Enhanced ubiquitination and proteasomal degradation of catalytically deficient human choline acetyltransferase mutants

Abstract

Choline acetyltransferase (ChAT) is essential for cholinergic neuron function as it mediates synthesis of the neurotransmitter acetylcholine. ChAT mutations have been linked to the neuromuscular disorder congenital myasthenic syndrome (CMS). One CMS-related ChAT mutation, V18M, reduces enzyme activity and cellular protein levels, and is positioned within a highly conserved proline-rich motif with the sequence 14 PKLPVPP 20 . We demonstrate that N-terminal truncation that includes this proline-rich motif, as well as mutation of prolines-17/19 together to alanine (P17A/P19A), dramatically reduces ChAT steady-state protein levels and cellular activity when expressed in cholinergic SN56 neural cells. The in vitro activity of bacterially expressed recombinant P17A/P19A-ChAT is also reduced, although this is not caused by changes in protein secondary structure or thermal stability. Treatment of SN56 cells with the proteasome inhibitor MG132 increases cellular P17A/P19A-ChAT steady-state protein levels, and by immunoprecipitation we found that ChAT is ubiquitinated and that polyubiquitination of P17A/P19A-ChAT is increased compared to wild-type (WT) ChAT. Using a novel fluorescentbiorthogonal pulse-chase protocol in SN56 cells, we determined that the protein half-life of P17A/P19A-ChAT (2.2 h) is substantially reduced compared to WT-ChAT (19.7 h). Lastly, we show that two CMS-related ChAT mutants (V18M and A513T) have enhanced ubiquitination, and that treatment with MG132 can partially restore both the steady-state protein levels as well as cellular activity of some CMS-mutant ChAT. These results identify a novel mechanism for regulation of ChAT through the ubiquitin-proteasome system that is influenced by the conserved N-terminal proline-rich motif of ChAT and may be implicated in CMS pathology.

Pedunculopontine Nucleus Cholinergic Deficiency in Cervical Dystonia

“…A decreased protein level may be the result of increased degradation or decreased production. Increased ChAT degradation occurs in congenital myasthenic syndrome because of mutations leading to increased ubiquitination . This is unlikely to be the case in CD because of normal staining in the putamen and the absence of ubiquitin inclusions.…”

“…Increased ChAT degradation occurs in congenital myasthenic syndrome because of mutations leading to increased ubiquitination. 32 This is unlikely to be the case in CD because of normal staining in the putamen and the absence of ubiquitin inclusions. The alternative explanation is decreased protein production, either by decreased messenger ribonucleic acid (mRNA) transcription or protein translation.…”

Ubiquitin-Proteasome Modulating Dolabellanes and Secosteroids from Soft Coral Clavularia flava

“…ChAT catalyzes the production of acetylcholine which is an essential neurotransmitter. Moreover, proteasome inhibitor MG132 stabilizes ChAT steady-state protein levels and increases the enzyme activity [10]. Therefore, we reason that stolonidiol may mediate the elevation of ChAT activity via both pathways in the inhibition of proteasome and the activation of protein kinase C [9][10][11].…”

“…Moreover, proteasome inhibitor MG132 stabilizes ChAT steady-state protein levels and increases the enzyme activity [10]. Therefore, we reason that stolonidiol may mediate the elevation of ChAT activity via both pathways in the inhibition of proteasome and the activation of protein kinase C [9][10][11]. Among the four dolabellanes-based compounds with stolonidiol (2) 5 µg/mL treatment, the EGFP-UL76 displayed the highest increase ratios of 2.12 and 1.75 of integrated and average intensity, respectively (Figure 2).…”

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

“…Residue Val 18 does not participate directly in acetyl-CoA binding, but is located within a highly-conserved, surface-exposed N-terminal proline-rich motif with sequence 14 P KL P V PP 20 that shares homology with the core PxxP binding-motif for SH3-binding domains (Kim et al, 2006 ; Kurochkina and Guha, 2013 ). We showed recently that ChAT protein stability is regulated by the ubiquitin-proteasome system and, importantly, that mutation of this proline-rich motif, including P17A/P19A and CMS-related V18M, as well as the CMS-related mutant A513T enhances the ubiquitination and proteasome-dependent degradation of ChAT protein (Morey et al, 2016 ). The mechanisms responsible for the basal regulation of ChAT protein stability and/or for the changes observed following mutation of ChAT, in particular disruption of the N-terminal proline-rich motif, have not been elucidated.…”