Biochemistry and Pharmacology - Theses
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ItemUnderstanding the molecular mechanism of AAA+ ATPase p97 in complex with different cofactors( 2023-10)p97 has emerged as an attractive target for treatment of cancer, neurodegenerative and infectious diseases. This enzyme is a highly conserved and abundant AAA+ ATPase in all eukaryotic organisms. p97 is composed of two rings with a central pore and containing six identical subunits. Each subunit contains an N-terminal domain, two ATPase domains (D1 and D2), linkers connecting the domains (N-D1 linker and D1-D2 linker), and a disordered C-terminus. p97 is a key element of the ubiquitin proteasome system and, in concert with various cofactor proteins, extracts and unfold damaged or misfolded substrates through ATP hydrolysis. p97, in complex with cofactors, plays a crucial role in maintaining organelle and protein homeostasis through both ubiquitin dependent and independent pathways. These cofactors control the substrate selection, subcellular localization and regulate p97 enzymatic functions . Dysfunction of p97 has been associated with several diseases, including cancer and neurodegenerative disorders. Binding and assembly of cofactors to p97 are essential for its function. Therefore, understanding how the p97-cofactor form a complex and process substrates can provide valuable insights to develop inhibitors for specific pathways. In this project, we explored five structurally and functionally p97 cofactors including p37, SAKS1, Ufd1-Npl4, OTUD2 and UBXD7 to understand the mode of interaction of these cofactors with p97 and determine how these cofactors impact p97’s ATPase and unfoldase activities. We combined structural biology methods with structural dynamics techniques to investigate the structure of p97-cofactor complex and the conformational changes that occur upon interaction between proteins. We also performed in silico studies to determine the mode of interaction between p37 and p97 at the atomic level. According to the HAWKDOCK, HDOCK, Arpeggio and MM-GBSA binding free energy calculations, we found multiple hydrophobic interactions as well as two hydrogen bonds between the p37 UBX protein and the p97 N-D1 domain. In addition, we observed that the residues of the p37 UBX protein predicted to participate in interactions with the p97 N-D1 domain interface are remarkably conserved across UBX cofactors This finding indicates the significance of these interacting residues among UBA-UBX cofactors in interaction with p97. In addition, the in silico study provided the first structural insights into the p37-p97 complex through methods such as homology modeling, protein-protein docking, and molecular dynamics simulation. This approach allowed us to identify critical residues involved in the interaction between p37 and p97. We conducted circular dichroism (CD), Size Exclusion Chromatography coupled with Multi-Angle Light Scattering (SEC-MALS), and analytical ultra centrifugation (AUC) to determine the secondary structures, molar mass and oligomeric state of cofactors, respectively. Our results from surface plasmon resonance (SPR) assays indicated that all these cofactors interact with high binding affinity to p97. The cross-linking mass spectrometry (XL-MS) data showed that all cofactors bind to at least two domains or linkers of p97. The ATPase and Unfoldase assays revealed both SAKS1 and UBXD7 enhance p97’s ATPase activity and enable it to unfold polyubiquitilated substrate in vitro. We also employed advanced techniques such as cryo-electron microscopy (cryo-EM), XL-MS and Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) techniques to understand how the interaction between SAKS1 and the p97 N domain leads to the conformational change in SAKS1 that promotes substrate recruitment. In addition, for the first time, we resolved a cryo-EM map of the p97-SAKS1 complex obtained from a pull-down assay, which reveals the unique conformation of p97. We also resolved two novel structures of p97 in the presence of ADP-BeFx. The first structure is p97 hexamer in which all the N domains are in up conformation. The second structure is a dodecamer form of p97 in which two hexamers of p97 attached to each other from the C-terminus and all p97 N domains are in Up conformations.
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ItemOrganellar translation and inhibition in Plasmodium falciparum( 2023-09)The malaria parasite Plasmodium falciparum has two prokaryote-derived organelles: the mitochondrion and a relic plastid known as the apicoplast. These contain their own distinct, reduced genomes which must be transcribed and translated to maintain parasite viability. The bacterial-like proteins and metabolic functions of these organelles make malaria parasites susceptible to many anti-bacterials. This study aims to investigate organellar translation in P. falciparum, including the expression of apicoplast-targeted translation enzymes, tracking the cellular consequences of apicoplast translation inhibition, and measuring active organellar protein synthesis. Aminoacyl tRNA synthetases are a family of essential enzymes required for protein translation in the cytosol, apicoplast and mitochondrion. Several of these enzymes are encoded by single genes, from which two protein isoforms are proposed to be generated by alternative translation initiation. One isoform contains an N-terminal apicoplast localisation sequence, while the other lacks this and is cytosolic. In this study we investigate the significance of the nucleotides surrounding canonical and proposed translation start sites and show that these are important for their recognition by translation machinery. Additionally, we verify one of these dual-localised enzymes - threonine aminoacyl tRNA synthetase - as the target of the potent anti-microbial agent borrelidin in P. falciparum. Most organelle translation inhibitors have a lethal, but slow phenotype, killing parasites in the cycle following their administration. This has been attributed to disruption of apicoplast translation, with parasite death due to the inability to continue synthesis of essential apicoplast-derived isoprenoid metabolites. The consequences of isoprenoid starvation has been partially characterised, implicating lipophilic prenyl and isoprene chains as important, however not all essential isoprenoid products have been identified. We therefore aimed to investigate other downstream consequences of apicoplast translation inhibitors in Plasmodium. We found that apicoplast isoprenoids are required for synthesis of the major parasite sugar anchor glycophosphatidylinositol. Following inhibition of apicoplast translation, proteins typically anchored via this glycoconjugate became untethered, resulting in parasite segmentation, egress, and invasion defects. Difficulty in detecting proteins derived from organellar genomes had made the verification of organellar translation inhibitors challenging. Here, we use a mass spectrometry approach to directly detect and measure organellar translation in P. falciparum. This has facilitated the confirmation of the anti-apicoplast mechanism of action for the clinically used anti-malarials doxycycline and clindamycin. In addition, doxycycline was determined to inhibit mitochondrial translation, which was found to affect the activity of the electron transport chain. Together, this work has confirmed both the direct mechanism of action and indirect cellular consequences of organellar translation inhibitors on P. falciparum. In verifying the essentiality of glycophosphatidylinositols for multiple processes during the asexual stages, we have highlighted the potential for designing therapies that directly target aspects of glycophosphatidylinositol maturation or their protein attachment. Furthermore, determining the secondary target of doxycycline to be the mitochondrion has important clinical implications and may influence which drugs can be safely recommended for combination treatments.
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ItemHexosamine-dependent growth and virulence in Leishmania major(University of Melbourne, 2010)
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ItemInsight into the early stages of protein folding from structural models of the unfolded state(University of Melbourne, 2007)
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ItemThe evolution of the structure and function of transthyretin-like protein(University of Melbourne, 2007)
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ItemFunctional roles of serum amyloid P component in amyloid diseases(University of Melbourne, 2006)
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ItemFunctional roles of serum amyloid P component in amyloid diseases(University of Melbourne, 2006)
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ItemThe mammalian grip domain proteins : a family of golgins localised to subdomains of the trans-Golgi network(University of Melbourne, 2006)
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ItemThe mammalian grip domain proteins : a family of golgins localised to subdomains of the trans-Golgi network(University of Melbourne, 2006)
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ItemCellular and molecular defence mechanisms against Legionella infection( 2023-04)The two most prevalent causes of Legionnaires’ disease are L. pneumophila and L. longbeachae. The rising incidence of the Legionnaires’ disease over the past two decades together with the increasing prevalence of L. longbeachae in the Northern hemisphere highlights the necessity to gain a more detailed insight into the L. longbeachae-induced immune response. The aim of this thesis was to investigate the cellular and molecular mechanisms required for protection, focussing on immune cell responses and intracellular host-bacterial interactions. By combining a murine mouse model, a novel fluorescent reporter to track L. longbeachae, and cell depletion experiments, I uncovered the differential contribution of tissue-resident alveolar macrophages (AM) and infiltrating neutrophils to the defence against L. longbeachae. Early during infection, AM contained most of the bacteria. AM numbers sharply decreased during infection, which was accompanied by a large influx of neutrophils that also internalized bacteria. Comparative analysis of bacterial viability revealed that neutrophils were more efficient at killing and clearing of L. longbeachae than AM. In contrast, the results presented here indicated that lung-resident AM promoted infection, most likely by serving as a replicative niche. Defence against L. pneumophila is known to require IFN-gamma but not IL-18, a strong IFN-gamma inducer. However, Il18r1–/– mice were less able to clear L. longbeachae and had significantly impaired IFN-gamma levels in the lung. Furthermore, IL-18R signalling was critical for the efficient bacterial killing by neutrophils via production of reactive oxygen species. Previous bone marrow chimera experiments in our laboratory suggested that IL-18R expression in epithelial cells was necessary and sufficient for protection against L. longbeachae. However, genetic in vivo experiments using the Cre-lox-system revealed that IL-18R+ NK cells and T cells were central players in the IL-18-dependent anti-L. longbeachae defence via IFN-gamma secretion, whereas IL-18R expression by ciliated bronchiolar epithelial cells did not confer protection. Finally, our results highlighted key mechanisms by which Legionella subverts host macrophages to form an intracellular endoplasmic reticulum (ER)-like vacuole as its intracellular replicative niche. Establishment of the Legionella-containing vacuole (LCV) by recruitment of ER-derived vesicles induces ER stress. Although the relevance of ER stress in this process is unclear, effector proteins secreted by L. pneumophila are known to inhibit onset of the ER stress response. Using the pharmacological ER stress inducer thapsigargin, we showed that ER stress induces a protective host response promoting the secretion of pro-inflammatory cytokines, limiting intracellular L. pneumophila replication, and improving host survival. Mechanistically, ER stress induced a novel non-canonical activation of the transcription factor STAT1 via the IRE1 kinase driving transcription of the IFN-gamma-induced chemokine CXCL10. These results highlighted a potential role of the host ER stress response in the initiation of a protective cellular immune response towards L. pneumophila.