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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ItemProteomic analysis of mHttex1 expression in Huntington’s disease( 2021)Huntington’s disease (HD) is a fatal neurodegenerative disorder caused by CAG trinucleotide repeat expansion in exon 1 of the Huntingtin (Htt) gene. This sequence encodes an abnormally elongated polyglutamine (polyQ) tract within the Huntingtin (Htt) protein that is directly involved in aggregation and Htt-mediated cytotoxicity. The key pathological signature of HD is the aggregation of mutant Htt protein into punctate aggregates. However, the mechanism by which polyQ-expanded mutant Httex1 (mHttex1) causes toxicity remains elusive. Previous research has indicated that mHttex1 can exert toxicity to cell models through two distinct phases. The first is when the protein is soluble and the second is when it is aggregated into inclusion bodies, which are the major pathological signature of HD brain. I hypothesized that apoptosis is caused by an unresolved quality control mechanism that oversees mHttex1 at synthesis. The goal of this project was to develop and implement a novel proteomics strategy to specifically detect the proteins that engage with mutant Htt during protein synthesis. I compared pathogenic huntingtin (Q97) and non-pathogenic huntingtin (Q25) using a proteomics-based approach. Firstly, a self-cleaving NS3 viral protease system called TimeSTAMP was employed, which can efficiently cleave epitopes from newly synthesized proteins and be potently inhibited using a viral protease inhibitor. The goal was to inhibit the cleavage across different-time windows to “pulse” label newly synthesized Htt and at the end of the pulse steps, proteins were crossed-linked with disuccinimidyl sulfoxide (DSSO) to preserve transient interactions. We also wanted to examine the changes in the global proteome and phosphoproteome across these mutant form and wild-type counterpart. After transfection of Neuro2a cells with TimeSTAMP-Httex1 constructs of differing poly-Q length, cells were lysed using RIPA lysis buffer. Proteins were then treated with a label-free relative quantitative phosphoproteomics workflow: i.e., samples were denatured (e.g., in 8 M urea), reduced and alkylated, then subjected to tryptic digestion. Next, a phosphopeptide enrichment step was performed before samples analyzed using LC-MS/MS. However, there was no significant difference observed between pathogenic and non-pathogenic Huntingtin, indicating a lack of polyQ-length dependence. To further probe the toxicity of the pathogenic huntingtin, I investigated its protein-protein interactions using a different proteomics-based approach. After transfection of Neuro2a cells with the Q25-GFPEm or Q97-GFPEm constructs, proteins were cross-linked with disuccinimidyl sulfoxide (DSSO) and then protein interactors were pulled down using anti-GFP VHH coupled magnetic agarose beads. We also examined the changes in the global proteome and phosphoproteome across the mutant form and wild-type counterpart. I did not find any significant difference between pathogenic and non-pathogenic Huntingtin which further indicates that the result was not polyQ dependent. Determination of these mechanisms are anticipated to be important for the design of new therapeutic strategies that mitigate toxicity of soluble mHttex1.