Barker, Eleanor, Milburn, Amy, Helassa, Nordine 
ORCID: 0000-0003-3743-1886, Hammond, Dean, Sanchez-Soriano, Natalia, Morgan, Alan ORCID: 0000-0002-0346-1289 and Barclay, Jeff
(2024)
Proximity labelling reveals effects of disease-causing mutation on the DNAJC5/cysteine string protein α interactome.
The Biochemical journal, 481 (3).
BCJ20230319-BCJ20230319.
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Abstract
Cysteine string protein α (CSPα), also known as DNAJC5, is a member of the DnaJ/Hsp40 family of co-chaperones. The name derives from a cysteine-rich domain, palmitoylation of which enables localization to intracellular membranes, notably neuronal synaptic vesicles. Mutations in the DNAJC5 gene that encodes CSPα cause autosomal dominant, adult-onset neuronal ceroid lipofuscinosis (ANCL), a rare neurodegenerative disease. As null mutations in CSP-encoding genes in flies, worms and mice similarly result in neurodegeneration, CSP is evidently an evolutionarily conserved neuroprotective protein. However, the client proteins that CSP chaperones to prevent neurodegeneration remain unclear. Traditional methods for identifying protein-protein interactions such as yeast 2-hybrid and affinity purification approaches are poorly suited to CSP, due to its requirement for membrane anchoring and its tendency to aggregate after cell lysis. Therefore, we employed proximity labelling, which enables identification of interacting proteins in situ in living cells via biotinylation. Neuroendocrine PC12 cell lines stably expressing wild type or L115R ANCL mutant CSP constructs fused to miniTurbo were generated; then the biotinylated proteomes were analysed by liquid chromatographymass spectrometry (LCMS) and validated by western blotting. This confirmed several known CSP-interacting proteins, such as Hsc70 and SNAP-25, but also revealed novel binding proteins, including STXBP1/Munc18-1. Interestingly, some protein interactions (such as Hsc70) were unaffected by the L115R mutation, whereas others (including SNAP-25 and STXBP1/Munc18-1) were inhibited. These results define the CSP interactome in a neuronal model cell line and reveal interactions that are affected by ANCL mutation and hence may contribute to the neurodegeneration seen in patients.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | PC12 cell, SNARE proteins, STXBP1, exocytosis, neurodegeneration, neuronal ceroid lipofuscinosis |
| Divisions: | Faculty of Health and Life Sciences Faculty of Health and Life Sciences > Institute of Systems, Molecular and Integrative Biology |
| Depositing User: | Symplectic Admin |
| Date Deposited: | 22 Jan 2024 09:34 |
| Last Modified: | 02 Feb 2024 17:02 |
| DOI: | 10.1042/bcj20230319 |
| Open Access URL: | https://doi.org/10.1042/BCJ20230319 |
| Related URLs: | |
| URI: | https://livrepository.liverpool.ac.uk/id/eprint/3177932 |
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