Barman Bahnisikha, Ramirez Marisol, Dawson T Renee, Liu Qi, Weaver Alissa M
Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee.
Vanderbilt Center for Extracellular Vesicle Research, Vanderbilt University, Nashville, Tennessee.
bioRxiv. 2023 Jul 19:2023.07.18.549588. doi: 10.1101/2023.07.18.549588.
Extracellular vesicles (EVs) influence cell phenotypes and functions via protein, nucleic acid and lipid cargoes. EVs are heterogeneous, due to diverse biogenesis mechanisms that remain poorly understood. Our previous study revealed that the endoplasmic reticulum (ER) membrane contact site (MCS) linker protein VAP-A drives biogenesis of a subset of RNA-enriched EVs. Here, we examine the protein content of VAP-A-regulated EVs. Using label-free proteomics, we identified down- and up-regulated proteins in sEVs purified from VAP-A knockdown (KD) colon cancer cells. Gene set enrichment analysis (GSEA) of the data revealed protein classes that are differentially sorted to SEVs dependent on VAP-A. STRING protein-protein interaction network analysis of the RNA-binding protein (RBP) gene set identified several RNA functional machineries that are downregulated in VAP-A KD EVs, including ribosome, spliceosome, mRNA surveillance, and RNA transport proteins. We also observed downregulation of other functionally interacting protein networks, including cadherin-binding, unfolded protein binding, and ATP-dependent proteins.
细胞外囊泡（EVs）通过蛋白质、核酸和脂质货物影响细胞表型和功能。由于多种生物发生机制仍未得到充分了解，EVs具有异质性。我们之前的研究表明，内质网（ER）膜接触位点（MCS）连接蛋白VAP-A驱动了一部分富含RNA的EVs的生物发生。在此，我们研究了VAP-A调节的EVs的蛋白质含量。使用无标记蛋白质组学，我们鉴定了从VAP-A敲低（KD）结肠癌细胞中纯化的小细胞外囊泡（sEVs）中下调和上调的蛋白质。对数据进行基因集富集分析（GSEA），揭示了依赖于VAP-A而被差异分选到SEVs中的蛋白质类别。对RNA结合蛋白（RBP）基因集进行STRING蛋白质-蛋白质相互作用网络分析，确定了在VAP-A KD EVs中下调的几种RNA功能机制，包括核糖体、剪接体、mRNA监测和RNA转运蛋白。我们还观察到其他功能相互作用的蛋白质网络的下调，包括钙黏蛋白结合、未折叠蛋白结合和ATP依赖性蛋白。
