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.

The M-transcript of human choline acetyltransferase (ChAT) produces an 82-kDa protein (82-kDa ChAT) that concentrates in nuclei of cholinergic neurons. We assessed the effects of acute exposure to oligomeric amyloid-β1–42 (Aβ1–42) on 82-kDa ChAT disposition in SH-SY5Y neural cells, finding that acute exposure to Aβ1–42 results in increased association of 82-kDa ChAT with chromatin and formation of 82-kDa ChAT aggregates in nuclei. When measured by chromatin immunoprecipitation with next-generation sequencing (ChIP-seq), we identified that Aβ1–42 -exposure increases 82-kDa ChAT association with gene promoters and introns. The Aβ1–42 -induced 82-kDa ChAT aggregates co-localize with special AT-rich binding protein 1 (SATB1), which anchors DNA to scaffolding/matrix attachment regions (S/MARs). SATB1 had a similar genomic association as 82-kDa ChAT, with both proteins associating with synapse and cell stress genes. After Aβ1–42 -exposure, both SATB1 and 82-kDa ChAT are enriched at the same S/MAR on the APP gene, with 82-kDa ChAT expression attenuating an increase in an isoform-specific APP mRNA transcript. Finally, 82-kDa ChAT and SATB1 have patterned genomic association at regions enriched with S/MAR binding motifs. These results demonstrate that 82-kDa ChAT and SATB1 play critical roles in the response of neural cells to acute Aβ -exposure.

Background: HSP70 inhibits heat-induced apoptosis by preventing NOXA accumulation. Results: Inhibition of CDK5 in heat-shocked cells reduces levels of miR-23a leading to increased NOXA abundance, which is prevented by HSP70. Conclusion: CDK5 regulates miR-23a expression and affects NOXA abundance. Significance: The prosurvival function of HSP70 can be attributed to its ability to prevent the stress-induced inhibition of CDK5.

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.

Assessing the stability and degradation of proteins is central to the study of cellular biological processes. Here, we describe a novel pulse-chase method to determine the half-life of cellular proteins that overcomes the limitations of other commonly used approaches. This method takes advantage of pulse-labeling of nascent proteins in living cells with the bioorthogonal amino acid L-azidohomoalanine (AHA) that is compatible with click chemistry-based modifications. We validate this method in both mammalian and yeast cells by assessing both over-expressed and endogenous proteins using various fluorescent and chemiluminescent click chemistry-compatible probes. Importantly, while cellular stress responses are induced to a limited extent following live-cell AHA pulse-labeling, we also show that this response does not result in changes in cell viability and growth. Moreover, this method is not compromised by the cytotoxicity evident in other commonly used protein half-life measurement methods and it does not require the use of radioactive amino acids. This new method thus presents a versatile, customizable, and valuable addition to the toolbox available to cell biologists to determine the stability of cellular proteins.