Pritchard, Catrin; Macip, Salvador Abstract: There is now a plethora of new precision medicines for B-cell malignancy including 'classical' kinase inhibitors, rationally designed inhibitors of anti-apoptotic proteins and antibody or antibody drug/toxin conjugates with functional properties. Some are showing spectacular single agent activity in early phase clinical studies and may reduce or, in combination, even obviate the need for chemotherapy. Nevertheless, significant problems remain if these medicines are to be introduced into routine clinical practice in a rational and affordable manner. Firstly, precision medicines must be carefully matched in a mechanistic fashion with specific subtypes of disease. Whilst sensitivity may be predicted by the detection of key mutations or by expression of target molecules, for therapies that depend on intact intracellular signalling pathways, functional assessment on viable primary malignant cells will be necessary using assays that faithfully mimic in vivo conditions. A second, but no less important challenge is to define mechanism-based synergistic combinations associated with minimal toxicities rather than simply adding new precision medicines to existing chemotherapeutic regimens. Finally, a closer, open, two-way interaction between academic medicine and the pharmaceutical industry will be necessary to achieve these aims. Implementing such changes would change radically how and where patients with B-cell malignancies are managed. 2015-02-03T09:29:29Z OFD1 and flotillins are integral components of a ciliary signaling protein complex organized by polycystins in renal epithelia and odontoblasts. http://hdl.handle.net/2381/31494 Title: OFD1 and flotillins are integral components of a ciliary signaling protein complex organized by polycystins in renal epithelia and odontoblasts. Authors: Jerman, S.; Ward, H. H.; Lee, R.; Lopes, Carla A. M.; Fry, Andrew M.; MacDougall, M.; Wandinger-Ness, A. Abstract: Mutation of the X-linked oral-facial-digital syndrome type 1 (OFD1) gene is embryonic lethal in males and results in craniofacial malformations and adult onset polycystic kidney disease in females. While the OFD1 protein localizes to centriolar satellites, centrosomes and basal bodies, its cellular function and how it relates to cystic kidney disease is largely unknown. Here, we demonstrate that OFD1 is assembled into a protein complex that is localized to the primary cilium and contains the epidermal growth factor receptor (EGFR) and domain organizing flotillin proteins. This protein complex, which has similarity to a basolateral adhesion domain formed during cell polarization, also contains the polycystin proteins that when mutant cause autosomal dominant polycystic kidney disease (ADPKD). Importantly, in human ADPKD cells where mutant polycystin-1 fails to localize to cilia, there is a concomitant loss of localization of polycystin-2, OFD1, EGFR and flotillin-1 to cilia. Together, these data suggest that polycystins are necessary for assembly of a novel flotillin-containing ciliary signaling complex and provide a molecular rationale for the common renal pathologies caused by OFD1 and PKD mutations. Description: PMCID: PMC4152239 2015-01-26T15:02:38Z Structure calculation, refinement and validation using CcpNmr Analysis http://hdl.handle.net/2381/31490 Title: Structure calculation, refinement and validation using CcpNmr Analysis Authors: Skinner, Simon P.; Goult, Benjamin T.; Fogh, Rasmus H.; Boucher, W.; Stevens, T. J.; Laue, E. D.; Vuister, Geerten W. Abstract: CcpNmr Analysis provides a streamlined pipeline for both NMR chemical shift assignment and structure determination of biological macromolecules. In addition, it encompasses tools to analyse the many additional experiments that make NMR such a pivotal technique for research into complex biological questions. This report describes how CcpNmr Analysis can seamlessly link together all of the tasks in the NMR structure-determination process. It details each of the stages from generating NMR restraints [distance, dihedral, hydrogen bonds and residual dipolar couplings (RDCs)], exporting these to and subsequently re-importing them from structure-calculation software (such as the programs CYANA or ARIA) and analysing and validating the results obtained from the structure calculation to, ultimately, the streamlined deposition of the completed assignments and the refined ensemble of structures into the PDBe repository. Until recently, such solution-structure determination by NMR has been quite a laborious task, requiring multiple stages and programs. However, with the new enhancements to CcpNmr Analysis described here, this process is now much more intuitive and efficient and less error-prone. 2015-01-26T11:36:17Z Stra6, a retinoic acid-responsive gene, participates in p53-induced apoptosis after DNA damage. http://hdl.handle.net/2381/31424 Title: Stra6, a retinoic acid-responsive gene, participates in p53-induced apoptosis after DNA damage. Authors: Carrera, Samantha; Cuadrado-Castano, S; Samuel, Jesvin; Jones, G. Don; Villar, E; Lee, S W; Macip, Salvador Abstract: Stra6 is the retinoic acid (RA)-inducible gene encoding the cellular receptor for holo-retinol binding protein. This transmembrane protein mediates the internalization of retinol, which then upregulates RA-responsive genes in target cells. Here, we show that Stra6 can be upregulated by DNA damage in a p53-dependent manner, and it has an important role in cell death responses. Stra6 expression induced significant amounts of apoptosis in normal and cancer cells, and it was also able to influence p53-mediated cell fate decisions by turning an initial arrest response into cell death. Moreover, inhibition of Stra6 severely compromised p53-induced apoptosis. We also found that Stra6 induced mitochondria depolarization and accumulation of reactive oxygen species, and that it was present not only at the cellular membrane but also in the cytosol. Finally, we show that these novel functions of Stra6 did not require downstream activation of RA signalling. Our results present a previously unknown link between the RA and p53 pathways and provide a rationale to use retinoids to upregulate Stra6, and thus enhance the tumour suppressor functions of p53. This may have implications for the role of vitamin A metabolites in cancer prevention and treatment. Description: PMCID: PMC3679452 2015-01-14T15:34:43Z The molecular enzymology of cytochromes P450 of biotechnological and medical interest http://hdl.handle.net/2381/29721 Title: The molecular enzymology of cytochromes P450 of biotechnological and medical interest Authors: Warman, Ashley James Abstract: This thesis reports the characterization of two members of a superfamily of enzymes, namely CYP51 from the human pathogen Mycobacterium tuberculosis and CYP116B1 from the soil bacterium Ralstonia metallidurans . CYP51 is one of twenty P450s encoded in the M. tuberculosis genome and has been demonstrated to demethylate sterol compounds. In this study, the spectroscopic and biophysical properties of CYP51 are extensively characterized, focussing on the unusual collapse of its carbon monoxy-ferrous form to a novel, protonated species, and interactions with a proposed physiological redox partner: a ferredoxin protein found adjacent to CYP51 in the M. tuberculosis genome. Additionally, the plausibility of this enzyme as a target for novel anti-tubercular agents, particularly azole-antifungal derivatives, is assessed. The study of CYP116B1 describes initial characterization of a novel fusion of a cytochrome P450 to its flavin mononucleotide- and iron sulphur cluster-containing redox partner protein, which is of potential biotechnological interest due to its proposed role in thiocarbamate herbicide degradation. Assessment of the spectroscopic properties of this enzyme are reported, including electron paramagnetic resonance and resonance Raman investigations of the heme macrocycle, in addition to kinetic studies of the electron transfer capabilities of the reductase component. Furthermore, the search for a substrate for this P450 and subsequent LCMS product analysis assays with the thiocarbamates EPTC and vernolate are detailed. The dissection of CYP116B1 into its constituent domains, in order to facilitate studies of the P450 and reductase components in isolation and further resolve structural, spectroscopic, and thermodynamic properties of this enzyme is also reported. 2014-12-15T10:32:36Z Solution structures of proteins and complexes involved in the regulation of eukaryotic gene expression http://hdl.handle.net/2381/29722 Title: Solution structures of proteins and complexes involved in the regulation of eukaryotic gene expression Authors: Waters, Lorna Catherine Abstract: The initial work focused on the interaction between the general coactivator CBP (SID domain) and members of the p160 family of coactivators (AD1 domain), which is a key step in the activation of transcription by nuclear receptors. The solution structure of the CBP SID / SRC1 AD1 complex described in this thesis shows that the two helical domains are intimately associated, with the first helix in SRC1 AD1 and the first three helices in CBP SID forming a four helix bundle, which is capped by the fourth helix of the AD 1 domain. Comparisons with the structure of the related CBP SID / ACTR AD 1 complex showed that while the CBP SID domain adopts a similar fold in complex with different p160 proteins, the topologies of the AD1 domains are strikingly different, a feature that is likely to contribute to functional specificity of these complexes. The second part of the work described here focused on the interaction between the MA-3 domains of the tumour suppressor Pdcd4 and the translation factor eIF4A, which has been shown to inhibit cap-dependent translation. The C-terminal MA-3 domain (Pdcd4 MA-3C) was shown to consist of three atypical HEAT repeats capped by a final helix. This domain was found to interact with the N-terminal domain of eIF4A through a conserved surface region. The comparison of NMR spectra obtained from Pdcd4 MA-3 C and the tandem MA-3 region strongly suggests that the tandem MA-3 region is composed of two equivalent domains connected by a semi-flexible linker. The high resolution structural information obtained provides important insights into the interactions and functional specificity of the protein complexes studied. 2014-12-15T10:32:36Z The supF assay for understanding DNA adduct-induced mutagenesis : traditional application and development of a site-specific version http://hdl.handle.net/2381/29719 Title: The supF assay for understanding DNA adduct-induced mutagenesis : traditional application and development of a site-specific version Authors: Liapis, Evagelos Abstract: A primary aim of this study was to establish and utilise the supF assay to investigate the mutagenicity of the cancer drug tamoxifen in target endometrial cells. In particular, the supF assay was employed to ascertain the mutations caused by two model reactive intermediates of tamoxifen, alpha-acetoxytamoxifen and 4-hydroxytamoxifen quinone methide (4-OHtamQM) in methylated pSP189 plasmid. The plasmid was methylated in vitro in order to allow application of the LwPy53 algorithm which enables in silico prediction of the G→T mutation distribution along the human p53 gene, using alpha-acetoxytamoxifen and 4-OHtamQM-induced mutation data from the supF assay.;Relative mutation frequencies increased proportionally with the adduct level for alpha-acetoxytamoxifen, up to ∼15 times the background frequency, while 4-OHtamQM failed to raise the mutation frequency above that of the untreated control. The majority of mutations in alpha-acetoxytamoxifen-treated plasmid were GC →TA transversions, while GC→AT transitions predominated in both 4-OHtamQM-treated plasmid and the untreated control. Based on the p53 G→T transversion predictions, alpha-acetoxytamoxifen induced damage resulted in two hotspots at positions 244 and 273; these mutations might be expected to occur in tamoxifen associated endometrial tumours if tamoxifen adducts play a role in cancer development.;The second part of the thesis was devoted to the development and validation of a novel site-specific assay. The assay, which is a site-specific version of the supF assay, was validated and utilized to investigate the mutagenic potential and types of mutations caused by individual O 6-MeG adducts situated in intact double stranded or gapped plasmids, within two different sequence contexts, in both E. coli and human cells. 2014-12-15T10:32:35Z STATs signalling in human bladder cancer : potential chemopreventive role for indoles http://hdl.handle.net/2381/29720 Title: STATs signalling in human bladder cancer : potential chemopreventive role for indoles Authors: Sun, Yiyang Abstract: In patient tissues, STAT1, STAT3and STAT5 were observed in normal bladder epithelium, while expression of phosphorylated proteins was essentially absent. In contrast, phospho-STAT3 in superficial tumours and phospho-STAT1, 3 and 5 in muscle invasive tumours were present. Thus their increased expression, activation and nuclear localisation, may be associated with bladder tumour progression. Three epithelial bladder cancer lines (RT112, RT4, HT1376) had constitutive phospho-STAT5, while three mesenchymal lines (J82, T24, UMUC3) had activated STAT3. Specific knockdown of STAT3 in J82 cells increased STAT1 expression and phosphorylation. STAT1 and/or STAT3 knockdown led to inhibition of cell-matrix adhesion to fibronectin or collagen and suppression of cell migration. The dietary molecules, Indole-3-carbinol (I3C) (≥100microM) or 3, 3'-diindolylmethane (DIM) (≥50microM) induced apoptosis in J82 and RT112 cells by 48 hours. DIM-induced apoptosis in J82 cells was accompanied by downregulation of STAT1, 5a and 5b and inhibition of phospho-STAT1 and phospho-STAT3. In RT112 cells, DIM inhibited STAT5a, 5b and phospho-STAT5, but had no effect on STAT3. DIM (≥10microM) inhibited J82 cell adhesion to fibronectin and collagen, accompanied by decreased N-cadherin, P-cadherin and beta-catenin expression. Furthermore, DIM (1microM) was able to significantly decrease cell adhesion following STAT1 and/or STAT3 knockdown, suggesting inhibition of cell adhesion by DIM may involve the STAT pathway. Phosphorylated STATs may be a potential marker of invasiveness in human bladder cancer. Elucidation of the effects of indoles and DIM in particular, at a molecular level has given an insight as to how these dietary agents may be useful in the therapy and/or prevention of bladder tumours. 2014-12-15T10:32:35Z Validating the Nima-related kinase Nek2 as a novel chemotherapeutic target http://hdl.handle.net/2381/29718 Title: Validating the Nima-related kinase Nek2 as a novel chemotherapeutic target Authors: Hayward, Daniel Abstract: Mitotic division in human cells prompts a dramatic rearrangement of cellular architecture chromosome condensation, nuclear envelope breakdown and bipolar spindle formation facilitates segregation of sister chromatids into daughter cells. Nek2 is a cell cycle-regulated serine/threonine kinase which is implicated in mitotic progression and establishment of a bipolar spindle. Overexpression of Nek2A in cultured human cells has resulted in an increased incidence of spindle abnormalities and aneuploidy, suggestive of defects in chromosome segregation. Currently there is considerable interest in determining whether centrosomal protein kinases that promote mitotic progression, such as Cdk1, Aurora-A, Plk1 and Nek2, are altered in tumour cells, are tumorigenic when upregulated and hence are viable chemotherapeutic targets. In this thesis I demonstrate that levels of Nek2 protein are upregulated in cell lines derived from leukaemia, breast and ovarian cancers. Importantly, I extended this analysis to primary human tumours and found Nek2 protein expression was increased in sixteen of twenty breast tumours studied, the first report of increased Nek2 protein in human neoplastic disease. I next established that the ablation of Nek2 by siRNA in cultured cervical carcinoma cells induced apoptosis. Lastly I present a series of small molecules that inhibit Nek2A in vitro and show the development of two cell-based assays of Nek2A activity, centrosomal disjunction and displacement of the inter-centrosomal component rootletin. Using stable cell lines inducibly expressing Nek2A I demonstrate ablation of Nek2 dependent phenotypes following treatment with a small molecule inhibitor. The ability to inhibit Nek2 in cells coupled with the observation that Nek2 depletion reduces the viability of cultured cells means Nek2 should now be considered a candidate for therapeutic intervention. 2014-12-15T10:32:34Z An investigation into whether telomerase can be used to identify stem cells in the mouse http://hdl.handle.net/2381/29717 Title: An investigation into whether telomerase can be used to identify stem cells in the mouse Authors: Aldridge, Victoria Selena Sarah Abstract: Telomeres are G-rich strands of DNA that protect chromosome ends from recognition as a DNA break. Telomeric DNA addition is catalysed by the reverse transcriptase telomerase which consists of a catalytic component; Tert and an RNA component Terc. In vitro and in vivo studies demonstrated that the primary mode of regulation of telomerase is through transcriptional regulation of the Tert gene. The mTert promoter driving a fluorescent reporter can recapitulate endogenous mTert expression in ES cells in vitro. Telomerase activity is a characteristic of stem cells. However, upon differentiation of stem cells, telomerase activity is switched off; resulting in the progressive shortening of telomeres until they are critically short and cellular senescence is triggered. Given that telomerase activity is determined by Tert promoter activity, it is hypothesised here that the Tert promoter could be used to mark stem cells in the adult.;A vector was generated in which the mTert promoter drives the expression of CFP. In vitro analysis of Tert-CFP ES clones revealed that CFP was expressed in undifferentiated cells but was down-regulated as ES cells were differentiated. Teratomas were generated from ES cells expressing the transgenic vector and CFP expression was found to correlate with regions of undifferentiation and intense proliferation.;Direct DNA microinjection generated four transgenic mouse lines containing the transgene within the germline. RT-PCR for endogenous mTert and CFP showed that their expression varied similarly from two transgenic lines. Histological analysis of testis and skin has shown expression in spermatogonia, basal layer and bulge cells, thus indicating activity of the reporter in these stem cells. However, reporter expression is also evident in spermatocytes of the testis and spinous and granular layers of the skin. Thus, the reporter not only identifies highly proliferative cells, it also identifies cells with a lower proliferative potential. 2014-12-15T10:32:34Z Regulation of gene expression during myocardial ischaemia and reperfusion http://hdl.handle.net/2381/29716 Title: Regulation of gene expression during myocardial ischaemia and reperfusion Authors: Hay, Joanna Abstract: The aim of these studies was to investigate the changes in gene expression that occur in H9c2 myoblast cells subjected to the metabolic stresses associated with myocardial ischaemia/reperfusion. The expression of Vascular Endothelial Growth Factor-A (VEGF-A) and Myocyte Stress 1 protein (ms1) were examined. VEGF-A has a physiological role in stimulating angiogenesis following cardiac ischaemia/reperfusion in vivo, whereas ms1 expression is induced following left ventricular hypertrophy, but has not been examined previously during ischaemia/reperfusion. No increase in VEGF-A, or ms1 mRNA was detected during simulated ischaemia but both were upregulated following reperfusion. The induction in VEGF-A mRNA was caused by an increase in mRNA stability regulated by ERK and JNK pathways. A reporter system was used to demonstrate that simulated ischaemia/reperfusion exerts its effect on both the 5' and 3'UTR of VEGF-A mRNA. The induction in ms1 mRNA was caused by an increase in transcription that was dependent on activation of the JNK pathway. An increase in the amount of ms1 protein expression was also detected during reperfusion. Regulation of translation also influences the expression of proteins during ischaemia/reperfusion. The effect of simulated ischaemia on message translation was investigated using cDNA microarrays. Simulated ischaemia inhibited CAP dependent translation and induced eIF2a phosphorylation. Some messages were more highly represented in the polysome fraction from ischaemic cells, compared to that from resting cells, including FAD oxidoreductase and a sub complex of NADH dehydrogenase, proteins involved in electron transport. 2014-12-15T10:32:33Z To investigate the subcellular localisation of CBP (CREB-binding protein) and its interacting proteins http://hdl.handle.net/2381/29715 Title: To investigate the subcellular localisation of CBP (CREB-binding protein) and its interacting proteins Authors: Ryan, Colm M. Abstract: CBP (Creb-binding Protein) is a histone acetyltransferase that plays an essential role in RNA-polymerase II-dependent transcription.;CBP acetylation of non-histone proteins such as p53, ACTR and Impa has been postulated to regulate their activity. The research described shows the interaction between CBP and the nuclear transport proteins CAS, Impa and the nucleoporin Nup93. We show that increasing global acetylation levels using HDAC inhibitors (HDACi) leads to a perturbation in the subcellular localisation of CAS, while the number of CBP-associated PML bodies increases. In addition, we demonstrate that CBP association with Impa increases in response to HDACi treatment. These results give us a novel insight into the regulation of non-histone proteins by HDACi. In addition, these findings suggest a functional link between chromatin regulation, acetylation and nuclear transport.;Mislocalisation of CBP from PML bodies has been implicated in the pathology of many diseases such as Huntingdon's disease and Acute Myeloid leukaemia expressing the fusion protein MOZ-TIF2. CBP mislocalisation has also been implicated in Kennedy's disease, a late onset neurodegenerative disorder, which expresses a mutant form of the Androgen Receptor (AR) with an expanded polyglutamine region. Reports in the literature suggest that the mislocalisation of CBP in Kennedy's disease may play a crucial role in its pathology. Here we describe experiments, which show that both CBP and PML are mislocalised in cell expressing AR with An expanded polyglutamine tract. Furthermore, we demonstrate that ARQ077-dependent transcriptional repression cannot be rescued by overexpression of CBP in neuronal and non-neuronal cell line. These results shed new light on the potential role of CBP and PML in Kennedy's disease. 2014-12-15T10:32:33Z Mechanistic and structural studies of a novel l-proline dehydrogenase identified from the hyperthermophile pyrococcus furiosus DSM 3638 http://hdl.handle.net/2381/29714 Title: Mechanistic and structural studies of a novel l-proline dehydrogenase identified from the hyperthermophile pyrococcus furiosus DSM 3638 Authors: Monaghan, Phillip James Abstract: The cloning of two open reading frames encoding a heterodimeric protein related in sequence to bacterial sarcosine oxidase and dimethylglycine oxidase from hyperthermophilic Pyrococcus furiosus DSM 3638 is described. the protein was overexpressed in E. coli, purified to homogeneity and identified as a flavoprotein by virtue of the enzymes UV-visible absorption spectrum showing characteristic lambdamax at 367 and 450 nm. The physiological substrate was identified as L-proline (t ½ = ∼105 s-1 for bleaching of flavoprotein spectrum at 450 nm). Additionally, the enzyme oxidises L-pipecolic acid ( t½ = ∼110.5 s-1) and, to a lesser extent sarcosine (t½ = ∼654 s-1). No reactivity with sodium sulfite was detected, consistent with the enzyme belonging to the flavoprotein dehydrogenase class. These data classified the enzyme as a novel hyperthermophilic L-proline dehydrogenase.;The crystal structure of PRODH at 3.3 A resolution shows the enzyme is a heterooctamer (alphabeta)4. The holoenzyme contains one mol each of FAD, FMN and ATP per mol of alphabeta complex. Isolation of monoflavinylated enzyme containing a single FAD cofactor permitted detailed redox potentiometry and pH-dependence studies of the reaction with L-proline. A bell-shaped dependence for kcat/K m as a function of pH was observed with macroscopic pK a values (7.0 +/- 0.2 and 7.6 +/- 0.2) attributed to residue ionisations in the free enzyme. The pH dependence of k cat is sigmoidal, with maximum activity realised in the alkaline region; the dependence is described by a macroscopic pK a value of 7.7 +/- 0.1 and by analogy with other flavoenzymes is tentatively attributed to the ionisation of L-proline in the Michaelis complex. Studies with H225A, H225Q and Y251F mutants ruled out the participation of these residues as catalytic bases. 2014-12-15T10:32:32Z The role of the global co-activator CREB-binding protein in transcription : a study of competitive interactions of diverse nuclear proteins with CBP http://hdl.handle.net/2381/29713 Title: The role of the global co-activator CREB-binding protein in transcription : a study of competitive interactions of diverse nuclear proteins with CBP Authors: Matsuda, Sachiko Abstract: The recruitment of the histone acetyltransferases CREB binding protein (CBP) and its homologue p300, is necessary for chromatin modification and assembly of the Pre Initiation Complex (PIC) at many gene promoters. CBP has multiple protein binding domains and we have focused our studies on the Steroid Receptor Coactivator-1 (SRC1) Interaction Domain (SID) of CBP. A novel conserved sequence motif is described which facilitates the binding of these nuclear proteins such as p160 proteins, Ets2, E1A and p53 to the CBP SID. We have confirmed the interaction of Ets2 by GST pull down with CBP SID and we show that SRC1, Ets2 and E1A full length proteins compete with each other to bind to the SID in vitro. We also show that the short peptide encompassing the SRC1 AD1 sequence is indeed sufficient to compete with SRC1, Ets2, E1A12S and p53 full length proteins on SID. Furthermore, we demonstrate that the transcriptional activity of Ets2 is inhibited by E1A 12S, but not by E1A Delta2-36 (CBP binding site) mutant in vivo. This inhibition by E1A was rescued with overexpression of CBP but not CBPDeltaSID.;In AML, the MOZ-TIF2 fusion protein transforms bone marrow cells in vitro and causes leukaemia in animal model. I have conducted the ChIP analysis of the effects of MOZ-TIF2 on chromatin modification and co-activator recruitment at the RAR beta2 promoter. I have successfully established the ChIP analysis of the RARbeta2 promoter in U20s, and demonstrated that ATRA treatment induces recruitment of RARbeta, CBP, p300 and to the RARbeta2 promoter with concomitant of acetylation of histone H3 K9 and K14 residues. I also have shown that MOZ-TIF2 is recruited to the RARbeta2 promoter and this correlates with the loss of CBP and p300. 2014-12-15T10:32:31Z Regulation of eukaryotic translational initiation in transformed and differentiation inducible haemopoietic cell lines http://hdl.handle.net/2381/29712 Title: Regulation of eukaryotic translational initiation in transformed and differentiation inducible haemopoietic cell lines Authors: Coles, Lucy Clare. Abstract: Mechanisms which regulate gene expression at the level of protein synthesis play an important role in the control of cell proliferation, and de-regulation of translation has been shown to cause cell transformation. Haemopoiesis is a complex and highly regulated process of cell proliferation, differentiation and apoptosis which is subject to a variety of neoplastic disorders. Control of translation is usually effected through modulation of the eukaryotic initiation factors (eIFs) and cultured cells of haemopoietic origin were chosen to study eIFs and their regulation.;The eIFs were examined in cell lines derived from patients with the B-cell tumours Burkitt's lymphoma and Multiple Myeloma. Study of the expression of the proto-oncogene c-myc and its transcriptional targets, eIF4E and eIF2 demonstrated no correlation between the eIFs and c-myc expression. Investigation of another component of the eIF4F complex revealed that eIF4G is cleaved into specific N- and C-terminal fragments in the Multiple Myeloma cell line GM2132. This suggests that in these cells there may be an advantage for mRNAs translated by internal ribosome entry.;HL60, a differentiation inducible leukaemic cell line, was used to investigate alterations in protein synthesis during granulocytic differentiation. A 2-fold increase in eIF4E phosphorylation was observed which does not affect the protein synthesis rate. This implies that the increase in eIF4F phosphorylation may specifically affect the translation of a sub-set of mRNAs. Differentiating HL60 cells were also used to study the relationship between c-myc, eIF4E and eIF2 expression. No correlation between expression of c-myc and eIF4E was found in this system. However, eIF2 expression did appear to be responsive to c-Myc during the initial stages of granulocytic differentiation.;Investigation of a serum response in HL60 cells demonstrated that association of eIF4G with eIF4E is modulated by alterations in eIF4G protein levels. This suggests that eIF4E is not a limiting factor in the initiation of translation in HL60 cells. 2014-12-15T10:32:31Z Phosphorylation site analysis of the centrosomal nek2 kinase and its substrates http://hdl.handle.net/2381/29710 Title: Phosphorylation site analysis of the centrosomal nek2 kinase and its substrates Authors: Baxter, Joanne Elizabeth Abstract: The duplication and separation of centrosomes is controlled in large part by protein phosphorylation. Nek2 is a protein kinase that localises to the centrosomes and has peak activity at the onset of mitosis. The aim of this thesis was to determine how the Nek2 protein is regulated and to identify phosphorylation sites both within the Nek2 kinase and its centrosomal substrates. Identification of a Nek2 phosphorylation concensus sequence should also facilitate the discovery of novel Nek2 substrates. Here, we have mapped eleven Nek2 autophosphorylation sites and identified those which are critical for Nek2 activity. These include sites within the activation loop of the catalytic domain and in the C-terminal non-catalytic domain. We have also identified a region within the Nek2 protein that may act as an auto-inhibitory domain. We also identify Nek2 phosphorylation sites within the centrosomal proteins, N1p and C-Nap1. N1p is localised to the mother centriole and may act as a novel microtubule anchoring protein. We have demonstrated that the localisation of N1p is dependent upon the Nek2 protein kinase. Others have previously identified P1k1 as a kinase involved in N1p regulation and we demonstrate here that Nek2 acts upstream of P1k1 and may be a P1k1 priming kinase. In addition, we identify Cdk1 as a possible P1k1 priming kinase on N1p. C-Nap1 is a protein involved in centriole cohesion and phosphorylation by Nek2 may regulate its interaction with other centriole linker proteins. This work substantially increases our knowledge of Nek2 regulation by phosphorylation and also extends our understanding of how it regulates centrosomes structure and function in a cell cycle dependent manner. 2014-12-15T10:32:31Z Functional analysis of the cytoskeletal protein talin2 using gene disruption http://hdl.handle.net/2381/29711 Title: Functional analysis of the cytoskeletal protein talin2 using gene disruption Authors: El Jai, Yasmine Abstract: Talinl is a large, ubquitously expressed cytoskeletal protein that couples integrins to the actin cytoskeleton. Analysis of genomic and EST databases shows the presence of a second Tin gene encoding a closely related protein (75% identity in human), Tln2. Disruption of both Tlnl alleles in mouse results in early embryonic lethality. Although both talinl and talin2 are very similar in sequence and most probably bind the same ligands, talin2 cannot compensate for the loss of talinl in early mouse development. To further understand the role of talin2, analysis of talin2 expression at the mRNA and protein level in mouse and human has been carried out. A genetrap cell line (BayGenomics) expressing a fusion protein talin2-p-galactosidase has been used to report on talin2 expression. This cell line was used to generate transgenic mice, which allowed the study of talin2 expression at different stages of embryonic development and in various adult mouse tissues. In order to study the role of talin2 in mammalian physiology and ontogeny, a conditional knockout approach based on a Cre-tri/ox system was used to inactivate Tln2 expression in mouse. The successful generation of a Tln2 conditional knockout mouse is reported. 2014-12-15T10:32:31Z The role and regulation of elF2[alpha] phosphorylation in pancreatic [beta]-cells http://hdl.handle.net/2381/29708 Title: The role and regulation of elF2[alpha] phosphorylation in pancreatic [beta]-cells Authors: Powell, Michael L Abstract: Incubation of pancreatic beta-cells or islets with glucose leads to acute increases in the rate of total protein synthesis, which are almost entirely regulated at the post-transcriptional level. However, the metabolic signals and the molecular mechanisms by which glucose stimulates increases in the rate of protein synthesis are poorly understood. In this thesis, I demonstrate that the mitochondrial metabolism of glucose is likely sufficient for glucose-stimulated protein synthesis. However, these increases are not regulated through changes in intracellular ATP content, an autocrine effect of secreted insulin, or by regulation of the activity of AMPK. I also demonstrated that glucose-stimulated protein synthesis in pancreatic beta-cells parallels the dephosphorylation of eIF2alpha and increases in the availability of the translational ternary complex (eIF2.GTP:Met-tRNAi). As with glucose-stimulated protein synthesis, the mitochondrial metabolism of glucose is sufficient for the regulation of eIF2alpha phosphorylation in response to glucose. However, these changes in eIF2alpha phosphorylation do not regulate glucose-stimulated protein synthesis in pancreatic beta-cells, but importantly are required for pancreatic beta-cell survival. I demonstrate that glucose-regulated eIF2alpha phosphorylation is dependent on the eIF2alpha kinase, PERK. Importantly, I show that the activity of PERK and increases in eIF2alpha phosphorylation closely parallel intracellular ATP content, and that ATP depletion activates PERK and stimulates the phosphorylation of eIF2alpha. Therefore, PERK is likely to act as an intracellular ATP sensor in pancreatic beta-cells, regulating the phosphorylation of eIF2alpha in response to changing glucose concentrations. 2014-12-15T10:32:30Z Kinetic and structural studies of wild-type and recombinant aromatic amine dehydrogenase http://hdl.handle.net/2381/29709 Title: Kinetic and structural studies of wild-type and recombinant aromatic amine dehydrogenase Authors: Abu-Khadra, Khalid Mohammad Abstract: TTQ-dependent aromatic amine dehydrogenase (AADH) from Alcaligenes faecalis has been expressed heterologously in Paracoccus denitrificans via the cloning of the aromatic amine dehydrogenase utilizing genes (aau) under the regulation of the mauF promoter, another TTQ-dependent methylamine dehydrogenase (MADH). The recombinant AADH obtained during this study is completely active indicating complete biosynthesis of the TTQ cofactor by restricted cloning of only five genes (aauBEDA and orf-2) of the nine known genes which encode AADH. Chemical and physical characterization as well as kinetic analysis, show that the recombinant AADH expressed in Paracoccus denitrificans is indistinguishable from the native AADH expressed in Alcaligenes faecalis. The catalytic small subunit of AADH has been isolated and studied in the absence of the large subunit. Separation from the holoenzyme was achieved by chemical denaturation. The separation small subunit was refolded, characterized and studied using a number of different kinetic approaches. The small subunit shows a fully integrated and completely redox active TTQ centre. The ability of the separated small subunit to oxidase amine substrate was demonstrated, despite the very slow reaction rates when compared to the holoenzyme. The altered catalytic potency of the TTQ-containing small subunit revealed the significant role of the large subunit in providing the enzyme's catalytic power. Structural X-ray crystallographic studies were performed on crystalline enzyme in an attempt to elucidate further details about the reaction mechanism of enzyme and specifically the mode of C-H bond breakage. Structures of AADH in complex with different para substituted benzylamines were obtained. These structures provide valuable datasets for future studies using computational methods to address specific aspects of the chemistry of TTQ reduction by amine substrates. 2014-12-15T10:32:30Z Characterisation of the binding specificity and functional roles of Mycobacterium tuberculosis complex CFP-10/ESAT-6 family proteins http://hdl.handle.net/2381/29707 Title: Characterisation of the binding specificity and functional roles of Mycobacterium tuberculosis complex CFP-10/ESAT-6 family proteins Authors: Lightbody, Kirsty Lynn Abstract: The secreted Mycobacterium tuberculosis complex proteins CFP-10 and ESAT-6 form a tight, 1:1 heterodimeric complex which is known to play an essential, but as yet undefined, role in tuberculosis pathogenesis. CFP-10 and ESAT-6 are members of a large protein family, including 23 members within the M. tuberculosis genome. Like CFP-10 and ESAT-6 the majority of family members are located in pairs within the genome. Yeast two-hybrid studies reported here show that the genome partners Rv0287/Rv0288 and Rv3019c/Rv3020c also form heterodimeric complexes, suggesting that all genome pairs within the CFP-10/ESAT-6 family are likely to form complexes. Further yeast two-hybrid analysis also revealed that closely related pairs, such as Rv0287/Rv0288 and Rv3019c/Rv3020c are able to bind to non-genome partners, however, distantly related proteins, including CFP-10 and ESAT-6, are unlikely to form complexes with family members other than their genome partner. The ability of some CFP-10/ESAT-6 family proteins to form complexes with non-genome partners greatly increases the number of potential complexes, and may have a significant effect on the functional diversity of this important protein family. The recently determined structure of the CFP-10˙ESAT-6 complex reveals that both proteins adopt an elongated helix-turn-helix hairpin structure and lie antiparallel to each other to form a stable four helix bundle. The surface and structural features of the complex suggest a role in binding one or more target proteins. fluorescence microscopy studies described here have demonstrated specific binding of the CFP-10˙ESAT-6 complex to the surface of monocyte and macrophage cells, suggesting the presence of a specific receptor on host cells and a possible cell role in pathogen-host cell signalling. A striking feature of the complex is the flexible C-terminal region of CFP-10, which was found to be essential for binding to host cells. 2014-12-15T10:32:30Z Signalling via vascular endothelial growth factor receptor complexes http://hdl.handle.net/2381/29705 Title: Signalling via vascular endothelial growth factor receptor complexes Authors: Roberts, Selene Karen Abstract: This study sought to investigate the role of various components of VEGF receptor signalling complexes using fluorescence microscopy to complement conventional biochemical techniques. Using DsRedII-Grp1 to visualise the product of the reaction catalysed by PI3K, activation of PI3K downstream of VEGF receptor-2 is shown. There was no obvious relocalisation of p85 to phosphorylated receptors however a small amount of GFP-p85 associates with, and is phosphorylated in response to, activated VEGF receptor-2. The Shc-related adaptor ShcB/Sck was localised to the plasma membrane in stimulated endothelial cells and associated with activated VEGF receptor-2. PTB and SH2 protein domains, contained within Sck, facilitated these events. Key tyrosine amino acids found within Grb2 binding motifs of ShcA and Sck were phosphorylated in response to VEGF. Tyrosine residues 315 and 316 of Sck were phosphorylated to a greater extent when compared to the corresponding residues of ShcA. Cells expressing Sck proteins lacking tyrosine phosphorylation sites showed a reduced amount of phospho-ERK and DNA synthesis. VEGF receptor-2 was internalised and degraded in response to VEGF. This occurred, at least in part, through the conventional endocytic pathway. VEGF receptor-2 is localised in caveolin-1 and EEA-1 containing vesicles, which is suggestive of internalisation via caveolae and/or early endosomes. Nedd4 co-localises with VEGF receptor-1 at regions of the plasma membrane and this association was confirmed by co-immunoprecipitation. The over-expression of Nedd4 enhanced the degradation of VEGF receptor-1. 2014-12-15T10:32:29Z Fluorescence and transient kinetic analysis of the dictyostelium myosin-II motor domain using green fluorescent protein (GFP) and its variants as probes http://hdl.handle.net/2381/29706 Title: Fluorescence and transient kinetic analysis of the dictyostelium myosin-II motor domain using green fluorescent protein (GFP) and its variants as probes Authors: Wakelin, Stuart Abstract: The determination of the structure of the myosin motor domain has made it possible to introduce fluorescent probes at defined sites, thereby allowing the resolution of the mechanochemical steps in greater detail. Here, modern genetic cloning techniques were utilised to create and express within Dictyostelium discoideum novel myosin-II motor fusion proteins containing various fluorescent probes, in an attempt to investigate conformational changes within the motor domain during the actin-bound stages of the crossbridge cycle. Stopped-flow analysis showed that the myosin ATPase of the single tryptophan myosin-II motor W501 was unaffected by N- and C-terminal YFP and CFP probes, whereas ATP-induced actomyosin dissociation was disrupted (potentially by the probes' propensity to dimerise in close proximity), thus rendering the system unsuitable for investigation of the actin-bound stages of the actomyosin ATPase. By combining total internal reflection microscopy with flash photolysis of an inert caged-ATP precursor, it was shown that kinetic information for the ATP-induced dissociation of fluorescently-labelled myosin motor domains may be achieved using only nanogram quantities, while simultaneously avoiding bundling artefacts common to solution kinetics. A slight adaptation of this process could yield a highly sensitive assay for the processivity of non-classical myosin types, yielding information on their rotational and lateral movement concurrently. Lever arm movement was assessed via the analysis of fluorescence resonance energy transfer (FRET) changes between the YFP (yellow fluorescent protein) and CFP (cyan fluorescent protein) probes. A FRET efficiency increase was observed upon nucleotide occupancy of the active site, in direct contrast to previous FRET studies. Anisotropy studies showed no change upon nucleotide binding, suggesting the FRET increase was due to a distance change, rather than a variation of relative dipole orientations. However, due to the high anisotropy (i.e. slow rotation in solution) of the protein, it was also shown that results from this type of FRET system are qualitative rather than quantitative. 2014-12-15T10:32:29Z Activation of a vinculin-binding site in the talin rod involves re-arrangement of a five helix bundle http://hdl.handle.net/2381/29704 Title: Activation of a vinculin-binding site in the talin rod involves re-arrangement of a five helix bundle Authors: Papagrigoriou, Evangelos Abstract: Talin is a conserved cytoskeletal protein consisting of two subdomains: the talin head and the talin rod. Talin physically connects integrins to the actin cytoskeleton and regulates the inside-out activation of integrins. Binding partners of the talin head include integrins, focal adhesion kinase (FAK) and phosphatidylinositol-4-phosphate 5 kinases type 1 gamma isoform (PIPK1gamma). Along the talin rod, two actin binding sites, one integrin binding site and a minimum of three vinculin-binding sites have so far been mapped.;Vinculin is a cytoskeletal protein, which localises to focal adhesions. It has a molecular weight of 120kDa and consists of two parts: the head and a C-terminal tail. The talin binding site is located in the vinculin head between residues 1-258 (Vh').;In this thesis, the crystal structures of talin rod fragments 482-655 and 482-789 are presented. The structures are classified and compared with structural homologues, demonstrating that the talin rod structural organisation resembles the vinculin tail, alpha-catenin and apolipoproteins A and E. Talin 482-655 is a five helix bundle and talin 656-789 a four helix bundle. All helices are amphipathic and the predicted vinculin-binding site 1 (VBS1) is found buried in the hydrophobic core of the 5-helix bundle of talin 482-655. With supporting evidence from the two structures, mutant binding studies, alanine substitution experiments, NMR spectroscopy, limited proteolysis and the crystal structure of Vh' in complex with talin 605-628, a mechanism involving extensive conformational changes for both talin and vinculin is proposed to be required, in order to achieve vinculin/talin interaction. 2014-12-15T10:32:29Z The P450 bioI redox system. http://hdl.handle.net/2381/29703 Title: The P450 bioI redox system. Authors: Lawson, Rachel Abstract: Cytochrome P450 BioI is isolated from the soil bacterium Bacillus subtilis. It is located in an operon that encodes genes involved in the synthesis of the vitamin biotin. From knock-out studies it was concluded that BioI catalyses an early step in the biosynthetic pathway, forming a precursor to the isolatable intermediate pimelic acid. This thesis covers the characterisation of this P450 and its various endogenous redox partners. The data presented here reports upon the bio-physical properties of the P450 including information on the redox potentials, how its environment affects its properties and the factors that govern substrate binding. To produce a catalytically active BioI redox system, two flavodoxins were cloned from B. subtilis and their over-expression and preparation is described. These flavodoxins have been characterised according to their redox and structural properties. The ability of the two flavodoxins to reconstitute the oxidase activity of BioI was examined by kinetic and spectroscopic techniques culminating in the analysis of the monooxygenation of various fatty acids. To complete the redox chain two NADPH-oxidoreductase candidates and a di-flavin reductase from B. subtilis were assessed and their ability to complement the P450 BioI-flavodoxin system examined. 2014-12-15T10:32:28Z Structural studies of cytochrome P450 BM3 and CPRK http://hdl.handle.net/2381/29702 Title: Structural studies of cytochrome P450 BM3 and CPRK Authors: Joyce, Michael Gordon Abstract: This work presents the crystal structure determination of the transcriptional regulator CprK and of individual domains of the multidomain cytochrome P450-BM3. The crystal structure of the A264E mutant heme domain was determined with and without substrate present. Surprisingly, the structures reveal the protein to exhibit a substrate bound conformation regardless of the presence of substrate. This has provided further evidence that substrate binding leads to a dramatic shift in the equilibrium of conformational states available to the protein. In addition, the crystal structure of the C773A mutant flavin binding domain has been determined both in presence and absence of NADP+. Together with the already available structures of the other domains, this now allows both modelling and further solution studies of the full length cytochrome P450-BM3 structure.;Dehalogenans sp. are capable of using a range of chlorophenolic compounds as terminal electron acceptors in a respiratory metabolism known as halorespiration. This process is under transcriptional control by CprK, a member of the CRP-FNR family of transcriptional regulators. The crystal structure of D. hafniense CprK in complex with o-chlorophenolacetic acid (OCPA) reveals tightly bound effector molecules. Binding of OCPA is analysed through both mutagenesis and fluorescence quenching binding studies. The results have led to the hypothesis that CprK uses the bound phenolic compound pKa as an additional mechanism to sense the presence of the chloride atom. The structure presents a structural framework for further studies of the mechanism of this family of transcriptional regulators and of CprK homologues in particular. 2014-12-15T10:32:28Z Characterisation of the Raf protein kinases by gene targeting http://hdl.handle.net/2381/29700 Title: Characterisation of the Raf protein kinases by gene targeting Authors: Hussain, Jahan Nurany Abstract: The role of B-Raf in cell growth and survival was investigated using primary MEFs lacking B-Raf. Cell growth and proliferation were found to be reduced and this coincided with alterations in the expression levels of various proteins involved in the G1 to S phase of the cell cycle. Analysis of B-raf cells upon serum withdrawal-induced apoptosis showed this was associated with decreased phosphorylation levels of phospho-T125-caspase 9. A B-raf allele containing a conditional kinase inactivating D594A mutation was generated. This was produced by designing a vector containing loxP sites flanking exons 15-18 of B-raf upstream of a second B-raf exon 15 containing the mutation. Upon gene targeting homologous recombination was successfully achieved for three ES clones and these were used to generate chimeric mice. Two of these targeted ES clones led to successful germline transmission. Mice carrying the mutation were mated with Cre-expressing mice, allowing the generation of mice heterozygous for the D594A mutation, B-raf+Lox-D594A. These mice were shown to have neoplasia due to splenomegaly and this was attributed to the elevated phospho-ERK levels observed. Intercrossing of B-raf+/Lox-D594A mice did not generate any viable B-rafLox-D594A/Lox-D594A mice, suggesting the D594A mutation was embryonic lethal. Analysis of B-rafLox-D594A/Lox-D594A MEFs, phospho-MEK and phospho-ERK levels were significantly elevated. This was attributed to increased C-Raf kinase activity observed within these cells. Further analysis indicated increased survival and increased cell growth and proliferation in these cells. 2014-12-15T10:32:27Z A study of c-myc translational regulation in multiple myeloma http://hdl.handle.net/2381/29701 Title: A study of c-myc translational regulation in multiple myeloma Authors: Paulin, Fiona E. M. Abstract: In cell lines derived from patients with multiple myeloma (MM), a B-cell neoplasia, a 10-20 fold elevation in the level of c-myc protein has been observed. This was not accompanied by a concomitant increase in c-myc mRNA levels and there was no alteration in the half life of the protein suggesting that a translational mechanism could be responsible for the elevated c-myc protein levels.;Sequence analysis of c-myc exon 1, a region previously implicated in translational control, revealed no gross sequence abnormalities of the c-myc gene in the MM cell lines. However, in 4 out of 5 MM cell lines examined, a consistent CT transition at position 2756 was observed. This mutation was not seen in any control cell lines or in peripheral blood samples derived from healthy patients implying that the sequence alteration is not merely a polymorphism of this locus and may be associated with the malignant phenotype. Additionally, the MM cell line which did not possess the mutation displayed atypical antigenic expression.;Examination of proteins binding to the c-myc 5'UTR revealed a large number of proteins with this capacity. In addition, the mutant sequence displayed enhanced binding affinity to proteins, specifically polypeptides of 98 and 38 kD. Differences in RNA binding factors were also detected between the control and MM cell lines.;c-myc exon 1 was found to inhibit the translation of heterologous reporter genes in vitro. However, this region was also found to be capable of promoting the internal initiation of ribosomes in dicistronic reporter constructs. Moreover, the mutant sequence displayed an augmented capacity for internal initiation of translation.;Both the mutation and altered protein factors in the multiple myeloma cell lines thus appear to affect the translational efficiency of c-myc mRNA and their combinatorial effects may be sufficient to account for the elevated levels of c-Myc observed. 2014-12-15T10:32:27Z Characterisacion of flavocytochrome P450 BM3 site directed mutants with novel heme ligation state http://hdl.handle.net/2381/29699 Title: Characterisacion of flavocytochrome P450 BM3 site directed mutants with novel heme ligation state Authors: Girvan, Hazel M. Abstract: Uniquely among cytochromes P450, family 4 P450s have a conserved glutamate covalently attaching heme to the protein backbone. In the related Bacillus megaterium P450 BM3, the corresponding residue is alanine (A264). The relevant mutation (A264E) was constructed and characterised spectroscopically, kinetically and structurally. Heme is not covalently ligated in A264E BM3. Instead, E264 coordinates directly to heme iron, generating novel Glu/Cys heme axial ligation. E264 coordination is promoted by fatty acid substrate binding. Both substrate-free and palmitoleate-bound A264E structures were solved and showed that one of two molecules in the substrate-free asymmetric unit had E264 ligation to heme iron, whilst the other had distal water. In all crystal forms, the structure was in a conformation only previously observed in substrate-bound wild-type enzyme. BM3 exists in a conformational equilibrium, and fatty acids bind preferentially to the "substrate-bound" conformation rather than substrate inducing conformational change per se. in light of A264E's novel heme axial ligation state, other A264 mutants were generated. A264H and A264K mutants showed His/Cys and His/Lys ligation. Both mutants showed completely coordinated by the amino acid sidechains in presence or absence of substrates, and were inactive. A264Q and A264M mutants had limited catalytic activity and partial Gln/Cys or Met/Cys ligation, whilst A264C also showed limited activity, and partial Cys/Cys ligation from spectroscopic studies (but none detected in the crystal). Atomic structures were solved for all mutants and spectroscopic analysis provided first characterisation of novel heme iron ligand sets. 2014-12-15T10:32:26Z An investigation of exon repetition http://hdl.handle.net/2381/29697 Title: An investigation of exon repetition Authors: Rigatti, Roberto Abstract: Exon repetition consists in the presence of tandemly repeated exons in mRNA. In all the cases reported so far, the trivial explanation that the phenomenon arises from a duplication of specific exons in the gene was ruled out by southern blot analysis. It is therefore assumed that exon repetition is the result of a post-transcriptional process involving two pre-mRNA molecules transcribed from the same gene. Exon repetition of SA is of particular interest, since it is both tissue and strain-specific. This at first suggested that repetition in SA might be caused by a tissue and strain-specific trans-acting factor. This work has shown that the property of exon repetition is allele-specific in the two best characterised examples, represented by the SA and COT (carnitine octanoyltransferase) genes in rat. Allele-specificity of exon repetition is therefore the underlying cause of strain-specificity of exon repetition in SA and also in COT.;The biological significance of exon repetition remains unknown. One possible function is the generation of new proteins. Quite often though, repetition of the exon that contains the AUG translational start codon creates a short upstream open reading frame (uORF) that precedes the ORF encoding the full length protein. The effect of this has been investigated and the results show that translation of the downstream open reading frame is significantly reduced by the presence of uORFs.;The mechanism that generates exon repetition is not well understood. It was initially proposed for the COT gene that the presence of a putative exonic splicing enhancer (ESE) in exon 2 conferred exon repetition. This work demonstrates that the presence of the ESE is not sufficient to cause exon repetition in-vivo.. 2014-12-15T10:32:25Z Mapping of the domains involved in B-MYB-partner protein interactions and characterisation of their structural properties and features http://hdl.handle.net/2381/29698 Title: Mapping of the domains involved in B-MYB-partner protein interactions and characterisation of their structural properties and features Authors: Dosanjh, Nuvjeevan Singh Abstract: In the work reported here, the minimal sites of interaction between B-Myb and its partner proteins p300 and cyclin D1 were mapped, and the structural properties and features of the interacting regions characterised in detail. A transactivation-associated central region of B-Myb was conclusively shown to bind to a region of p300 corresponding to the TAZ2, zinc ion binding domain. As is common for many transcriptional activation domains, the B-Myb transactivation region is intrinsically disordered in isolation but exhibits coupled folding upon binding p300. The specific B-Myb-p300 complex formed was found to adopt a 1:1 stoichiometry with a measured dissociation constant of less than ~3.0 X 10-8 M. An adjacent region of B-Myb containing part of exon 9a, was also found to interact with the preceeding ZZ region of p300, but substantially weaker than observed for theB-Myb-TAZ2 interaction. These findings clearly show that the conserved cysteine/histidine rich region 3 (C/H3) of p300 provides an extensive binding site for B-Myb.;In parallel work, a large central part of B-Myb containing both the transactivation region and exon 9a was found to show no binding to full-length cyclin D1. Together with previous work, this suggests that residues in the N-terminal DNA binding domain of B-Myb are also involved in cyclin D1 binding. Overall, this work suggests that the inhibition of cooperativity between B-Myb and p300 by cyclin D1 is not due to competition for a common binding site on B-Myb. 2014-12-15T10:32:25Z Protein engineering and mechanistic studies of morphinone reductase http://hdl.handle.net/2381/29694 Title: Protein engineering and mechanistic studies of morphinone reductase Authors: Messiha, Hanan Latif Fahmi Abstract: The thesis describes mechanistic studies of the bacterial protein morphinone reductase (MR) using mutagenesis, kinetic and spectroscopic methods. The roles of seven active site residues were investigated. Cys-191 was initially predicted to be an active site acid; however, mutagenesis studies revealed that Cys-191 does not act in such role. His-186 and Asn-189 were shown to be involved in ligand binding. Mutation of His-186 to alanine has dramatic effects on the enzyme in the steady-state and in the oxidative half-reaction suggesting that His-186 could be an active site acid; however, pH dependence and NMR spectroscopic studies proved that His-186 could not play such role. No charge-transfer complex was observed between the H186A mutant and b-NADH in the reductive half-reaction. Mutation of Asn-189 affects enzyme kinetics and results in a slower rate of flavin oxidation. Faster rates for flavin reduction in the N189A mutant suggest that mutation of Asn-189 favours orientation of the flavin and b-NADH for hydride transfer. Neither Tyr-72 nor Tyr-356 is an active site acid. Mutation of Tyr-72 does not substantially affect enzyme kinetics. Mutations of Tyr-356 and Trp-106 had pronounced affects on enzyme kinetics, suggesting that each mutation alters the active site geometry. In the seven MR mutants studied, the flavin redox potentials are not altered compared to wild-type enzyme except for the T32A mutant where a 50 mV decrease is observed. Mutation of Thr-32 produced enhanced activity in the oxidative half-reaction and a slower activity in the reductive half-reaction effect of copper on MR was investigated. His-186 is shown to be involved in copper binding. 1-Nitrocyclohexene is a potential substrate for MR and the underlying mechanism requires further study. 2014-12-15T10:32:24Z Characterization of NLP, a novel centrosomal substrate of the NEK2 kinase http://hdl.handle.net/2381/29696 Title: Characterization of NLP, a novel centrosomal substrate of the NEK2 kinase Authors: Rapley, Joseph. Abstract: The centrosome is a non-membraneous organelle composed of two centrioles surrounded by pericentrolar material. The primary function of the centrosome is to act as the dominant microtubule organizing centre in animal cells. It therefore contributes to formation of both the interphase cytoskeleton and bipolar mitotic spindle. Both centrosome structure and microtubule organization are controlled in a cell cycle dependant manner by protein phosphorylation. The centrosomal kinase Nek2 regulates centrosome organization, mitotic progression and bipolar spindle assembly. However, the only core centrosomal substrate of this kinase so far identified is C-Napl, a structural protein required for centriolar cohesion. The aims of this project were therefore to isolate a Xenopus laevis homologue of C-Napl in order to study its function using Xenopus based cell free assays and identify and characterize novel centrosomal substrates of Nek2. By database screening, we identified a Xenopus protein of high similarity to C-Napl called rootletin, while using the yeast two hybrid system we identified the Xenopus Nip protein as a novel substrate of Nek2. Human Nip is a recently characterized centrosomal protein involved in microtubule organization and which is regulated by another centrosomal kinase, Plkl. Antibodies were raised to Xenopus Nip and used to confirm subcellular localization to the centrosome in Xenopus cells. Further localization and expression studies revealed that Nip is a mother centriole specific protein that is displaced from the centrosome, but not degraded, during mitosis. These data suggest that Nip is involved in interphasic microtubule anchorage. By transfection into Xenopus and human cells, we found that although Nek2 and Plkl phosphorylate Nip at distinct sites, they can both trigger Nip displacement from the centrosome at the onset of mitosis. Finally, data were obtained raising the possibility that Nek2 may act as a novel priming kinase for recruitment of Plkl to its substrate Nip. 2014-12-15T10:32:24Z Investigations into the cardioprotective properties of resveratrol http://hdl.handle.net/2381/29695 Title: Investigations into the cardioprotective properties of resveratrol Authors: Poolman, Toryn Abstract: In this study the ability of resveratrol to inhibit the activation of the monocyte respiratory burst was investigated.;Differentiated U937 (dU937) cells were pre-treated with resveratrol before stimulation with f-met-leu-phe (fMLP), phorbol 12-myristate-13-acetate (PMA) or arachidonic acid (AA). The extra-cellular and total production of reactive oxygen species (ROS) were measured by isoluminol and luminol chemiluminescence (CL). Superoxide production was measured using lucigenin. Resveratrol was found to inhibit ROS production induced by all three stimuli. Measurement of ROS production was also confirmed using with two used, 2',7'-dichlorofluorescein (DCF) and dihydrorhodamine (DHR). Again resveratrol inhibited both responses. There were significant between the inhibitory effects of resveratrol on peroxidase-dependent (isoluminol, luminol, DCF and DHR) and independent (lucigenin- ROS measuring principles. Moreover, resveratrol was found to be oxidised by the horseradish peroxidase/hydrogen peroxide system.;The cell signal transduction pathways activated by fMLP, PMA and AA were investigated. Only fMLP was found to activate phosphatidylinositol-3-kinase (PI3K) and Akt, using specific inhibitors of both kinases. Resveratrol inhibited PI3K activity with little direct effect on other kinases shown to regulate the respiratory burst, including Akt, extra-cellular regulated protein kinase (ERK); and protein kinase C (PKC). Akt and ERK were found to be activated by fMLP.;In conclusion, resveratrol was found to be a potent inhibitor of ROS production, particularly if a peroxidase-dependent measuring principle was used. Resveratrol can be oxidised by peroxidases, which inhibit the oxidation of the redox probe. Use of a detection method that did not require peroxidase revealed that resveratrol was still a potent inhibitor of fMLP-induced ROS. Moreover, this inhibitory dose of resveratrol correlated with its ability to inhibit the PI3K-Akt pathway, one of the major regulatory pathways of fMLP-induced ROS production. Modulation of these cell signalling intermediates by resveratrol might represent an important anti-inflammatory pathway and further add to its potential cardioprotective properties. 2014-12-15T10:32:24Z Mechanistic studies of the explosive degrading enzyme, pentaerythritol tetranitrate (PETN) reductase http://hdl.handle.net/2381/29693 Title: Mechanistic studies of the explosive degrading enzyme, pentaerythritol tetranitrate (PETN) reductase Authors: Khan, Huma Abstract: PETN reductase is a member of the old yellow enzyme (OYE) family of flavoproteins. The enzyme is able to utilise diverse substrates such as a/b-unsaturated compounds, nitrate esters and nitroaromatic explosives, which makes it of potential interest of phytoremediation purposes. This thesis describes the characterisation of PETN reductase and investigates the role of four active site residues. Trp 102 is shown to influence the binding and transformation of TNT/picric acid. PETN reductase transforms TNT via two competing pathways; (i) nitro group reduction and (ii) direct ring reduction leading to hydride-Meisenheimer complex formation. Mutation of Trp 102 significantly affects the stability of the hydride-Meisenheimer complex, which is sufficient to affect the concentration distribution and/or nature of final TNT degradation products. The ability to produce the TNT hydride-Meisenheimer complex is abolished in the H181A and H184A mutant enzymes indicating that His 181 and His 184 determine the initial route of TNT transformation. These residues are also crucial in ligand binding. Hydride transfer to 2-cyclohexenone and nitrocyclohexene is compromised in the H181A and H184A PETN reductases, owing to the altered mode of substrate binding. Tyr compounds, however, studies presented herein reveal that his is not the case. Trp 102 and Tyr 186 are also shown to play a key role in GTN reduction. Studies on the reductive half-reaction reveal that the rate of NADPH-enzyme complex formation occurs too fast to be measured with Y186F enzyme. Conversely, no complex formation between NADPH and the H181A and H184A mutant enzymes is observed, attributed to the perturbation in NADPH binding, which favours hydride transfer. Collectively, the research described proposes interesting implications into the underlying mechanism of TNT and 2-cyclohexneone reduction by the OYE family of enzymes. 2014-12-15T10:32:23Z Studies on UV-induced DNA damage and repair to human DNA http://hdl.handle.net/2381/29692 Title: Studies on UV-induced DNA damage and repair to human DNA Authors: Karakoula, Aikaterini. Abstract: The induction and repair of DNA damage has been shown to occur heterogeneously throughout the mammalian genome. As a consequence, analysis of these parameters at a global genome level may not reflect important gene-level events. Few techniques have been established to explore quantitatively gene-specific DNA damage and repair. Most of these are PCR-based assays and are relatively insensitive, relying on decreased PCR amplification arising from damage in template DNA. In this study, a quantitative assay that combines specific immunocapture of damaged DNA by an antiserum specific for thymine dimers (IgG479), with PCR amplification of a 149bp fragment of the human H-ras proto-oncogene was established. Quantification of DNA damage was based upon proportionality between the amount of the PCR product and the initial amount of damage. Detection of thymine dimers was possible with nanogram amounts of genomic DNA and increased in a linear, dose-responsive manner. Using this assay, gene-level induction of thymine dimers was shown to be directly proportional to levels induced in the global genome of UVC-and-UVB-irradiated genomic DNA, as measured by gas chromatography-mass spectrometry and enzyme-linked immunosorbent assay, respectively. This result suggests that global damage assessments do indeed reflect gene-level events. However, preferential repair of UVB-induced T<>T from the human H-ras proto-oncogene than the overall genome of human keratinocyte cells was detected. These findings demonstrate the suitability of this approach to the detection of UVR-induced DNA damage and repair at the level of individual genes. 2014-12-15T10:32:23Z Multiple ligand binding and kinetic isotope effects in methanol dehydrogenase http://hdl.handle.net/2381/29691 Title: Multiple ligand binding and kinetic isotope effects in methanol dehydrogenase Authors: Hothi, Parvinder Abstract: The reaction of PQQ-dependent methanol dehydrogenase (MDH) has been studies by steady-state and stopped-flow kinetic methods, with particular reference to multiple ligand binding and kinetic isotope effects (KIEs). Phenazine ethosulphate (PES; an artificial electron acceptor) and cyanide (a suppressant of endogenous activity), but not ammonium (an activator of MDH), compete for binding at the catalytic methanol-binding site. MDH activity is dependent on activation by ammonium, bit is inhibited at high ammonium concentrations. Glycine ethyl ester (GEE; an alternative activator) does not inhibit enzyme activity to the same degree as ammonium. Data suggest a close spatial relationship between the Ks and Ki activator-binding sites such that binding of GEE to the Ks site sterically impairs binding of GEE to the Ki site. Cyanide is less effective at suppressing endogenous activity with GEE, which is attributed to impaired binding of cyanide at the catalytic site through steric interaction with GEE at the Ks site. Combined, this data suggests that the catalytic methanol-binding site and the K s and Ki sites are in close proximity. PES and substrate influence the stimulatory and inhibitory effects of activator through competitive binding, giving rise to unusual KIEs as a function of activator and PES concentration. The KIEs are independent of temperature, consistent with the proposed competitive binding model. Stopped-flow studies indicate that at high ammonium concentrations, the oxidative half-reaction becomes more rate-limiting. With GEE, the oxidative half-reaction makes a larger contribution to rate limitation, stopped-flow method has enabled study of the reductive half-reaction of MDH. A kinetic mechanism for the reaction cycle of MDH is proposed. Furthermore, this study emphasises the need for caution in using KIEs, and the temperature dependence of KIEs, as a probe for enzymatic hydrogen tunnelling. 2014-12-15T10:32:22Z Subcellular distribution and funciton of the neuronal JNK signalling pathway scaffold protein JIP3 http://hdl.handle.net/2381/29689 Title: Subcellular distribution and funciton of the neuronal JNK signalling pathway scaffold protein JIP3 Authors: Caswell, Patrick Tomas Abstract: The JNKs have been shown to regulate cellular processes ranging from apoptosis to differentiation and survival, depending on cell-tissue type and cell context. JIP3 was identified as a putative scaffold protein functioning in the JNK signalling pathway. JIP3 is predominantly expressed in neurons, and is thought to have a role in neuronal vesicular transport. The function of JIP3 in the context of JNK signalling has, however, not been established.;A polyclonal JIP3 antiserum was generated in the course of this study, which was suitable for western blotting, immunprecipitation and indirect immunofluorescence microscopy experiments. This antiserum showed greater efficacy than any other available antisera, and was essential in subsequent experiments.;Biochemical fractionation and immunofluorescence microscopy experiments identified the association of endogenous JIP3 with an unconventional vesicular species. The majority of JIP3 was found to localise predominantly in growth cone regions of differentiated N1E-115 and PC-12 cells. In these growth cone regions, significant co-localisation was identified between JIP3 and JNK pathway components, but was not apparent in other subcellular compartments. Immunoprecipitation experiments showed increased association of JIP3 with JNK pathway components upon differentiation of PC-12 cells.;The proposed function of the JNK pathway in neurite outgrowth led to the investigation of the potential for JIP3 regulation of this neuronal differentiation process. Overexpression of JIP3, which acts as a partial inhibitor of JNK pathway signalling, was found to significantly suppress neurite outgrowth in N1E-115 and PC-12 cells. Moreover, re-constitution of a JIP3 mediated JNK signalling module was sufficient to induce neurite outgrowth in PC-12 cells.;Finally, JIP3 co-localised with the cytoskeletal regulator paxillin specifically within growth cone regions of differentiated PC-12 cells. This suggests that JIP3 may direct JNK pathway signalling towards a growth cone associated substrate, paxillin, and thus regulate neurite outgrowth. 2014-12-15T10:32:21Z The proteasome and its regulators http://hdl.handle.net/2381/29690 Title: The proteasome and its regulators Authors: Murray, Rachael Zoe. Abstract: The proteasome, a multisubunit high molecular weight complex, is involved in lysosomal degradation. A number of endogenous cellular proteins that modulate proteasome function have been identified, such as the regulatory complex of the 26S proteinase and PA28. The 26S proteinase has been implicated in both the ubiquitin-dependent and - independent pathway of protein degradation, while PA28, along with proteasomes containing the -IFN inducible subunits LMP2 and LMP7, has been implicated in antigen presentation. In this study 26S protinease has been purified to homogeneity from rat liver and subunit specific antibodies produced. A comparison of the 20S and 26S proteasomes showed significant differences in the rate of peptide substrate hydrolysis. Mass spectrometry and immunoblot analysis have identified many of the 26S proteinase subunits separated by two dimensional PAGE. Using electron microscopy subunits S4 and S12 have been found to be situated on the ends of the 26S proteinase.;The regulatory subunits S3, P45, S10 and S12 have been found to exist predominantly as part of the 26S proteinase in rat liver, whereas TBP1 was found to exist freely in the cell and as part of a complex other than 26S proteinase. Subunit specific antibodies have been used to study the levels of 26S proteinase in various rat tissues, the lowest level being found in muscle and the highest in thymus. Localisation studies suggest that subcomplexes of the 26S proteinase exist. These differences may reflect different functions of the complex. Although proteasomes have been found in the nucleus and the cytoplasm, the LMP2 and LMP7 containing proteasomes were found to be associated with the ER further implicating them in antigen presentation. PA28 was found to be localised in the cytoplasm. 2014-12-15T10:32:21Z Preparaion and characterisation of P450 3A4 http://hdl.handle.net/2381/29688 Title: Preparaion and characterisation of P450 3A4 Authors: Ward, Richard James Abstract: This study demonstrates that highly pure P450 3A4 can be prepared utilising either of the detergents Emulgen 911 (E911) or CHAPS as the solubilising agent, and that CHAPS, a steroidal detergent, binds to P450 3A4 but is less inhibitory than E911, most likely because of its low affinity. In addition this work highlights, for the first time, that CHAPS-solubilised P450 3A4 is not a homogeneous preparation, and is in fact a mixture of oligomers, the size of the oligomeric state being affected by the amount of detergent used to purify the enzyme. Despite this aggregation, however, the preparations of P450 3A4 were shown to be active with respect to binding carbon monoxide in the reduced for, oxidising a profluorescent substrate (7-BQ), and producing an optical spin state change upon substrate binding. Optical binding studies have suggested a correlation between a compound's affinity for P450 3A4 and its solubility, i.e. the more soluble the substrate the weaker it will bind to this protein, as has been suggested in the past. These studies have also indicated that, (i) the affinity of a substrate may be enhanced by the presence of a nitrogen that can coordinate to the iron, (ii) the active site is unlikely to be merely a large cavity where substrates can bind, (iii) the cooperative behaviour of testosterone and amitriptyline with P450 3A4 is dependent upon the buffer conditions used and (iv) one or more substrate molecules may be interacting with this protein at once, and at distinct binding sites, although the nature of these sites cannot be determined. In addition, NMR relaxation studies were carried out in an attempt to determine haem iron to substrate proton distances for P450 3A4. However, it was not possible to achieve this because the systems under investigation were all in slow/intermediate exchange on the NMR time scale, possibly because the protein samples were aggregated. 2014-12-15T10:32:20Z The leukaemogenic fusion protein MOZ-TIF2 inhibits nuclear receptor-mediated transcription and mislocalises CBP http://hdl.handle.net/2381/29687 Title: The leukaemogenic fusion protein MOZ-TIF2 inhibits nuclear receptor-mediated transcription and mislocalises CBP Authors: Troke, Philip J. F. Abstract: Chromosomal rearrangements associated with the M4/M5 subtype of Acute Myeloid Leukaemia include fusions of the gene encoding the histone acetyltransferase (HAT) MOZ with genes encoding the transcription factor coactivators TIF2, CBP or p300. The physiological roles of MOZ in vivo remain to be established. However, TIF2 functions to recruit the HATs CBP/p300 to ligand-bound Nuclear Receptors (NR), thus leading to transcription activation. Studies investigating the role of NRs and their ligands in haematopoiesis suggest that they aid the regulation of myeloid cell differentiation. The research described in this thesis therefore investigated the effect of the MOZ-TIF2 fusion protein on NR-mediated transcription. Expression of MOZ-TIF2 resulted in an inhibition of ligand-dependent transcription by the Retinoid X Receptor a, Retinoic Acid Receptor a and Estrogen Receptor a in the C0S1 cell line. Further studies showed that MOZ-TIF2 also inhibited transcription mediated by endogenous NRs in the haematopoietic cell line U937. Investigation of protein interactions demonstrated that MOZ-TIF2 bound CBP through its Activation Domain 1 (AD1) and that deletion of this region resulted in the inability of the protein to inhibit NR transcription. Immunofluorescence analysis showed that MOZ-TIF2 formed a mesh-like nuclear pattern in contrast to the speckled nuclear localisations of MOZ, TIF2 and CBP. In addition, in cells expressing MOZ-TIF2, but not the AD1 deletion mutant, endogenous CBP displayed a diffuse staining pattern. Thus, the experiments described here show that MOZ-TIF2 inhibits NR-mediated transcription and mislocalises CBP, and that both of these activities are dependent upon its AD1. Further experiments investigating the effect of MOZ-TIF2 on p53 and AML1 -mediated transcription have indicated that MOZ-TIF2 also has the ability to affect the function of transcription factors in addition to NRs. Thus, expression of the MOZ-TIF2 fusion protein may result in the formation of leukaemia through the misregulation of normal haematopoietic transcription. 2014-12-15T10:32:19Z Structural, functional and mechanistic analysis of the Bag-1 internal ribosome entry site http://hdl.handle.net/2381/29686 Title: Structural, functional and mechanistic analysis of the Bag-1 internal ribosome entry site Authors: Pickering, Becky M. Abstract: Bag-1 is an anti-apoptotic protein involved in the regulation of a number of cellular processes, notably as a co-chaperone for the 70kDa heat shock proteins. At least four protein products of Bag-1 have been isolated, p50, p46, p36 and a minor isoform, p29. The 5' UTR of the p36 isoform of Bag-1 has been shown to contain an internal ribosome entry segment (IRES). The internal ribosome entry mediated mechanism of translation has been shown to maintain Bag-1 expression when cap- dependent translation is compromised during heat shock. Many IRESes require trans-acting protein factors for optimal IRES activity. Bag-1 IRES activity is cell-type specific and is inefficient in cell lines with low endogenous levels of the /r<ms-acting factors poly (rC) binding protein 1 (PCBP1) and polypyrimidine tract binding protein 1 (PTB1). Activity of the Bag-1 IRES can be stimulated in vitro and in vivo by overexpression of PTB and PCBP1. PTB and PCBP1 bind specifically to the minimal active Bag-1 IRES element. A secondary structural model of the minimal Bag-1 IRES was obtained by chemical and enzymatic probing of IRES RNA in vitro. Addition of PTB and PCBP1 modulates the secondary structure of the Bag-1 IRES in the ribosome-landing region. Overexpression of Bag-1 proteins in cells subjected to genotoxic stress has been shown to protect cells from stress-induced growth inhibition and cell death. The Bag-1 IRES is functional in heat-shocked cells and cells treated with chemotherapeutic agents and this correlates with a redistribution of PTB and PCBP1 from the nucleus of the cell to the cytoplasm. A model for the mechanism of action of the Bag-1 IRES and the influence of PTB and PCBP1 is proposed. 2014-12-15T10:32:19Z