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

IF 4.4 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Biochemical Journal Pub Date : 2024-01-09 DOI:10.1042/BCJ20230319
Eleanor Barker, Amy Milburn, Nordine Helassa, Dean Hammond, Natalia Sanchez-Soriano, Alan Morgan, Jeff Barclay
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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.

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近距离标记揭示了致病突变对DNAJC5/半胱氨酸串联蛋白α相互作用组的影响。
半胱氨酸串联蛋白α(CSPα)又称 DNAJC5,是 DnaJ/Hsp40 协同伴侣蛋白家族的成员。其名称源于一个富含半胱氨酸的结构域,该结构域的棕榈酰化作用可使其定位到细胞内膜,特别是神经元突触囊泡。编码 CSPα 的 DNAJC5 基因发生突变会导致常染色体显性、成人发病型神经细胞类脂膜炎(ANCL),这是一种罕见的神经退行性疾病。在苍蝇、蠕虫和小鼠中,CSP编码基因的无效突变同样会导致神经退行性病变,因此CSP显然是一种进化保守的神经保护蛋白。然而,目前仍不清楚 CSP 可通过与哪些客户蛋白结合来防止神经退行性变。传统的蛋白质-蛋白质相互作用鉴定方法,如酵母双杂交和亲和纯化方法,都不太适合 CSP,因为它需要膜锚定,而且细胞裂解后容易聚集。因此,我们采用了近距离标记法,通过生物素化在活细胞中原位鉴定相互作用的蛋白质。我们生成了稳定表达与 miniTurbo 融合的野生型或 L115R ANCL 突变型 CSP 构建物的神经内分泌 PC12 细胞系;然后用液相色谱-质谱联用仪(LCMS)分析了生物素化的蛋白质组,并用 Western 印迹法进行了验证。这证实了几种已知的 CSP 相互作用蛋白,如 Hsc70 和 SNAP-25,但也发现了新的结合蛋白,包括 STXBP1/Munc18-1。有趣的是,一些蛋白质的相互作用(如 Hsc70)不受 L115R 突变的影响,而另一些(包括 SNAP-25 和 STXBP1/Munc18-1)则受到抑制。这些结果确定了神经元模型细胞系中的CSP相互作用组,揭示了受ANCL突变影响的相互作用,因此可能导致患者的神经退行性变。
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来源期刊
Biochemical Journal
Biochemical Journal 生物-生化与分子生物学
CiteScore
8.00
自引率
0.00%
发文量
255
审稿时长
1 months
期刊介绍: Exploring the molecular mechanisms that underpin key biological processes, the Biochemical Journal is a leading bioscience journal publishing high-impact scientific research papers and reviews on the latest advances and new mechanistic concepts in the fields of biochemistry, cellular biosciences and molecular biology. The Journal and its Editorial Board are committed to publishing work that provides a significant advance to current understanding or mechanistic insights; studies that go beyond observational work using in vitro and/or in vivo approaches are welcomed. Painless publishing: All papers undergo a rigorous peer review process; however, the Editorial Board is committed to ensuring that, if revisions are recommended, extra experiments not necessary to the paper will not be asked for. Areas covered in the journal include: Cell biology Chemical biology Energy processes Gene expression and regulation Mechanisms of disease Metabolism Molecular structure and function Plant biology Signalling
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