Biochemistry and Pharmacology - Theses
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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.