Biochemistry and Pharmacology - Theses

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    Profiling the Lipidome Hallmarks of Colorectal Cancer
    Rustam, Yepy Hardi ( 2021)
    Altered lipid metabolism is associated with carcinogenesis and metastatic capacity in human cancers, including colorectal cancer (CRC). Elucidating the composition of dysregulated lipids is therefore essential to understanding CRC biology, to identify diagnostic or prognostic biomarkers, and to highlight novel therapeutic targets for CRC. Here, a mass spectrometric and bioinformatic analysis strategy was developed and employed for comprehensive lipidome analysis of a series of established CRC cell lines, and primary tumour tissues. A lipidomic analysis workflow employing ultra-high resolution and accurate mass spectrometry (UHRAMS) was developed and optimised to enable high-throughput lipid identification and semi-quantitative analysis at the 'sum composition' level from both CRC cell lines and clinical tissue samples. In addition, an ultraviolet photodissociation tandem mass spectrometry (UVPD-MS/MS) fragmentation technique was developed to localise double bond (unsaturation) position(s) in unsaturated fatty acids and fatty acyl chain containing lipid species. Finally, in order to facilitate robust lipid identification, new software and analytical tools were developed to perform lipidome identification, quantification, statistical analysis, and data visualisation. Systematic lipidomic profiling of 43 CRC cell lines revealed a highly heterogeneous profile of 755 lipid species across four categories, 14 classes, and 36 subclasses. Statistical analyses, including differential abundance analysis and hierarchical clustering, were performed to test for associations of lipidome profiles with molecular and histopathological CRC subtypes, and mutation status of major CRC driver genes including APC, KRAS, BRAF and PIK3CA. Lipidome profiles at the 'sum composition' level were found to be significantly associated with both microsatellite instability (MSI) status and cancer grade. Detailed characterisation of lipid structure revealed additional lipidome remodelling at the fatty acyl chain level including alterations in the chain length and unsaturation, and the isomeric compositions of 18:1(n-7) and 18:1(n-9) fatty acyl containing lipids as a function of CRC subtypes. However, no associations were identified between lipidome profiles and cancer gene mutations after adjustment for cell line MSI status, although power to detect such relationships was limited given our sample size. To further examine the potential correlation between lipid metabolism and cancer gene mutation status, the lipidomes of isogenic DLD1 and HCT116 cell lines with KRAS mutant (pro-oncogenic activation) or KRAS wildtype (pro-oncogenic deactivation) genotypes were investigated. For both sets of isogenic CRC cell lines, significant alteration in lipid composition across multiple classes and subclasses were observed for KRAS mutated cells as compared to wildtype cells. However, the patterns of lipidome deregulation between the two cell lines were inverse, suggesting that these were not a direct output related to oncogenic KRAS, but rather a function of differential cellular responses. Multi-omic comparisons were also performed to investigate the correlation between lipidome and mRNA levels of each of the CRC cell lines. A significant positive correlation was identified between the levels of triacylglycerides (TG) and Diacylglycerol O-Acyltransferase 2 (DGAT2) transcript, suggesting a potential functional relationship. Moreover, the changes in transcript levels in the DLD1 and HCT116 isogenic cell lines were also reflected in the lipidome profiles alteration due to KRAS mutation. The established lipidomic analysis workflow was then applied to perform a pilot translational study examining a series of 32 CRC tumour and a subset of 11 patient matched normal tissue samples. These results revealed distinct lipidomic profiles that distinguished tumour from normal tissues, including lipid species within GL, GP, SP and ST categories. Differences in lipid profiles between tumour and normal tissues are not only observed at the lipid class or subclass level, but also in the fatty acyl chain properties (i.e., chain length and unsaturation). Analysis of a larger number of patient samples will be required to validate and expand these findings. A comparison of lipidome profiles between CRC tumour tissues and cell lines relative to normal tissues revealed that CRC cell lines broadly reflect the lipidome patterns of the primary cancers. This study is the first to provide a systematic and comprehensive overview of the lipidomic landscape of CRC across a wide range of commonly used CRC cell lines, providing novel insights into the role of aberrant lipid metabolism on CRC biology. The pilot lipidomic study on CRC patient tissue samples establishes the foundation for the discovery of lipidomics-based diagnostic and prognostic biomarkers, and potential novel therapeutic targets for the disease.
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    Probing the immunopeptidome: enhanced epitope discovery through sHLA technology and bioinformatics
    Scull, Katherine Elise ( 2018)
    Human leukocyte antigen (HLA) molecules are cell-surface glycoproteins that present peptides, derived from diverse protein antigens, for surveillance by T lymphocytes. This immunosurveillance seeks signs of disease or abnormality, facilitating the eradication of infected or malignant cells. Collectively, the vast array of HLA-bound peptides (including immunogenic epitopes) is termed the immunopeptidome. Many research groups seek to identify the peptides which comprise the immunopeptidome of particular HLA allomorphs or cell types using tandem mass spectrometry. However, the nature of the immunopeptidome presents particular challenges for such discovery studies; the peptides are not only hugely diverse both qualitatively and quantitatively, but their complex biological origins render standard proteomic methodologies problematic. For example, conventional analysis software typically identify peptides by matching tandem mass spectra with sequences from genome-based protein databases, but HLA-bound peptides can have sequences not found in such databases, causing the software to ignore or misidentify such peptides. My thesis aims to facilitate epitope discovery in two ways. Firstly, I investigated an existing experimental technique, secreted HLA (sHLA) technology, in which a secreted form of a chosen HLA allele is transfected into cells, allowing straightforward and specific purification of the HLA allotype of choice. I validated the use of sHLA technology by showing that sHLA resembles natural membrane-bound HLA in terms of the molecules’ maturation kinetics and peptide repertoires. Secondly, I used computational methods and novel bioinformatics to aid immunopeptidomics studies. This involved the development and implementation of several computer programs. The major program is ‘Mmers’, which aids identification of peptides with non-standard sequences, in addition to those with sequences found in standard genome-based protein databases. Mmers generates comprehensive ‘artificial databases’ which include all of the possible permutations of amino acids for peptides of a given length, then searches spectra using Mascot-based scoring. To test Mmers, I obtained tandem mass spectrometric data from complex SW480 and SW620 colon cancer immunopeptidome samples, and searched for peptides of 8-11 amino acids in length using Mmers, alongside conventional software. I showed that Mmers can identify many sequences in agreement with Mascot, ProteinPilot and PEAKS DB. Furthermore, despite statistical challenges necessitating more manual inspection than desired, Mmers allowed me to identify four novel peptides with non-standard sequences, which I validated in comparison to synthetic peptides by MRM. I wrote two minor programs, Shifty and Spliceprot, to start investigating the possible biological origins of the four peptides. I found that two of the peptides could derive from unusual transcription or translation from the WAPL oncogene and the PTEN tumour suppressor gene, respectively. In conclusion, my thesis confronts the challenging complexity of the immunopeptidome, and seeks to provide novel tools to help researchers understand it more fully, with potential benefits for developing cancer immunotherapies and vaccines, and for determining the causes of autoimmune disease.
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    High-content screening of antimalarial drugs using metabolomic approaches
    Chua, Hwa Huat ( 2016)
    Malaria continues to have an appalling health and economic impact on many of the poorest countries in the world, with more than 214 million cases and 438,000 deaths a year. While considerable progress has been made in reducing the overall impact of malaria, the emergence of clinical resistance to current first-line antimalarial drugs threatens to roll back these advances. The reliance on drug treatments is further exacerbated by the limited efficacy of recent vaccine trials. Malaria is caused by mosquito-transmitted protists belonging to the genus Plasmodium. Of these, P. falciparum is the cause of the most serious form of malaria. In order to identify new lead compounds, a number of high-throughput live cell-based screens using P. falciparum have been undertaken with millions of compounds derived from the GlaxoSmithKline (GSK), St. Jude Children's Research Hospital, and Novartis compound libraries. These screens identified thousands of new drug-like compounds which selectively kill asexual intraerythrocytic stages of P. falciparum. In order to promote research on the modes of action of these compounds the Medicines for Malaria Venture (MMV), assembled a subset of 400 compounds into the Malaria Box which has been made freely available to the research community for further characterisation. The major goal of this study was to develop a generic metabolomic approach for investigating the mode of action of the Malaria Box compound library and other lead compounds identified in live-cell screens. A high content metabolomics screen was developed to allow measurement of several hundred intracellular metabolites using gas chromatography-mass spectrometry (GC-MS). GC-MS was chosen as it allows highly reproducible coverage of many metabolites in parasite central carbon metabolism. Initial studies demonstrated that GC-MS profiling could readily differentiate between uninfected red blood cell (uRBC) cultures and P. falciparum infected RBC (iRBC) cultures (at 10% parasitaemia) without fractionation of the infected cultures (i.e. separation of infected RBC from 90% uninfected RBC). A screen was subsequently developed in which synchronised P. falciparum-infected RBC cultures in a 24-well plate format was treated with drugs of interest (1 µM concentration) for 12 hours when they reached mid-trophozoite stage (32-hour post-invasion). Extensive method development resulted in an optimised protocol for rapid quenching of parasite/RBC metabolism and metabolite extraction. This screen was validated using metabolic inhibitors with known modes of action, as well as several front-line antimalarial drugs. Atovaquone, an antimalarial that is thought to primarily target the cytochrome bc1 complex in P. falciparum asexual erythrocytic stages was found to induce changes in a limited number of mitochondrial metabolites including fumarate and γ-aminobutyric acid (GABA). In contrast, drugs such as artemisinin and chloroquine that kills the parasites induced changes in a large number of metabolites across many metabolic pathways. This screen was subsequently used to profile the impact of the 80 most potent Malaria Box compounds on the metabolome of mid-trophozoite stage (1 µM concentration, 12 hours of exposure). Hierarchical cluster analysis (HCA) was used to triage these compounds into groups with putatively similar modes of action. Strikingly, this analysis allowed clustering to many of the 80 compounds into two major groups: In particular, 31 Malaria Box compounds, as well as chloroquine, resulted in characteristic changes in a range of metabolites, including a signature decrease (> 1.5-fold) in the metabolite pipecolate (a putative intermediate in lysine degradation). Another 22 Malaria Box compounds clustered together, and resembled atovaquone-induced metabolic phenotype, that included a > 1.5-fold accumulation of fumarate. Three additional metabolic phenotypes were also identified in this initial screen, highlighting its utility in broadly grouping drugs with known antimalarial activity and potentially similar modes of action. To further characterise the mode of action for the 22 Malaria Box compounds that induced atovaquone-like metabolic perturbations, the effect of these compounds on the mitochondrial electron transport chain and pyrimidine biosynthesis were investigated. These processes are linked as one of the key enzymes in pyrimidine biosynthesis, dihydroorotate dehydrogenase (DHODH), is dependent on the respiratory chain for activity. The impact of the Malaria Box compounds on pyrimidine biosynthesis was assessed by measuring changes in early intermediates in this pathway using liquid chromatography-mass spectrometry (LC-MS) and by metabolic labelling studies with H13CO3- which is incorporated into these intermediates. Eight of the 22 Malaria Box compounds inhibited pyrimidine biosynthesis in the same way as atovaquone, leading to the accumulation of N-carbamoyl aspartate and depletion of downstream metabolite uridine monophosphate (UMP). Further differences in the mode of action of these Malaria Box compounds and atovaquone were indicated by the finding that episomal-expression of the yeast DHODH, which by-passes the need for a mitochondrial respiratory activity in asexual erythrocytic stages, conferred resistance to fourteen of these compounds. In contrast, expression of yeast DHODH leads to effective resistant to atovaquone. Finally, none of the Malaria Box compounds exhibited synergistic activity with proguanil, indicating that they either target different sites on the cytochrome bc1 complex or other proteins altogether. Studies were also undertaken to investigate the function of pipecolate, which appears to be part of a parasite specific metabolic pathway, as it was only found in P. falciparum infected RBC and was selectively depleted when infected RBC were treated with several first-line antimalarials (artemisinin and chloroquine) and 31 Malaria Box compounds. Several lines of evidence suggested that pipecolate is generated during catabolism of lysine, which in turn is produced during haemoglobin degradation in the parasite food vacuole. In particular, inhibition of haemoglobin degradation led to concomitant decreases in both lysine and pipecolate, while metabolic labelling studies with 13C-lysine demonstrated conversion to 13C-pipecolate in vivo. Further, in vitro studies using positionally labelled lysine suggested the operation of an unanticipated lysine catabolic pathway involving the initial conversion of lysine to saccharopine. Pipecolate, but not lysine, is actively secreted by P. falciparum-infected RBC, and its synthesis may constitute an important mechanism for removing and/or detoxifying excess lysine generated by constitutive haemoglobin degradation. Further studies are needed to determine whether lysine degradation is essential in P. falciparum, which could be further explored as a potential novel drug target. Finally, the 24-well plate screen was further extended to measure macromolecule biosynthesis using heavy water, 2H2O labelling. Incubation of cells in the presence of low concentrations of 2H2O leads to the incorporation of deuterium (2H) into a wide range of metabolic intermediates that are subsequently used for macromolecule biosynthesis. A workflow was developed for measuring the turnover of DNA, RNA, proteins and lipids in P. falciparum-infected RBC following labelling of cells in 5% 2H2O and GC-MS analysis of constituent components of these macromolecules (i.e. deoxyribose, ribose, amino acids and fatty acids). Incorporation of 2H enrichment in DNA (deoxyribose) and RNA (ribose) at different stages of intraerythrocytic parasite development indicated stage-specific expression of DNA and RNA throughout the 48-hour cycle. As expected, incorporation into these molecules was inhibited by aphidicolin (DNA synthesis inhibitor) or actinomycin (RNA synthesis inhibitor), respectively. Interestingly 2H-enrichment in protein and lipids was minimal or not detected indicating that precursors for these molecules are largely derived from the host cell. Screening of late ring stage parasites (18-hour post-invasion) with 240 of the Malaria Box compounds (1 µM concentration, 12 hours of exposure) led to the identification of 47 compounds that inhibited DNA biosynthesis by > 50% DNA synthesis, with concomitant partial or strong inhibition of RNA biosynthesis. Detailed stage-specific analysis of these compounds provided further insights into different modes of DNA/RNA biosynthesis. Collectively, these studies have led to the development of two screens for investigating the mode of action of compounds that have been shown to have antimalarial activity in live-cell screens. The high content GC-MS based metabolomic screen demonstrated that many of the Malaria Box compounds can be grouped based on the metabolic responses that they generated. This raises the prospect that many of the top hits identified in these screens may target relatively few processes in the parasite (or the host cell). It will be of interest to extend these studies to the next tier of compounds which may exhibit lower malarial activity but allow identification of other drug targets. These studies have also highlighted unanticipated metabolic pathways in P. falciparum, such as lysine degradation, which may be important for virulence and potential drug targets.
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    Mechanisms regulating ribosome biogenesis by AKT
    Woods, Simone Jacinta ( 2015)
    Ribosomes are essential for cell growth and proliferation, and their biogenesis is a highly energy consuming process that requires exquisite regulation. Aberrant ribosome biogenesis underlies diseases of ribosomes, the so-called “ribosomopathies”. Deregulated ribosome biogenesis is now considered a characteristic of transformed cells, presenting a specific target for cancer treatment. One global regulator of ribosome biogenesis is the transcription factor c-MYC, which selectively transcribes an RNA Polymerase I (Pol I) specific regulon required for synthesis of the ribosomal RNA (rRNA), a key rate-limiting step in ribosome biogenesis. Recently it has been demonstrated that the kinase AKT also mediates Pol I-driven ribosomal DNA (rDNA) transcription and ribosome biogenesis to a similar extent as c-MYC. Importantly, maximal activation of rDNA transcription, ribosome biogenesis, and cell growth is achieved through the co-operative activities of both c- MYC and AKT. While previous work has outlined c-MYC’s major regulatory role as a transcription factor, the mechanism(s) by which AKT alone or in co-operation with c-MYC regulates rDNA transcription by Pol I remains unclear. This thesis aims to identify and elaborate upon potential mechanisms of AKT-driven ribosome biogenesis regulation. I hypothesise that AKT phosphorylates and subsequently activates Pol I complex components and associated factors, some of which may be c-MYC transcriptional targets. To test this hypothesis, the Pol I complex and associated proteins were immunoprecipitated and high sensitivity mass spectrometry was used to characterise the members of the complex and to identify phosphorylated proteins, providing insight into potential regulators of Pol I function. I found that the endogenous nucleolar protein treacle associates within the Pol I complex, providing insight into this protein previously implicated in promoting rDNA transcription by Pol I through an unknown mechanism, and which may be transcriptionally regulated by c- MYC. Further mass spectrometric analysis of phosphorylated proteins associated with the Pol I complex consistently identified p-peptides corresponding to treacle, including its putative AKT phosphorylation site (S1350 (human)), also identified in a bioinformatics analysis using Scansite. Furthermore, immunoprecipitated treacle could be phosphorylated by purified AKT in vitro demonstrating that treacle is a direct AKT substrate. In order to establish phosphorylation of treacle as a possible mechanism by which AKT regulates rDNA transcription by Pol I, I examined the effects of creating a phosphoinhibitory mutation in treacle at the AKT consensus phosphorylation site (S1191 (mouse)), replacing the serine with an alanine (S1191A). I found that while wild type treacle localised to the nucleolus (the site of rDNA transcription by Pol I), the phosphoinhibitory S1191A treacle mutant was dispersed throughout the cytoplasm. This suggests that AKT phosphorylation of treacle at S1191/S1350 is required for treacle’s nucleolar localisation and potentially its ability to promote rDNA transcription by Pol I there. Subsequent phosphoproteomic analysis further identified the inverse relationship between this phosphorylation site and phosphorylation of treacle at another site (S853) and further mass spectrometric analysis of immunoprecipitated samples also reveal treacle’s additional affiliation with components of the pre-rRNA methylation complex. This study has therefore uncovered aspects of key new regulatory mechanisms by which AKT can drive rDNA transcription and ribosome biogenesis through treacle, thereby revealing novel potential therapeutic targets for addressing dysregulated ribosome biogenesis.
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    The relationship between the plasma lipidome and cardiovascular disease outcomes in diabetes
    Alshehry, Zahir Hasan ( 2015)
    People with T2D have a two-fold increased risk of developing cardiovascular disease (CVD) compared to non-diabetic individuals. However, risk stratification in this group is challenging. Moreover, lifestyle factors i.e. smoking, alcohol consumption and sedentary life style are additional risk factors for CVD. Traditional lipid measurements (elevated cholesterol, triglycerides and/or lowered HDL-C) do not show the full complexity of the altered lipid metabolism associated with T2D or CVD. Lipidomics enables the quantification of hundreds of individual lipid species in biological samples in a single analytical process. Previous lipidomic analyses of plasma have revealed information regarding the pathogenesis of these diseases and identified novel biomarkers for disease diagnosis and risk assessment. In this study, a high throughput extraction method using 1-Butanol:Methanol (1:1, v:v) was developed to facilitate efficient extraction of a wide range of lipids from plasma. The method is fast, simple, uses non-halogenated solvents and is suitable for high throughput liquid chromatography electrospray ionization-tandem mass spectrometry (LC ESI-MS/MS). The method showed high recovery (>90%) and reproducibility (CV% < 20%). The simplicity of the method makes it amenable to an automated approach. Comprehensive lipidomic analysis was then performed on sub-cohort (n=3,779) from the ADVANCE (Action in Diabetes and Vascular disease: preterAx and diamicroN-MR Controlled Evaluation) study in a case/cohort design. The sub-cohort contained 698 participants who had a cardiovascular event (CVE) in the five-year follow-up period. Weighted Cox regression was performed to identify lipid species associated with future CVE, CVD death, stroke and myocardial infarction (MI). Multivariable models containing traditional risk factors with or without lipid species were developed using Cox regression. Features were selected and ranked using the Akaike information criteria. C-statistics, net reclassification index (NRI) and integrated discrimination improvement (IDI), were computed within a 5-fold cross-validation framework (200 iterations) to evaluate the ability of the models to reclassify and discriminate five-year risk. Sphingolipids, phospholipids, sterols and glycerolipids were associated with future CVE (31 species), CVD death (37 species) and stroke (5 species). Compared to base models to predict CVE, CVD death, stroke and MI (incorporating 14, 14, 8 and 10 risk factors, respectively), combined models (11, 15, 14 and 17 lipids and risk factors, respectively) resulted in an increase of C-statistics (0.698 (1.8%), 0.768 (2.8%), 0.766 (18.3%) and 0.707 (7.0%), respectively) and NRIs (5.2%, 13.4%, 13.0% and 10.1%, respectively) based on categorical models of <10, 10-15, and >15% 5-year risk. Finally, logistic regression analyses were performed to identify lipid species associated with smoking, alcohol consumption and exercise that may mediate CVE risk in T2D. The ADVANCE sub-cohort contained (565, 1557 and 1822, participants) of smokers, alcohol consumers and exercise active, respectively. Sphingolipids, phospholipids, sterols and glycerolipids were associated with smoking and alcohol consumption and may represent mediators of disease pathogenesis and so potential therapeutic targets. In summary, these studies improve our understanding of the lipid metabolism associated with CVD risk and the interactions with lifestyle factors in the T2D population. Importantly, we have demonstrated the ability of plasma lipids to improve upon traditional risk factors for the prediction of future CVE and identified potential therapeutic targets to modify that risk.
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    Proteomic screening of disease-affected tissues in a mouse model of multiple sclerosis
    Dagley, Laura Franscesca ( 2013)
    Multiple sclerosis is chronic demyelinating disorder characterized by auto-immunity towards myelin and the subsequent infiltration of auto-reactive immune cells from the periphery into the central nervous system resulting in axonal injury and neuronal cell death. Myelin is the insulating membrane that surrounds axons (or nerve fibers), allowing for the efficient and rapid propagation of electrical signals. In multiple sclerosis, this protective coating is targeted for destruction, resulting in a slowing of action potentials and the onset of neurological symptoms. Whilst there is currently no cure for the disease, there are a range of disease-modifying therapies with the majority targeting the immunological features of the disease and very few exerting neuroprotective effects. Using a high-precision 18O-based stable isotope labeling approach I have quantitatively mapped the region-specific changes in membrane proteins isolated from myelin-enriched fractions within select regions of the healthy adult mouse brain, specifically those that are susceptible to lesion development in multiple sclerosis, including the brain stem, cerebellum and remaining cerebral hemispheres. Approximately 90 statistically significant changes in relative abundance were identified thus representing putative region-specific brain markers. Protein abundance data were then compared with online digital brain atlases of expression and previously published literature with a subset of markers subjected to validation by western blot analysis. The central nervous system myelin proteome that was generated by performing this quantitative proteomics consisted of ~1800 unique proteins, with over 1000 proteins that have yet to be described in any other mammalian myelin proteome characterized to date. Subsequently, a comparison of this myelin proteome was then made to an existing transcriptome database containing messenger ribonucleic acid abundance profiles mapped during oligodendrocyte differentiation. Oligodendrocytes are the myelin-forming cells of the central nervous system. This mapping process provided a means of validating whether the new members of the myelin proteome have putative roles in myelin biology, thus implicating them as myelin-associated proteins. Experimental autoimmune encephalomyelitis represents the best characterized animal model of multiple sclerosis as common clinical, histological and immunological features are recapitulated. The application of quantitative proteomics approaches to disease-affected tissues harvested from the animal model represents an unbiased screening method ideally suited for the detection of novel biomarkers. To this end, a dual MS1- and MS2-based label-free quantitative proteomics approach was used to delineate the profile of differentially expressed proteins in a comparative analysis of actively-induced C57BL/6 experimental autoimmune encephalomyelitis mice and sham-induced (‘vehicle’ control) counterparts at the disease peak. I identified novel, disease-specific and membrane-associated proteins within experimental autoimmune encephalomyelitis-affected tissues, including the spinal cord and membrane protein-enriched fraction of peripheral blood mononuclear cells. More than half of these observed putative biomarkers have not previously been linked to multiple sclerosis or its animal model. The biological significance of all candidate disease markers were elucidated through rigorous literature searches, pathway analysis and validation studies including western blot analysis. The sensitivity and specificity of novel disease biomarkers were further assessed through targeted mass spectrometry analyses using a scheduled multiple reaction monitoring approach which confirmed the differential abundance of ~200 candidate markers at a ~70% success rate.
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    Characterization of c-Jun N-terminal kinase 1 (JNK1) – subcellular localization, post-translational modifications and protein complexes under control and stress conditions
    MISHEVA, MARIYA ( 2013)
    c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein kinase (MAPK) subfamily of protein kinases involved in a number of biological processes such as programmed cell death, cell survival, development and the inflammatory response. The role of JNKs in this diverse range of processes has continued to attract attention. The numerous investigations undertaken since their discovery in the early 1990s have led to the identification of multiple substrates localized in the nucleus, the cytoplasm and the mitochondria, as well as regulators of the JNK signaling pathway (scaffolds, upstream kinases and downstream phosphatases). In addition, the involvement of JNKs in both physiological and pathological processes in the cell has been investigated in more detail by studies in knockout animals. The accumulated evidence points to the different JNK isoforms having both redundant and specific functions in a number of biological processes under basal conditions and upon stimulation. The function of the different JNK isoforms may therefore be both cell type- and stress stimulus-specific. In addition, the reported data implies that JNK1 is the main contributor to the development of pathophysiological processes. However, despite this growing knowledge on the JNK signaling pathway, the precise mechanisms underlying their regulation in the cell still requires more detailed evaluation. Thus, this thesis has explored specifically the subcellular localization, the post-translational modifications and the proteins associated with JNK1 in the absence of stress and following stress treatment. This work presents the first live cell imaging and fluorescence recovery after photobleaching (FRAP) analyses for JNK, and has revealed that JNK1α1 is exchanged between and within the nucleus and the cytoplasm under basal conditions. Contrary to expectations for JNK1 as a stress-activated kinase, hyperosmotic stress treatment slows the rate of kinetics of JNK1 movement within the cell. These observations led to further evaluation by mass spectrometry of the regulation of JNK1α1. Selected reaction monitoring (SRM) analyses indicated that there are numerous post-translational modifications present on JNK1 in the absence of stress, and that hyperosmotic stress treatment did not change this state of JNK1 but rather enhanced the phosphorylation of JNK1 Ser129, Thr183, Tyr185, and Thr188. SRM also has led to the identification of a new dual phosphorylated form of JNK1α1 comprising of phospho-Tyr185 in combination with phospho-Thr188. The significance of this new form of JNK will require further evaluation in future more detailed studies, but as both residues are within the activation loop of JNK1 the possibility that this represents an alternatively regulated form of JNK1 will warrant more detailed evaluation, particularly in testing whether this dual-phospho form is also an activated form of JNK1. Finally, the application of standard mass spectrometry to the identification of proteins co-immunoprecipitating with JNK1 showed that a number of proteins involved in different processes are associated with JNK1α1. The precise roles of these new protein partners in JNK1 signaling should form the basis for future studies evaluating the diversity of JNK functions and provide further evidence supporting the complexity of actions of JNK1 in signal transduction events in the cell. This work presents the first live cell imaging and fluorescence recovery after photobleaching (FRAP) analyses for JNK, and has revealed that JNK1α1 is exchanged between and within the nucleus and the cytoplasm under basal conditions. Contrary to expectations for JNK1 as a stress-activated kinase, hyperosmotic stress treatment slows the rate of kinetics of JNK1 movement within the cell. These observations led to further evaluation by mass spectrometry of the regulation of JNK1α1. Selected reaction monitoring (SRM) analyses indicated that there are numerous post-translational modifications present on JNK1 in the absence of stress, and that hyperosmotic stress treatment did not change this state of JNK1 but rather enhanced the phosphorylation of JNK1 Ser129, Thr183, Tyr185, and Thr188. SRM also has led to the identification of a new dual phosphorylated form of JNK1α1 comprising of phospho-Tyr185 in combination with phospho-Thr188. The significance of this new form of JNK will require further evaluation in future more detailed studies, but as both residues are within the activation loop of JNK1 the possibility that this represents an alternatively regulated form of JNK1 will warrant more detailed evaluation, particularly in testing whether this dual-phospho form is also an activated form of JNK1. Finally, the application of standard mass spectrometry to the identification of proteins co-immunoprecipitating with JNK1 showed that a number of proteins involved in different processes are associated with JNK1α1. The precise roles of these new protein partners in JNK1 signaling should form the basis for future studies evaluating the diversity of JNK functions and provide further evidence supporting the complexity of actions of JNK1 in signal transduction events in the cell.
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    Targeted discovery of T cell epitopes: high energy, stable isotope differentiated precursor ion scanning
    Reilly, Charles B. ( 2012)
    The cellular arm of the acquired immune system relies on the activity of T lymphocytes and their ability to recognize peptide epitopes. These peptides, collectively referred to as the immunopeptidome, are bound by molecules of the major histocompatibility complex (MHC) and are found on the surface of antigen presenting cells. The vast complexity of the immunopeptidome reflects the diversity of a cells proteome and provides a window into the cells overall ‘system’. The complexity of the immunopeptidome presents enormous challenges for characterization. It is estimated that this set of peptides may comprise of up to one hundred thousand or more unique peptides found over a large dynamic range of abundances on the surface of any single cell. All peptides bound to a particular MHC molecule contain common biophysical characteristics that complicate analysis of individual peptides. A major challenge arises in the attempt to identify peptides derived from known T cell stimulating antigens, due to this complex background of constitutively presented peptides and the incredible sensitivity of T cells, which in some circumstances are purported to recognize a single MHC-peptide complex on the surface of target cells. Here a strategy is developed for the identification of candidate antigen specific T cell epitopes. Furthermore, this strategy is able to identify target breakdown products of any proteinaceous compound, even when these targets are found in low abundance and in extremely complex biological matrices. An example of this kind of application is in the drug metabolism and pharmacokinetic (DMPK) studies of peptide/protein based therapeutics, of which there are over 200 currently approved. Monitoring the metabolism of these increasingly common biotherapeutics is challenging due to the large number of potential metabolites that can be generated in vivo. Initially in this study, a technique is developed where immonium ions are isotopically differentiated by the incorporation of 15N . The shift in m/z caused by isotopic labeling is exploited in the screening of target compounds from within complex biological matrices by way of precursor ion scanning. The immonium ions used are found in the low mass region of a peptides MS/MS spectra and their presence is reported to be largely representative of their corresponding amino acid residues presence in a parent ion. Through development of the precursor ion scanning strategy in this study, a common low mass ion produced by all peptides undergoing collision activated dissociation (CAD) at high energy was identified. This ion of C4H8N chemical formula, with an m/z of 70.1 can be shifted by incorporation of 15N in to the peptide analyte to m/z 71.1. Importantly, there is negligible production of any ion at 71.1 in high energy CAD of unlabeled compounds. This therefore enables the use of 71.1 as a ubiquitous diagnostic ion at high energy CAD for all 15N labeled compounds. The fact that every amino acid in the context of a peptide will facilitate production of an ion of the chemical formula C4H8N, means that an accumulative effect contributes to a very strong signal in all peptides at m/z 70.1 or at m/z 71.1 for 15N-labeled species. This results in the C4H8N ion always being one of the most intense ions in product ion spectra at collision energies approximately equivalent to five times normal proteomic type intensities (~150 eV). This diagnostic ion was incorporated into a precursor ion scanning based workflow using triple quadrupole instruments with ion trap functionality. These instruments are capable of information dependent acquisition (IDA) of product ion spectra at alternate energies. Thus allowing the acquisition of a ‘normal’ proteomic MS/MS spectrum for peptide sequencing, following target detection with the high-energy ,scan. Precursor ion scanning with the use of high energy and a diagnostic target ion of 71.1 was employed in identification of MHC bound peptides derived from 15N proinsulin (PI). PI is a known T cell auto-antigen involved in the early onset of Type 1 Diabetes (T1D). Here we identified known and novel PI peptides that were presented by the HLA DR3/DQ2 haplotype which is highly associated with susceptibility to T1D. Of the peptides identified, some contained three cysteine residues and were found to be present in multiple intramolecular disulphide bonded forms. Many of these post translationally modified (PTM) peptides originated from the A-chain region of insulin, a region known as a 'hot spot' for T cell epitopes.