title: 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 PKLPVPP20 . 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 fluorescent-biorthogonal 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. Choline acetyltransferase (ChAT) synthesizes acetylcholine in cholinergic neurons. In this study we find that steady-state protein levels of human 69-kDa ChAT are regulated by the ubiquitin-proteasome system. Mutation of a highly conserved N-terminal proline-rich motif in human 69-kDa ChAT reduces both cellular ChAT protein levels, through enhanced ubiquitination and proteasomal degradation, and enzyme activity. Ubiquitination of catalytically deficient congenital myasthenic syndrome (CMS)-mutant ChAT is increased in cells, and importantly proteasome inhibition partially restores steady-state protein levels as well as cellular activity of some CMS-mutant ChAT proteins.
authors.affiliation: Molecular Medicine Research Group, Robarts Research Institute, Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, University of Western Ontario, London, Ontario, Canada.
authors.forename: Trevor M
authors.initials: TM
authors.lastname: Morey
authors.affiliation: Molecular Medicine Research Group, Robarts Research Institute, Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, University of Western Ontario, London, Ontario, Canada.
authors.forename: Shawn
authors.initials: S
authors.lastname: Albers
authors.affiliation: Department of Biochemistry, Schulich School of Medicine & Dentistry, University of Western Ontario, London, Ontario, Canada.
authors.forename: Brian H
authors.initials: BH
authors.lastname: Shilton
authors.affiliation: Molecular Medicine Research Group, Robarts Research Institute, Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, University of Western Ontario, London, Ontario, Canada.
authors.forename: R Jane
authors.initials: RJ
authors.lastname: Rylett
mesh_terms: D000818:Animals; D002384:Catalysis; D002478:Cells, Cultured; D002795:Choline O-Acetyltransferase / Q000235:genetics / Q000378:metabolism*; D059329:Cholinergic Neurons / Q000378:metabolism; D006801:Humans; D051379:Mice; D009154:Mutation / Q000502:physiology*; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism*; D059748:Proteolysis; D054875:Ubiquitination / Q000502:physiology*
chemical_list: D002795:Choline O-Acetyltransferase; D046988:Proteasome Endopeptidase Complex
keywords: Choline acetyltransferase (ChAT); click chemistry; proteasome; protein degradation; recombinant protein expression; ubiquitin