Laboratory of Structural Neuropathology, Doshisha University Graduate School of Brain Science, Kyoto, Japan.
Molecular Mechanisms of Brain Development, RIKEN Center for Brain Science, Saitama, 351-0198, Japan.
Sci Rep. 2020 May 6;10(1):7610. doi: 10.1038/s41598-020-64517-z.
An ER transmembrane protein, vesicle-associated membrane protein-associated protein B (VAPB), binds to several organelle-resident membrane proteins to mediate ER-organelle tethering. Mutation in amyotrophic lateral sclerosis (ALS) induces protein misfolding and aggregation, leading to ER disorganization. Gain or loss of function is suggested for VAPB mutation, however comprehensive study focusing on VAPB-ER domain has yet been performed. We here conducted proteomic characterization of the ER containing VAPB and its ALS-linked P56S mutant. For this purpose, we first optimized the proteomics of different ER domains immuno-isolated from cultured cells, and identified ER sheet- and tubule-specific proteomes. By using these as references, we found that VAPB-ER proteome had intermediate ER domain properties but its tubular property was specifically decreased by its mutation. Biochemical, immunofluorescence and proximity ligation assays suggested this was mediated by delocalization of VAPB from ER tubules. The VAPB-ER proteomics further suggested reduced incorporation of multiple proteins located in different organelles, which was confirmed by proximity ligation assay. Taken together, our proteomics-based approach indicates altered ER domain properties and impaired ER-organelle tethering by VAPB mutation.
一种内质网跨膜蛋白，囊泡相关膜蛋白相关蛋白 B（VAPB），与几种细胞器驻留膜蛋白结合，介导内质网与细胞器的连接。肌萎缩侧索硬化症（ALS）中的突变诱导蛋白质错误折叠和聚集，导致内质网紊乱。VAPB 突变被认为是获得或丧失功能的原因，但尚未对 VAPB-ER 结构域进行全面研究。我们在这里对含有 VAPB 的内质网及其与 ALS 相关的 P56S 突变体进行了蛋白质组学特征分析。为此，我们首先优化了从培养细胞中免疫分离的不同内质网区域的蛋白质组学方法，并鉴定了内质网片层和小管的特异性蛋白质组。使用这些作为参考，我们发现 VAPB-ER 蛋白质组具有中间内质网结构域特性，但由于其突变，其管状特性特异性降低。生化、免疫荧光和邻近连接测定表明，这是通过 VAPB 从内质网小管的位置转位介导的。VAPB-ER 蛋白质组学进一步表明，多种位于不同细胞器的蛋白质的掺入减少，这通过邻近连接测定得到了证实。总之，我们基于蛋白质组学的方法表明，VAPB 突变导致内质网结构域特性改变和内质网-细胞器连接受损。
