Proteomics-Based Approach Identifies Altered ER Domain Properties by ALS-Linked VAPB Mutation
Proteomics-Based Approach Identifies Altered ER Domain Properties by ALS-Linked VAPB Mutation
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DOI：
10.1038/s41598-020-64517-z
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发表时间：
2020-05
影响因子：
4.6
通讯作者：
T. Yamanaka;R. Nishiyama;T. Shimogori;N. Nukina
中科院分区：
文献类型：
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作者：
T. Yamanaka;R. Nishiyama;T. Shimogori;N. Nukina
一种内质网跨膜蛋白，囊泡相关膜蛋白B(VAPB)，与几种细胞器驻留的膜蛋白结合，介导内质网-细胞器系留。肌萎缩侧索硬化症(ALS)中的突变导致蛋白质错误折叠和聚集，导致内质网解体。VAPB突变的功能获得或丧失被认为是可能的，但针对VAPB-ER结构域的全面研究尚未完成。我们在这里对含有VAPB的内质网及其ALS连锁的P56S突变体进行了蛋白质组学研究。为此，我们首先优化了从培养细胞中免疫分离的不同ER结构域的蛋白质组学，并鉴定了ER片状和小管特异的蛋白质组。以此为参考，我们发现VAPB-ER蛋白质组具有中间的ER结构域属性，但其管状属性因其突变而特异性降低。生化、免疫荧光和邻近结扎分析表明，这是通过VAPB从内质网小管上移位所介导的。VAPB-ER蛋白质组学进一步表明，位于不同细胞器的多个蛋白质的掺入减少，这一点通过邻近连接实验得到了证实。综上所述，我们基于蛋白质组学的方法表明，VAPB突变改变了ER结构域的性质，损害了ER细胞器的连接。
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.