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 localisation 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 the 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 chromatography–mass spectrometry 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.

中文翻译：

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邻近标记揭示了致病突变对 DNAJC5/半胱氨酸串蛋白 α 相互作用组的影响

半胱氨酸串蛋白 α (CSPα)，也称为 DNAJC5，是 DnaJ/Hsp40 共伴侣家族的成员。该名称源自富含半胱氨酸的结构域，其棕榈酰化能够定位到细胞内膜，特别是神经元突触小泡。编码 CSPα 的 DNAJC5 基因突变会导致常染色体显性遗传、成人发病的神经元蜡样脂褐质沉着症 (ANCL)，这是一种罕见的神经退行性疾病。由于果蝇、蠕虫和小鼠中 CSP 编码基因的无效突变同样会导致神经变性，因此 CSP 显然是一种进化上保守的神经保护蛋白。然而，CSP 分子伴侣预防神经变性的客户蛋白仍不清楚。识别蛋白质-蛋白质相互作用的传统方法（例如酵母 2-杂交和亲和纯化方法）不太适合 CSP，因为它需要膜锚定且在细胞裂解后易于聚集。因此，我们采用了邻近标记，这使得能够通过生物素化在活细胞中原位识别相互作用的蛋白质。生成了稳定表达野生型或与 miniTurbo 融合的 L115R ANCL 突变体 CSP 构建体的神经内分泌 PC12 细胞系；然后通过液相色谱-质谱分析生物素化的蛋白质组，并通过蛋白质印迹进行验证。这证实了几种已知的 CSP 相互作用蛋白，例如 Hsc70 和 SNAP-25，但也揭示了新的结合蛋白，包括 STXBP1/Munc18-1。有趣的是，一些蛋白质相互作用（例如 Hsc70）不受 L115R 突变的影响，而其他蛋白质相互作用（包括 SNAP-25 和 STXBP1/Munc18-1）则受到抑制。这些结果定义了神经元模型细胞系中的 CSP 相互作用组，并揭示了受 ANCL 突变影响的相互作用，因此可能导致患者出现神经退行性变。