Biochemistry and Pharmacology - Theses
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ItemReaction hijacking tyrosyl-tRNA synthetase as a new anti-infectives strategy( 2022)Malaria is a deadly disease of humans, with Plasmodium falciparum responsible for the most cases. Disappointingly, drug resistance is observed against current front-line therapies; thus, new drugs with novel mechanisms are urgently needed. ML901, a nucleoside sulfamate derivative, has been shown to possess good antimalarial efficacy and to specifically target P. falciparum tyrosyl-tRNA synthetase (PfYRS). PfYRS is a pivotal enzyme that participates in the protein synthesis pathway, in which tyrosine-charged tRNA is formed. ML901 appears to target PfYRS via a novel reaction hijacking mechanism in which PfYRS catalyzes the synthesis of a Tyr-ML901 adduct, which in turn poisons the enzyme. Human YRS is not susceptible to the reaction hijacking mechanism. This project sought to understand the molecular basis for the potency and specificity of ML901 and to determine if reaction hijacking could be exploited more widely. All YRS sequences harbor a conserved motif, referred to as “KMSKS”, in a loop that is reported to change conformation to facilitate ATP binding and the aminoacylation reaction. Sequence alignment across species reveals that most pathogenic parasites, including P. falciparum, possess a KMSKS motif, whereas higher eukaryotes possess an equivalent KMSSS motif. Structural analysis revealed that the motif in human YRS is part of a flexible (unstructured) loop while the equivalent loop is structured in PfYRS. Here we examined the role of the second lysine (K250) in determining loop flexibility and activity of PfYRS as well as the susceptibility of the mutant enzyme to reaction hijacking. Surprisingly, the X-ray crystal structure of recombinant PfYRS harboring the K250S mutation (PfYRSK250S) showed that the KMSSS loop is even more stable than the wildtype KMSKS loop. PfYRSK250S was found to consume substantively less ATP in the initial activation step. However, the weakly active PfYRSK250S is still susceptible to reaction hijacking by ML901. This study shows that the flexibility of the loop is not determined simply by the K250. Moreover, it shows that K250 plays an important role in enzymatic mechanism. Further investigations are required to understand the important factors that contribute to the particular susceptibility of PfYRS to reaction hijacking by ML901. Broad specificity nucleoside sulfamates, such as adenosine sulfamate (AMS), have previously been shown to have inhibitory activity against Gram-positive and Gram-negative bacteria. The equivalent of the KMSKS motif in the Escherichia coli YRS sequence is KFGKT. Here we explored the possibility that bacterial YRS might also be susceptible to inhibition via a reaction hijacking mechanism. A screen of a range of bacterial species revealed that AMS inhibits growth of E. coli and Enterococcus faecium. Targeted mass spectrometry confirmed the production of a range of amino acids adducts upon treatment in E. coli with AMS. Recombinant EcYRS was purified and expressed and shown to be inhibited via the reaction hijacking mechanism by AMS. These data suggest that bacterial amino acyl tRNA synthetases may be exciting new targets for reaction-hijacking nucleoside sulfamates.
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ItemUbiquitination in the malaria parasite Plasmodium falciparum( 2022)Ubiquitin is a post-translational modification that plays a role in many cellular processes, including protein degradation, trafficking, and signaling. The ubiquitination machinery includes E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes, E3 ubiquitin ligases, ubiquitin-binding domain-containing proteins, and deubiquitinases. In the malaria parasite P. falciparum, only a few ubiquitination proteins have been characterised and <10 more have been implicated in drug resistance. Post-translational mechanisms are known to be important in sexual development in Plasmodium, and so we investigated the role of selected ubiquitination proteins in differentiation into sexual forms called gametocytes. Using a CRISPR/Cas9 knockout strategy, we initiated the characterisation of selected ubiquitination genes that are upregulated in gametocytes compared to asexual parasites. We found two ubiquitination genes, encoding for a polyubiquitin binding protein and an E2 ubiquitin-conjugating enzyme, that play an important role on the regulation of sex-specific differentiation and stage development. Loss of the polyubiquitin binding protein produced gametocytes that reached late stages but lack a defined sex. Loss of the E2 ubiquitin-conjugating enzyme produced gametocytes with a morphological defect in the late stages and lack a defined sex. We also investigated the role of Kelch 13 (K13), a protein mutated in artemisinin-resistant parasites and hypothesised to be a ubiquitination protein and demonstrate that it is required for normal parasite uptake of haemoglobin. This work furthers our knowledge on the role of ubiquitination and of K13 in P. falciparum.
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ItemNo Preview AvailableUnderstanding virulence protein trafficking in the P. falciparum infected red blood cell( 2022)After invading the red blood cell (RBC), the malaria-causative parasite P. falciparum traffics an adhesin, erythrocyte membrane protein 1 (here referred to as EMP1), to the host cell surface. EMP1 is essential for parasite survival in vivo as it prevents splenic clearance. Moreover, variants of EMP1 that confer cytoadherence to cerebral or placental tissue can lead to fatal complications of the disease. In addition to EMP1, ~500 other parasite proteins are exported into the host cell compartment, localizing to parasite- induced membrane-bound and membraneless structures and knob-like protrusions at the RBC surface. These exported proteins do not resemble canonical trafficking machinery (e.g., ESCRT, SNARE, and Rab machineries), and there is no remnant secretory machinery for the parasite to co-opt, so how EMP1 is transported through the host cell cytoplasm remains unclear. Here we present functional characterization of two exported proteins with potential roles in EMP1 transport. First focusing on the gametocyte exported protein 7 (GEXP07), we found that GEXP07 localizes to the Maurer’s clefts, an intermediate compartment for EMP1 trafficking. In the absence of GEXP07, the clefts segment into smaller membrane- bound structures, the knobs are larger and clustered, and EMP1 transport to the host cell surface is reduced. The work confirms the critical role of Maurer’s clefts in EMP1 trafficking and reveals a previously unappreciated link between EMP1 transport and host cell remodeling. We also characterized the PfEMP1 trafficking protein 7 (PTP7). We found that PTP7 localizes to the Maurer’s cleft and associated structures, including vesicles and membraneless structures called J-dots. In the absence of PTP7, the clefts become decorated with budding vesicles - seemingly stalled in the process of fission. The knobs morphology is altered and forward trafficking of EMP1 from the cleft to the infected RBC surface is ablated. We show that the poly-asparagine repeat-containing C-terminal domain of PTP7 is essential for its function. The work leads to the intriguing suggestion that low complexity domains might be important for the function of these non-canonical trafficking proteins. We sought to further understand the physical processes that might drive trafficking of exported proteins and explored the role of intrinsically unstructured protein domains, which have been shown to drive some trafficking events in other organisms. As mobile, membrane-less structures, the J-dots resemble the phase separated biomolecular condensates observed in other eukaryotes. Here, we used an optogenetic technique in a heterologous mammalian cell system to explore the condensate-forming potential of full-length J-dot proteins and protein domains. We identified the central region of a protein called 0801 as having a propensity to phase separate. We generated sequence variants of this protein region and determined the molecular grammar responsible for condensate formation. This work points to the possibility that intrinsically unstructured protein domains could play a previously unrecognized role in protein trafficking and/or protein sequestration in P. falciparum. Overall, the work presented in this thesis adds new insights to our understanding of EMP1 trafficking. Also, in the context of the RBC cytoplasm which lacks canonical trafficking machinery, our preliminary findings regarding the phase separation capacity of some exported proteins may inform the wider field of protein transport. If validated in P. falciparum, our findings prompt a re-evaluation of the molecular requirements for coordinated protein transport in eukaryotes more broadly.