Biochemistry and Pharmacology - Theses
Permanent URI for this collection
Search Results
1 - 3 of 3
-
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.
-
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.
-
ItemNo Preview AvailableInvestigation of alternative splicing in apicomplexan parasites( 2021)Alternative splicing is the phenomenon by which coding and non-coding regions of pre-mRNA molecules can be differentially spliced to yield multiple mRNA isoforms from a single gene. In metazoans, alternative splicing occurs to a substantial degree, contributing to protein diversity and the post-transcriptional regulation of gene expression. However, to what extent this occurs in apicomplexan parasites is much less understood. This thesis examines the landscape, regulators and function of alternative splicing in two apicomplexan parasites, T. gondii and P. falciparum. Technological advances in the short read sequencing of nucleic acids at unprecedented depths have enabled deep profiling of the transcriptome. However, the short reads present a limitation in the analysis of complex splicing events that span beyond the length of the reads. We evaluated the capability of a third generation long read sequencing technology, Oxford Nanopore Technologies (ONT) sequencing, in sequencing full-length native mRNA from T. gondii and P. falciparum, and established a method to analyse the alternative splicing landscape from the long reads. We successfully identified full-length transcripts spanning annotated and non-annotated junctions, implying a suitability in exploring complex splicing events. The analysis reveals an unusually high level of intron retained transcripts with premature terminating codons (PTCs). This suggests that most alternative splicing events in T. gondii and P. falciparum are unlikely to be productive. Alternative splicing in metazoans is modulated by alterative splicing factors, most notably the SR (serine-arginine–rich) protein family. We characterised the suite of SR proteins and two putative kinases/regulators of SR proteins in T. gondii. The proteins were found localised to sub-nuclear compartments characteristic of splicing factors. We demonstrated through genetic ablation and whole-transcriptome sequencing that the SR proteins modulate distinct but overlapping subsets of mostly non-productive alternative splicing events, as well as impacting transcript abundance. Alternatively spliced junctions were also enriched in characteristic SR binding motifs. The putative kinases of SR proteins were found to be essential to parasite survival and modulate extensive splicing events, but the events poorly mirrored that modulated by the SR proteins. This suggests a complex system of splicing regulation that do not conform to other eukaryotic models. The targeting of non-productive alternatively spliced transcripts for degradation through the nonsense mediated decay (NMD) pathway is one mechanism by which metazoans post-transcriptionally regulate gene expression. To explore if this was the case for T. gondii, we characterised the three core NDM proteins- UPF1, UPF2 and UPF3. The three proteins were found to co-immunoprecipitate with one another, implying a conservation of the core NMD complex. However, when we conditionally ablated the UPF proteins, parasite growth and survival was not impacted. We sequenced the parasite mRNA and found that only UPF2 impacted global intron retention rates. Moreover, a link between intron retention and gene expression regulation could not be established. Our results show that the fitness cost of mis- splicing determines intron retention rates, rather than targeted regulation. Hence, this thesis has shown that although non-productive alternative splicing is widespread and regulated in T. gondii, it is not a mechanism for post-transcriptional regulation of gene expression through the NMD pathway.