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Proximity labelling reveals effects of disease-causing mutation on the DNAJC5/cysteine string protein α interactome.

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.

SUBMITTER: Barker E

PROVIDER: S-EPMC10903463 | biostudies-literature | 2024 Jan

REPOSITORIES: biostudies-literature

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Proximity labelling reveals effects of disease-causing mutation on the DNAJC5/cysteine string protein α interactome.

Barker Eleanor E Milburn Amy A Helassa Nordine N Hammond Dean D Sanchez-Soriano Natalia N Morgan Alan A Barclay Jeff J

The Biochemical journal 20240109

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 r ...[more]

PMID: 38193346

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Project description:BackgroundCysteine string protein-α (CSPα) is a chaperone to ensure protein folding. Loss of CSPα function associates with many neurological diseases. However, its function in modulating regulated exocytosis remains elusive. Although cspα-knockouts exhibit impaired synaptic transmission, overexpression of CSPα in neuroendocrine cells inhibits secretion. These seemingly conflicting results lead to a hypothesis that CSPα may undergo a modification that switches its function in regulating neurotransmitter and hormone secretion. Previous studies implied that CSPα undergoes phosphorylation at Ser10 that may influence exocytosis by altering fusion pore dynamics. However, direct evidence is missing up to date.Methodology/principal findingsUsing amperometry, we investigated how phosphorylation at Ser10 of CSPα (CSPα-Ser10) modulates regulated exocytosis and if this modulation involves regulating a specific kinetic step of fusion pore dynamics. The real-time exocytosis of single vesicles was detected in PC12 cells overexpressing control vector, wild-type CSPα (WT), the CSPα phosphodeficient mutant (S10A), or the CSPα phosphomimetic mutants (S10D and S10E). The shapes of amperometric signals were used to distinguish the full-fusion events (i.e., prespike feet followed by spikes) and the kiss-and-run events (i.e., square-shaped flickers). We found that the secretion rate was significantly increased in cells overexpressing S10D or S10E compared to WT or S10A. Further analysis showed that overexpression of S10D or S10E prolonged fusion pore lifetime compared to WT or S10A. The fraction of kiss-and-run events was significantly lower but the frequency of full-fusion events was higher in cells overexpressing S10D or S10E compared to WT or S10A. Advanced kinetic analysis suggests that overexpression of S10D or S10E may stabilize open fusion pores mainly by inhibiting them from closing.Conclusions/significanceCSPα may modulate fusion pore dynamics in a phosphorylation-dependent manner. Therefore, through changing its phosphorylated state influenced by diverse cellular signalings, CSPα may have a great capacity to modulate the rate of regulated exocytosis.

Dataset Information

Proximity labelling reveals effects of disease-causing mutation on the DNAJC5/cysteine string protein α interactome.

Publications

Proximity labelling reveals effects of disease-causing mutation on the DNAJC5/cysteine string protein α interactome.

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