Biochemistry and Pharmacology - Theses
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ItemDoxycycline has a dual mode of action against malaria parasites( 2021)Traditionally used as a broad-spectrum antibiotic, doxycycline is frequently used for malaria prophylaxis and treatment - the latter in combination with artemisinin derivatives. Its mechanism of action in Plasmodium spp. has not yet been fully elucidated, though there is substantial evidence that ribosomes in the apicoplast - a relict plastid - are the primary target, with doxycycline causing delayed death (a phenotype associated with inhibitors of apicoplast housekeeping). Inhibition of the apicoplast depletes isoprenoids, synthesised via a pathway housed in the organelle, perturbing the prenyl-dependent trafficking mechanism for haemoglobin uptake and trafficking. This same uptake of haemoglobin is required for activation of artemisinin derivatives. Here, we show that apicoplast-targeting antibiotics, such as doxycycline, reduce the abundance of the catalyst of artemisinin activation (free haem) in P. falciparum, likely through diminished haemoglobin digestion. We demonstrate antagonism between dihydroartemisinin and these antibiotics, likely because apicoplast inhibitors reduce artemisinin activation. Separately, we identify a secondary, more immediate target of doxycycline that exists at clinically relevant concentrations. We show that supplementation with the apicoplast-derived isoprenoid precursor, isopentenyl pyrophosphate, only rescues parasites from delayed death, demonstrating independence of the first cycle target from the relict plastid. Instead, we show that doxycycline depletes mitochondrial electron transport and selectively reduces the abundance of proteins encoded by the mitochondrion in the related apicomplexan parasite, Toxoplasma gondii, suggesting that inhibition of mitochondrial protein synthesis could underpin the immediate death phenotype caused by doxycycline. These data have potential clinical significance when considering the reliance on - and widespread use of - doxycycline and other apicoplast-targeting antibiotics in malaria endemic regions. They reinforce the strategic importance of rational choice of antimalarial combinations; and lay the groundwork for further exploration of the underlying mechanisms of drug resistance in Plasmodium parasites.
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ItemThe human TIM22 complex: functions, substrates, and connections to disease( 2021)Mitochondria are hubs of metabolic activity in cells. In addition to supporting ATP production through nutrient catabolism, mitochondria also house many crucial biosynthetic pathways, including iron-sulphur cluster synthesis and heme synthesis. The crucial role of mitochondria in cellular function means that their dysfunction is associated with a variety of human pathologies, including cancer, neurodegenerative disease, and mitochondrial disease. The diversity of mitochondrial functions necessitates a large and versatile proteome, which in human cells contains over 1000 proteins. Targeting and import of mitochondrial proteins to the correct mitochondrial subcompartment is mediated by multi-subunit complexes called translocases. Insertion of proteins with multiple transmembrane domains into the inner mitochondrial membrane is mediated by a translocase called the Translocase of the Inner Membrane 22 (TIM22) complex. The human TIM22 complex contains two subunits, AGK and Tim29, which are not present in the more extensively characterised Saccharomyces cerevisiae TIM22 complex. AGK had been previously described as a lipid kinase, although its kinase activity is dispensable for its function at the TIM22 complex. Mutations in the AGK gene cause a mitochondrial disease called Sengers syndrome, characterised by congenital cataracts, hypertrophic cardiomyopathy, lactic acidosis, and exercise intolerance. Despite recent advances in understanding of AGK function, the extent to which the functions of AGK in lipid and protein biogenesis contribute to the pathogenesis of Sengers syndrome were unclear, and the impact of AGK mutations on protein function had not been assessed. We set out to define the impact of AGK and TIM22 complex dysfunction on mitochondrial biology to provide much needed information on how perturbations at the TIM22 complex cause mitochondrial disease. Using unbiased proteomics approaches we characterised multiple Sengers syndrome patient fibroblast and disease-model cell lines, identifying mitochondrial one-carbon metabolism as a dysregulated pathway and potential therapeutic target in Sengers syndrome. One-carbon metabolism supports redox homeostasis and translation within the mitochondria, and its dysregulation might contribute to the pathology of Sengers syndrome. This analysis also identified Sideroflexins as novel substrates of the TIM22 complex. Sideroflexins are serine transporters required for one-carbon metabolism, providing a direct link between AGK/TIM22 complex dysfunction and the observed one-carbon metabolism defect. We also used mitochondrial proteomics and biochemistry to analyse several AGK mutations, revealing that AGK mutations vary in their functionality, and that partial functionality of certain mutations might underpin phenotypic variability observed for Sengers syndrome. Our focus on sideroflexins as TIM22 complex substrates drew our attention to SFXN4, a poorly characterised and divergent sideroflexin that cannot transport serine and causes a mitochondrial disease when mutated. We demonstrated that deletion of SFXN4 results in a severe isolated complex I defect, and that SFXN4 interacts with extensively with complex I assembly factors, establishing it as a bona-fide complex I assembly factor. Overall, this project demonstrates the power of unbiased approaches for defining protein function and identifying targetable pathways in mitochondrial disease. This study highlights the importance of fundamental biology for generating foundational knowledge that forms the basis of our understanding of human disease.
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ItemTargeting the mitochondrion in Coxiella burnetii infection( 2020)Mitochondria are essential organelles, fundamental to eukaryotic cell function and survival. Perhaps best known for their role in energy production, mitochondria are also central to many cellular processes, including calcium homeostasis, immune and cell death signalling. With such diverse cellular roles, it is no surprise that microbial virulence factors target the mitochondrion during infection. Coxiella burnetii is a unique intracellular bacterial pathogen and the causative agent of Q fever. The bacterium infects alveolar macrophages and replicates within a highly acidic, lysosome-like vacuole, termed the Coxiella-containing vacuole (CCV). C. burnetii translocates over 150 bacterial effector proteins into the host cytosol via a Type 4 Secretion System (T4SS). Effector proteins translocated into the cell modulate cellular functions to facilitate CCV development and bacterial replication. This study has aimed to deepen our understanding of the host-pathogen interactions occurring between C. burnetii and the host cell mitochondrion during infection. In doing so, we have developed new methods and adapted current technologies to the study of this fascinating bacterial pathogen. We uncovered the effector protein CBU0077 (later renamed Mitochondrial Coxiella effector A (MceA)) that localised to the mitochondrial network during infection. This established the mitochondrion is a bona fide target during C. burnetii infection. To investigate whether additional C. burnetii proteins associate with the organelle during infection, we analysed mitochondria purified from infected cells by high-sensitivity mass spectrometry. This unbiased, proteomic approach identified an additional 7 effector proteins associated with mitochondria in the context of infection and we further went on to biochemically characterise CBU1425. We were able to demonstrate that CBU1425 is imported into the mitochondria and interacts with key components of the organelle quality control pathway, revealing a new aspect of C. burnetii biology. A complex interplay exists between the host cell and the pathogenic agent during infection. To disentangle the impact of C. burnetii infection on the host cell and the mitochondrion, we utilised SILAC quantitative proteomics to investigate changes to the cellular and organellar proteome. This revealed a global effect on cellular and mitochondrial pathways. Our findings compare and contrast these organelle-wide changes between cell types and another distinct pathogen, providing a deeper understanding of the role of the mitochondrion during C. burnetii infection and, more broadly in the host cell response to this assault. This research has contributed comprehensive new insights into our understanding of the interaction between the host cell mitochondria and C. burnetii during infection. With a foundational knowledge of the host-pathogen interactions occurring during infection, we can begin to further probe the molecular requirements and outcomes of our own cellular pathways in both health and disease.