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.
Morey, Trevor M., "Regulation of Human 69-kDa Choline Acetyltransferase Protein Stability and Function by Molecular Chaperones and the Ubiquitin-Proteasome System" (2017). Electronic Thesis and Dissertation Repository. 5218.
Available for download on Sunday, March 01, 2020