Biochemistry and Pharmacology - Theses
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ItemProteomic analysis of mHttex1 expression in Huntington’s disease( 2021)Huntington’s disease (HD) is a fatal neurodegenerative disorder caused by CAG trinucleotide repeat expansion in exon 1 of the Huntingtin (Htt) gene. This sequence encodes an abnormally elongated polyglutamine (polyQ) tract within the Huntingtin (Htt) protein that is directly involved in aggregation and Htt-mediated cytotoxicity. The key pathological signature of HD is the aggregation of mutant Htt protein into punctate aggregates. However, the mechanism by which polyQ-expanded mutant Httex1 (mHttex1) causes toxicity remains elusive. Previous research has indicated that mHttex1 can exert toxicity to cell models through two distinct phases. The first is when the protein is soluble and the second is when it is aggregated into inclusion bodies, which are the major pathological signature of HD brain. I hypothesized that apoptosis is caused by an unresolved quality control mechanism that oversees mHttex1 at synthesis. The goal of this project was to develop and implement a novel proteomics strategy to specifically detect the proteins that engage with mutant Htt during protein synthesis. I compared pathogenic huntingtin (Q97) and non-pathogenic huntingtin (Q25) using a proteomics-based approach. Firstly, a self-cleaving NS3 viral protease system called TimeSTAMP was employed, which can efficiently cleave epitopes from newly synthesized proteins and be potently inhibited using a viral protease inhibitor. The goal was to inhibit the cleavage across different-time windows to “pulse” label newly synthesized Htt and at the end of the pulse steps, proteins were crossed-linked with disuccinimidyl sulfoxide (DSSO) to preserve transient interactions. We also wanted to examine the changes in the global proteome and phosphoproteome across these mutant form and wild-type counterpart. After transfection of Neuro2a cells with TimeSTAMP-Httex1 constructs of differing poly-Q length, cells were lysed using RIPA lysis buffer. Proteins were then treated with a label-free relative quantitative phosphoproteomics workflow: i.e., samples were denatured (e.g., in 8 M urea), reduced and alkylated, then subjected to tryptic digestion. Next, a phosphopeptide enrichment step was performed before samples analyzed using LC-MS/MS. However, there was no significant difference observed between pathogenic and non-pathogenic Huntingtin, indicating a lack of polyQ-length dependence. To further probe the toxicity of the pathogenic huntingtin, I investigated its protein-protein interactions using a different proteomics-based approach. After transfection of Neuro2a cells with the Q25-GFPEm or Q97-GFPEm constructs, proteins were cross-linked with disuccinimidyl sulfoxide (DSSO) and then protein interactors were pulled down using anti-GFP VHH coupled magnetic agarose beads. We also examined the changes in the global proteome and phosphoproteome across the mutant form and wild-type counterpart. I did not find any significant difference between pathogenic and non-pathogenic Huntingtin which further indicates that the result was not polyQ dependent. Determination of these mechanisms are anticipated to be important for the design of new therapeutic strategies that mitigate toxicity of soluble mHttex1.
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ItemInvestigating the Epitranscriptome of Plasmodium falciparum( 2019)Recent years have seen an increasing identification and awareness of post-transcriptional modifications of RNA. Nucleotide modifications have long been known to be important in diverse non-coding RNAs, but post-transcriptional modifications of mRNA are now also recognised as playing an important role in regulation of gene expression. One of the most abundant mRNA modifications is N6-methyladenosine (m6 A), the presence of which guides mRNA metabolism including maturation, nuclear export, translation and decay. In model eukaryotes, m6A is produced by the action of METTL-family methyltransferases and recognised by a family of YTH proteins. These enzymes play a key role in cellular differentiation and development. I have identified several putative METTL methyltransferases and several YTH reader proteins in the Plasmodium falciparum genome, and have investigated the m6A modification in the transcriptome of P. falciparum. I have characterised two methyltransferase proteins, Mettl3 and Mettl14-like proteins and two reader YTHDC1-like proteins (PfYTHDC1-a and PfYTHDC1-b)in P. falciparum through localisation, overexpression experiments and knock-sideways studies. Initial studies using epitope tagging and GFP fusion proteins revealed a predominantly nuclear localisation for both the methyltransferase and reader proteins, with some signal in the cytoplasm. Knock-sideways studies revealed complete inducible mis-localisation by 24 hours for PfMETTL3 and within 7 hours for PfYTHDC1-a. Additionally, under induced mis-localisation, both transfectant lines underwent morphological changes as evident by microscopy, with clear spots within the digestive vacuole, changes not seen in the 3D7 parental line. A pilot RNA-seq study was conducted, wherein differential transcript expression was analysed as a means to characterise the impact of perturbing m6A modifications by overexpressing and disrupting METTL methyltransferases. Future experiments should analyse the distribution of m6A in mRNA by nanopore direct-RNA sequencing. Additional investigations are also warranted on the influence of the METTL and YTHDC1-like proteins on splicing patterns in the parasite as a means to test the role of these modifications on proliferation and differentiation.
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ItemSequence-based typing of IncA/C and IncI1 plasmids in the era of high-throughput sequencing( 2016)Conjugative plasmids are able to disseminate antimicrobial resistance genes amongst a broad range of bacterial hosts, including pathogens that cause infections in humans and animals. To investigate the diversity of IncA/C and IncI1 plasmids amongst Gram-negative bacteria, plasmid and whole-genome sequence data were analysed via multi-locus sequence typing and phylogenetic analysis of core and common genes. The data show these two plasmid types, which both contribute to dissemination of antimicrobial resistance, differ in their geographical distribution and bacterial host range.
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ItemInvestigating the structure, function and inhibition of DHDPS from an intracellular pathogen( 2014)Enzymes of the diaminopimelate (DAP) pathway have attracted much attention in the past two decades as potential antimicrobial targets. The end products of this pathway, meso-DAP and lysine, are essential components in bacterial cell walls and protein synthesis. One key enzyme catalysing the rate-limiting step in the DAP pathway is dihydrodipicolinate synthase (DHDPS). DHDPS has been extensively characterised from plant and bacterial species. However, little information is available on the enzyme from intracellular bacteria. As such, this thesis examines the structure and function of DHDPS from the intracellular, Gram-negative pathogen Legionella pneumophila. DHDPS from L. pneumophila was cloned from gDNA, followed by expression and purification of recombinant protein to homogeneity. Identity of the purified product was confirmed using mass spectrometry and the overall fold was similar to a classical DHDPS enzyme, as determined by CD spectroscopy. Kinetic analyses also showed that L. pneumophila DHDPS functioned in a similar capacity to E. coli DHDPS, with a kcat of 101 s-1, KM of 0.24±0.01 mM for pyruvate and KM of 0.19±0.01 mM for (S)-ASA. At the commencement of this research project, DHDPS enzymes existed predominantly as homotetramers. The formation of DHDPS tetramers is thought to facilitate catalysis by restricting movement of key active site residues. Through use of X-ray crystallography, small angle X-ray scattering and analytical ultracentrifugation, it is shown that L. pneumophila DHDPS forms the less frequently observed dimeric structure. Examination of the 1.65 Å L. pneumophila DHDPS crystal structure reveals a greater number of contacts at the interface between both DHDPS monomers relative to the E. coli homolog. This lends weight to the theory of an alternate evolutionary solution for limiting flexibility of active site residues, as previously proposed for the MRSA DHDPS dimer. Work presented in this dissertation also challenges the currently accepted paradigm of allosteric inhibition in DHDPS enzymes. To date, all plant and Gram-negative DHDPS respond to feedback inhibition by lysine. A key finding of the work described here is the lack of allosteric regulation displayed by L. pneumophila DHDPS. This represents the first example of a DHDPS enzyme from a Gram-negative pathogen to remain insensitive to lysine inhibition. Examination of the allosteric site in the crystal structure shows substitutions of key lysine-binding residues. An overlay of the DHDPS allosteric sites from L. pneumophila and E. coli reveal these substitutions to either hinder the binding of lysine or prevent formation of critical bonds with lysine. Importantly, the observed lack of inhibition may be linked to the intracellular lifestyle of L. pneumophila. A high demand for meso-DAP and lysine in this bacterium, coupled to their low availability in human cells, likely reflects the absence of regulation in L. pneumophila DHDPS. Alternatively, L. pneumophila DHDPS may be regulated by other means. Availability of high resolution X-ray data for the L. pneumophila DHDPS structure enables visibility of two conformations of the critical catalytic residue Tyr106. The conformation with higher occupancy is within hydrogen-bonding distance of other key active site residues. In contrast, the positioning of the lower occupant reveals disruption to a vital proton relay. This suggests a previously unidentified mechanism for DHDPS inhibition that may replace allosteric regulation in intracellular forms of the enzyme. Findings from this work are expected to broaden knowledge of key sites within the enzyme, thereby aiding in the future design of inhibitor molecules against DHDPS. Further development of such molecules may lead to a new class of antimicrobials that will help combat the impending issue of antibiotic resistance in bacteria.
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ItemAutophagy: a multifaceted pathway in immunity( 2014)Autophagy is an evolutionary conserved pathway of protein degradation. In recent years, autophagy has been shown to play numerous critical roles in the immune system, contributing to both immune cell homeostasis and survival, and the initiation of adaptive immune responses through the putative delivery of antigen to MHCI and MHCII antigen presentation pathways. The objective of this thesis was to further elucidate autophagy in both T cell maintenance and survival and in MHCII antigen presentation and MHCI antigen cross-presentation. To begin to assess autophagy in T cells, it was demonstrated that both naïve and in vitro activated T cells utilised the autophagy pathway. In addition, it was shown that atg5 was essential for an intact naïve T cell compartment and efficient T cell proliferation in vitro. As T cells switch metabolism profiles to meet changing energetic demands, it was hypothesised that T cells regulate autophagy in response to these cues. To monitor autophagy regulation within T cells, the GFP-LC3 mouse model was utilised. However, the GFP-LC3 mouse model was proven to be inappropriate for the study of autophagy regulation due to differential expression patterns of endogenous LC3B and transgenic GFP-LC3 in activated T cells. To extend upon findings that autophagy was involved in MHCII antigen presentation and potentially in MHCI antigen cross-presentation, antigen presentation assays were performed in the presence of the autophagy inhibitor 3-MA. It was demonstrated that 3-MA inhibition resulted in reduced MHCII antigen presentation and MHCI antigen cross-presentation of exogenous antigen. It was hypothesised that the CD11cCre x Atg7F/F mouse model could be employed to further investigate autophagy in antigen presentation. However, at the conclusion of this study, the CD11cCre x Atg7F/F mouse model was undergoing optimisation.
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ItemStructural basis of tumor-associated phosphopeptides bound to the MHC molecule HLA A2 and stabilization studies( 2011)Phosphorylation of cellular proteins is significantly different in cancerous cells compared to normal tissue. But also for cancerous cells the usual MHC class I processing pathway takes place. Proteins are degraded through the proteasome, peptides are transported to the endoplasmic reticulum where they are loaded onto empty MHC class I molecules. The loaded MHC class I molecule is then transported to the cell surface where the peptide antigens are presented to cytotoxic T cells. Due to the different phosphorylation status of cellular proteins, the degraded phosphopeptide antigens, especially those found on different cancer cell lines, are potential candidates for anticancer vaccination. For this study three cancer specific phosphopeptides presented by the common caucasian HLA A2 molecule were chosen for structural and biophysical studies to understand the molecular nature of phosphopeptide presentation to T cells. To further investigate the impact of the phospho- group on the binding to the MHC molecule HLA A2, phosphopeptide-MHC complexes were generated, crystallized and X-ray crystallography was undertaken. The phosphopeptide- MHC structures showed in all three cases that the phospho- group was solvent exposed and thus accessible to the T cell receptor for recognition. Apart from this fact the phospho- group was involved in intrapeptide interactions as well as interactions with the HLA A2 heavy chain in an epitope dependent manner. In one case the phosphopeptide-MHC structure showed in comparison to the nonphosphorylated counterpart a change in peptide-MHC complex conformation, this is an example of transformed self. In addition to the detailed structural studies, stabilization and peptide binding experiments were undertaken using recombinant HLA A2 and cell surface HLA A2 molecules respectively. But only in one case the phospho- group of the peptide stabilized the phosphopeptide- MHC complex and enhanced peptide binding. This suggests that phosphorylation will not always enhance MHC binding as previously reported. Finally dephosphorylation experiments were performed on the free peptide and the HLA A2-phosphopeptide complexes. These analyses indicated that in all cases the phospho- group of the peptide was protected by the MHC molecule from dephosphorylation, consistent with the structural analysis which showed extensive interactions of the phosphate group with the HLA heavy chain. These findings show that the phosphopeptide-MHC complexes are stable and underpin observations that the phosphorylated peptides are presented to cytotoxic T cells in cancer and normal cell lines. Given the solvent accessibility of the phosphate group, the uniqueness of certain phosphopeptides to cancer cells and the ability of the phosphate to mediate changes in peptide binding affinity, stability and conformation these phosphopeptides are excellent vaccination targets for cancer immunotherapy.
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ItemThe role of small G proteins in the trans-Golgi network( 2011)Small G Proteins play a major role in the regulation of membrane traffic at the trans-Golgi network (TGN) and include members of the Rab, Arf and Arl families. These small G proteins regulate the recruitment of a wide range of effector molecules, including one important class of long coiled-coil proteins, the golgins. A major aim of this thesis was centred on investigating the recruitment of TGN-localised golgins by small G proteins. The TGN golgins p230/245 and golgin-97 are known to interact with the Arf-like GTPase Arl1, enabling the C-terminal GRIP domain of each golgin to interact with the TGN membrane, while other mammalian TGN golgins such as GCC88 and GCC185 have different membrane binding properties. The small G-protein partners responsible for their recruitment have not been identified. A group of Arl GTPase family members were screened for their potential to localise to the Golgi apparatus in mammalian cells. Two novel members of the Arl family associated with the Golgi were identified, Arl5a and Arl5b. The putative GTP binding Arl5a(Q70L) and Arl5b(Q70L) mutants localised to the Golgi in close proximity to TGN golgins, while the putative GDP binding mutant Arl5b(T30N) localised throughout the cytosol and at high levels showed some perturbation of the Golgi. A stable HeLa cell line expressing Arl5b(Q70L)-GFP showed an increased rate of transport of the membrane cargo TGN38 from endosomes to the Golgi. In addition, depletion of Arl5b by RNAi in cultured cells resulted in disruption of the localisation of mannose 6 phosphate receptor (M6PR). Depletion of Arl5b reduced the rate of TGN38 transport, as well as the retrograde transport of Shiga toxin (STxB), between endosomal compartments and the Golgi, while anterograde transport of E-cadherin from the TGN to the cell surface was unaffected in the absence of Arl5b. These data suggested that Arl5b is localised to the TGN and is involved in the regulation of endosome-to-TGN trafficking. In response to a paper which proposed that both Rab6 and Arl1 were required for GCC185 to localise to the Golgi (Burguete et al., 2008), the interaction between the golgin GCC185 and the small G proteins Rab6A/A’ and Arl1 in vivo was investigated. There was minimal co-localisation of Rab6 with endogenous GCC185 on Golgi membranes. Yeast two-hybrid analyses failed to detect an interaction between Rab6A/A’ and the C-terminal domains of GCC185. Depletion of both Rab6A/A’ and Arl1 had no effect on the localisation of endogenous GCC185 or the isolated GRIP domain of GCC185. It was therefore concluded that the localisation of GCC185 to the Golgi was independent of these two small G proteins.
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ItemStathmin, a novel JNK substrate( 2010)Mammalian cells can initiate intracellular signalling pathways that activate pro-survival changes to maintain their integrity following their exposure to a range of extracellular stresses. One group of changes preserves cellular integrity through the regulation of cytoskeletal organization. Despite the recognised importance of maintaining microtubule (MT) networks, the specific mechanisms regulating cytoskeleton organisation in response to stress remain relatively poorly explored. Among the numerous proteins that regulate MT organisation, stathmin (STMN) is a key MT destabilising protein that regulates MT disassembly through its ability to bind tubulin dimers. The actions of STMN can be regulated by a number of growth factor-activated and cell cycle regulatory protein kinases. In preliminary work, our studies suggest the potential regulation of STMN by c-Jun N-terminal Kinase (JNK) in cells exposed to stress. Specifically, we observed changes in STMN phosphorylation which were coordinated with JNK activation. This project has explored the contribution of stress-activated c-Jun N-terminal Kinase (JNK) to STMN phosphorylation observed during osmotic stress. More detailed in vitro biochemical analysis has revealed that JNK directly phosphorylates STMN. In addition, we have compared STMN phosphorylation by different MAPK family member. In particular, our results illustrated that JNK predominantly phosphorylate STMN on serine residue 38 (S38) whereas ERK most likely targeted STMN S25. By examining specifically the phosphorylation of the four regulatory serine residues in vitro, we proposed a model of hierarchical phosphorylation among STMN serine residues. Specifically, our results demonstrated that phosphorylation of S38 was a pre-requisite for S25 phosphorylation by JNK in vitro. Furthermore, our results also demonstrated the impacts of JNK binding domain (JBD) and tubulin on STMN phosphorylation in vitro. Overall, this project identified STMN as a novel JNK substrate. The results have broadened our understanding on the JNK-mediated STMN phosphorylation as the first step to provide deeper insights into the different functions of JNK in the mammalian stress response.