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.
Highlights
Choline O-acetyltransferase (ChAT, EC2.3.1.6) catalyzes synthesis of the neurotransmitter acetylcholine (ACh) using the substrates acetyl-CoA and choline (Oda, 1999; Abreu-Villaca et al, 2011)
We show that Choline acetyltransferase (ChAT) interacts with and is a client for the heat shock proteins HSC/HSP70 and HSP90, where inhibition of HSC/HSP70 protein interactions results in accumulation of insoluble ChAT protein, and inhibition of HSC/HSP70 and HSP90 activity led to reduced cellular ChAT activity, along with enhanced ChAT ubiquitination and proteasome-dependent loss of ChAT protein
Though we show that ChAT interacts with the HSP-associated E3 ubiquitin ligase Cterminus of heat shock cognate kDa protein (HSC70)-interacting protein (CHIP), Small-Interfering RNA (siRNA)-mediated knock-down of CHIP had no effect on ChAT protein levels
Summary
Choline O-acetyltransferase (ChAT, EC2.3.1.6) catalyzes synthesis of the neurotransmitter acetylcholine (ACh) using the substrates acetyl-CoA and choline (Oda, 1999; Abreu-Villaca et al, 2011). In humans and non-human primates only, the M-transcript encodes for an 82-kDa ChAT protein due to the presence of a unique in-frame translation initiation codon located 5′ to the common translation initiation site for 69-kDa ChAT (Oda et al, 1992; Chireux et al, 1995; Misawa et al, 1997; Ohno et al, 2001) This primate-specific 82-kDa ChAT protein contains a nuclear localization signal (NLS) located within its extended N-terminus that directs it to the nucleus of cholinergic neurons in human brain and spinal cord (Resendes et al, 1999; Gill et al, 2003, 2007). Though our lab has shown that nuclear 82-kDa ChAT can synthesize ACh (Resendes et al, 1999; Gill et al, 2003) and alter gene expression (Albers et al, 2014; Winick-Ng et al, 2016), the function/s of this ChAT protein remain under investigation
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