Chaperone-Mediated Regulation of Choline Acetyltransferase Protein Stability and Activity by HSC/HSP70, HSP90, and p97/VCP

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 14PKLPVPP20. 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.

RETRACTED: STUB1 suppresseses tumorigenesis and chemoresistance through antagonizing YAP1 signaling

“…*P < .05; **P < .01 promote the ubiquitination and degradation of proteins through cooperative interaction with the E3 ubiquitin ligase STUB1. 32 Here, we used 17-AAG to treat cells and found significantly reduced YAP1 ubiquitination ( Figure 2C). To identify the specific lysine sites in the YAP1 protein with ubiquitination modification, we used UbPred software (http://www.ubpred.org/).…”

Congenital Myasthenic Syndrome Caused by a Novel Hemizygous CHAT Mutation

“…This mutation causes a structural change in the substrate-binding site (Figures 2C,D). Mutations located near the active-site tunnel impair substrate binding and result in more severe phenotypic effects in patients with CHAT-related CMS (12)(13)(14). Glutamine possesses features of polarity compared with non-polar leucine due to the amide group in the residue side chain.…”

“…To date, 48 CHAT gene mutations have been identified as related to CMS (http://www.hgmd.cf.ac.uk/ ac/dipx.php). Mutations located near the active-site tunnel, impairing substrate binding, result in more severe phenotypic effects in patients with CHAT-related CMS (12)(13)(14). Fiftytwo cases of CMS caused by CHAT gene mutations have been reported, and only two of them have been identified as hemizygous mutations (12,13,(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29).…”

SPAAC Pulse-Chase: A Novel Click Chemistry-Based Method to Determine the Half-Life of Cellular Proteins

“…Our previously published results showed that inhibition of proteasome function by MG132 treatment, but not lysosomal function by chloroquine treatment, resulted in increased steady-state protein levels of both wild-type and P17A/P19A-ChAT ( Morey et al, 2016 , 2017 ). Furthermore, we have shown that MG132 treatment resulted in stabilization of ubiquitinated wild-type and P17A/P19A-ChAT, suggesting that ChAT protein degradation is regulated through the proteasome.…”