Biochemistry and Pharmacology - Theses
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ItemMapping the trafficking and assembly of Plasmodium falciparum virulence complex( 2024-01)The ability of the human malaria parasite Plasmodium falciparum to remodel its host red blood cell (RBC) is central to the parasite’s ability to survive within the circulation of its host and cause disease. Following invasion, the parasite exports proteins into the RBC cytoplasm and membrane where they remodel the membrane skeleton and alter the cell’s biophysical properties. To facilitate this remodelling the parasite builds structures called Maurer’s clefts in the cytoplasm which act as intermediate compartments for the trafficking of proteins to the RBC membrane especially the major virulence protein, PfEMP1. PfEMP1 is trafficked to protrusions on the RBC membrane called the knob. Knobs displaying PfEMP1 form the P. falciparum virulence complex which in turn mediates cytoadhesion to capillary walls throughout the body resulting in severe disease. Despite the importance of this structure, the mechanism of PfEMP1 trafficking to the surface and the organisation and assembly of the knobs are unknown. In this thesis, I address this gap in our understanding of virulence protein trafficking through the functional characterisation of three previously uncharacterised proteins that locate at and function at the Maurer’s clefts and RBC membrane skeleton. During the remodelling process the Maurer’s clefts become immobilised at the underside of the RBC membrane. This process was hypothesised to be controlled by a tube-like structure called the tether which appears to connect the Maurer’s clefts to the RBC membrane. The function and formation of the tether is unknown. I conditionally knocked out the only known tether protein, MAHRP2 (PF3D7_1353200). I show that MAHRP2 localises to the Maurer’s cleft periphery and tether structures. I show that conditional deletion of MAHRP2 results in a loss of tether formation and a change in Maurer’s cleft architecture. This results in reduced immobilisation of the Maurer’s clefts and a decrease in PfEMP1 surface trafficking. Next, I characterised a protein - PTP8 (PF3D7_0113200) associated with a newly identified Maurer’s cleft and J-dot protein complex. We show that PTP8 localises to the periphery of the Maurer’s cleft. Conditional knockdown of PTP8 decreases iRBC cytoadherence and loss of Maurer’s cleft anchoring. Our data suggests an extended network of Maurer’s cleft peripheral proteins acting as accessory proteins for Maurer’s cleft anchoring and PfEMP1 onward export from the clefts. The third protein characterised was a member of the PHIST family- ESAP1 (PF3D7_0532300). Localisation studies showed that ESAP1 is localised to the Maurer’s clefts early in development, before being trafficking to the RBC membrane skeleton where it appears as a homogeneous labelling pattern. Deletion of this protein leads to large extended, lobed, and smaller vesicle structures at the RBC membrane. Examination of these abnormal structures using Airyscan microscopy shows a reorganisation of the RBC membrane skeleton components. We also show that ESAP1 interacts with host myosin and that this interaction is essential for normal knob formation. The data suggests a possible role for ESAP1 in facilitating and organising the knob structure. Using quantitative mass spectrometry, we identified several uncharacterised PHIST proteins that may be involved in knob biogenesis. We were able to add to the virulence protein trafficking network and suggest an extensive interplay between protein involved in the trafficking of PfEMP1 and assembly of knobs
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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)