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| Item Type: | Article |
|---|---|
| Title: | Chaperone-mediated regulation of choline acetyltransferase protein stability and activity by HSC/HSP70, HSP90, and p97/VCP |
| Creators Name: | Morey, T.M., Winick-Ng, W., Seah, C. and Rylett, R.J. |
| Abstract: | Choline acetyltransferase (ChAT) synthesizes the neurotransmitter acetylcholine in cholinergic neurons, and mutations of this enzyme are linked to the neuromuscular disorder congenital myasthenic syndrome (CMS). 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 (14)PKLPVPP(20). We showed previously that disruption of this proline-rich motif by either proline-to-alanine mutation (P17A/P19A) or mutation of residue Val18 (V18M) enhances ubiquitination and degradation of these mutant ChAT proteins expressed in cholinergic SN56 cells by an unknown mechanism. In this study, using proximity-dependent biotin identification (BioID), co-immunoprecipitation and in situ proximity-ligation assay (PLA), we identified the heat shock proteins (HSPs) HSC/HSP70 and HSP90 as novel ChAT protein-interactors. These molecular chaperones are well-known for promoting the folding and stabilization of cellular proteins. Thus, we found that inhibition of HSPs by treatment of cells with either the HSC/HSP70 inhibitors 2-phenylethynesulfonamide (PES) or VER-155008, or the HSP90 inhibitor 17-AAG reduced cellular ChAT activity and solubility, and enhanced the ubiquitination and proteasome-dependent loss of ChAT protein. Importantly, the effects of HSP inhibition were greater for mutant ChAT proteins (P17A/P19A-ChAT and CMS-related V18M- and A513T-ChAT) compared to wild-type ChAT. HSPs can promote ubiquitination and degradation of terminally misfolded proteins through cooperative interaction with the E3 ubiquitin ligase CHIP/Stub1, and while we show that ChAT interacts with CHIP in situ, siRNA-mediated knock-down of CHIP had no effect on either wild-type or mutant ChAT protein levels. However, inhibition of the endoplasmic reticulum (ER)- and HSP-associated co-chaperone p97/VCP prevented degradation of ubiquitinated ChAT. Together, these results identify novel mechanisms for the functional regulation of wild-type and CMS-related mutant ChAT by pro-stabilizing HSPs and the pro-degradative co-chaperone p97/VCP that may have broader implications for ChAT function during cellular stress and disease. |
| Keywords: | Choline Acetyltransferase, BioID, Heat Shock Proteins, Ubiquitination, CHIP/Stub1, Proximity-Ligation Assay, Cdc48/p97/VCP |
| Source: | Frontiers in Molecular Neuroscience |
| ISSN: | 1662-5099 |
| Publisher: | Frontiers Media SA |
| Volume: | 10 |
| Page Range: | 415 |
| Date: | December 2017 |
| Official Publication: | https://doi.org/10.3389/fnmol.2017.00415 |
| PubMed: | View item in PubMed |
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