Organelle proteomics
Organelle proteomics
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DOI：
10.1074/mcp.m600365-mcp200
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发表时间：
2007-05-01
影响因子：
7
通讯作者：
Hewitt, Eric W.
中科院分区：
文献类型：
--
作者：
Casey, Tammy M.;Meade, Josephine L.;Hewitt, Eric W.
自然杀伤 (NK) 细胞和细胞毒性 T 淋巴细胞消除病毒感染和转化的细胞。靶细胞杀伤是由分泌性溶酶体的受调节的胞吐作用介导的，其将穿孔素和促凋亡颗粒酶递送至感染或转化的细胞。然而，尽管分泌性溶酶体胞吐作用在病毒和肿瘤的免疫反应中发挥着核心作用，但人们对调节这种细胞器与质膜对接和融合的分子机制知之甚少。为了识别这种胞吐机制的潜在成分，我们使用蛋白质组学来定义 NK 细胞分泌溶酶体膜的蛋白质组成。通过亚细胞分级分离从 NK 细胞系 YTS 中分离分泌溶酶体，使用 Triton X-114 富集整合膜蛋白和膜相关蛋白，并通过 SDS-PAGE 分离，并通过 LC ESI-MS/MS 鉴定胰蛋白酶肽。在分泌性溶酶体膜部分中总共明确鉴定了 221 种蛋白质，其中 61% 预计是整合膜蛋白或膜相关蛋白。已鉴定的大部分蛋白质在囊泡运输中发挥作用，包括 Rab GTP 酶和可溶性 N-乙基马来酰亚胺敏感因子附着蛋白受体 (SNARE) 和蛋白质家族的成员。这些蛋白质包括 Rab27a 和 SNARE 囊泡相关膜蛋白 7，两者都富含分泌性溶酶体部分，代表了调节 NK 细胞中该细胞器胞吐作用的机制的潜在组成部分。
Natural killer (NK) cells and cytotoxic T lymphocytes eliminate virally infected and transformed cells. Target cell killing is mediated by the regulated exocytosis of secretory lysosomes, which deliver perforin and proapoptotic granzymes to the infected or transformed cell. Yet despite the central role that secretory lysosome exocytosis plays in the immune response to viruses and tumors, little is known about the molecular machinery that regulates the docking and fusion of this organelle with the plasma membrane. To identify potential components of this exocytic machinery we used proteomics to define the protein composition of the NK cell secretory lysosome membrane. Secretory lysosomes were isolated from the NK cell line YTS by subcellular fractionation, integral membrane proteins and membrane-associated proteins were enriched using Triton X-114 and separated by SDS-PAGE, and tryptic peptides were identified by LC ESI-MS/MS. In total 221 proteins were identified unambiguously in the secretory lysosome membrane fraction of which 61% were predicted to be either integral membrane proteins or membrane-associated proteins. A significant proportion of the proteins identified play a role in vesicular trafficking, including members of both the Rab GTPase and soluble N-ethylmaleimide-sensitive factor attachment protein receptor ( SNARE) and protein families. These proteins include Rab27a and the SNARE vesicle-associated membrane protein-7, both of which were enriched in the secretory lysosome fraction and represent potential components of the machinery that regulates the exocytosis of this organelle in NK cells.