Electronic Thesis and Dissertation Repository


Doctor of Philosophy




Rylett, R. Jane


The enzyme choline acetyltransferase (ChAT) mediates synthesis of the neurotransmitter acetylcholine required for cholinergic neurotransmission. ChAT mutations are linked to congenital myasthenic syndrome (CMS), a rare neuromuscular disorder. One CMS-related mutation, V18M, reduces ChAT enzyme activity and cellular protein levels, and is located within a highly-conserved N-terminal proline-rich motif at residues 14PKLPVPP20. It is currently unknown if this motif regulates ChAT function. In this thesis, I demonstrate that disruption of this proline-rich motif in mouse cholinergic SN56 cells reduces both the protein levels and cellular enzymatic activity of mutated P17A/P19A- and V18M-ChAT. The cellular loss of mutant ChAT protein appears to be a result of increased proteasome-dependent degradation due to enhanced ChAT ubiquitination. Using a novel fluorescent-biorthogonal pulse-chase protocol, I determined that the cellular protein half-life of P17A/P19A-ChAT (2.2 h) is substantially reduced compared to wild-type ChAT (19.7 h), and that proteasome inhibition by MG132 treatment increases the half-life and steady-state levels of ChAT protein. By proximity-dependent biotin identification (BioID), co-immunoprecipitation, and in situ proximity-ligation assay (PLA), I identified the heat shock proteins HSC/HSP70 and HSP90 as novel ChAT protein-interactors that are enriched in cells expressing mutant P17A/P19A-ChAT. Pharmacological inhibition of these HSPs by treatment with the HSC/HSP70 inhibitors 2-phenylethynesulfonamide (PES) or VER-155008, or the HSP90 inhibitor 17-AAG reduced cellular ChAT activity and solubility, and enhanced ubiquitination and proteasomal loss of ChAT protein. The effects of HSP inhibition were greatest for mutant P17A/P19A- and V18M-ChAT. While I observed that ChAT interacts in situ with the HSP-associated E3 ubiquitin ligase CHIP, siRNA-mediated knock-down of CHIP had no effect on ChAT protein levels. Inhibition of HSC/HSP70 by PES treatment sensitized ChAT to H2O2-induced insolubilization, and ChAT ubiquitination was enhanced following H2O2 treatment. Lastly, inhibition of the endoplasmic reticulum (ER)- and HSP-associated co-chaperone Cdc48/p97/Valosin-containing protein (VCP) prevented the degradation of ubiquitinated ChAT. Together my results identify novel mechanisms for the functional regulation of wild-type and CMS-related mutant ChAT by multiple molecular chaperones and the ubiquitin-proteasome system that, importantly, may have broader implications for ChAT function during cellular stress and disease.