Affinity Purification Strategy to Capture Human Endogenous Proteasome Complexes Diversity and to Identify Proteasome-interacting Proteins
Affinity Purification Strategy to Capture Human Endogenous Proteasome Complexes Diversity and to Identify Proteasome-interacting Proteins
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DOI：
10.1074/mcp.m800193-mcp200
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发表时间：
2009-05-01
影响因子：
7
通讯作者：
Monsarrat, Bernard
中科院分区：
文献类型：
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作者：
Bousquet-Dubouch, Marie-Pierre;Baudelet, Emilie;Monsarrat, Bernard
开发了一种亲和纯化策略来表征人类蛋白酶体复合物的多样性以及内源性蛋白酶体相互作用蛋白（PIPs）。这个单步程序最初用于20s蛋白酶体的纯化，后来被用于纯化所有现有的与各种调节复合物及其相互作用伙伴相关的生理蛋白酶体复合物。该方法适用于从人红细胞中纯化蛋白酶体复合物及其pip，但也可用于从任何人类样品中纯化蛋白酶体作为起始材料。通过比较纯化蛋白酶体和两种方案（有或没有甲醛交联）中鉴定的蛋白质的状态，研究了体内甲醛交联作为蛋白质-蛋白质相互作用稳定剂的好处。随后的蛋白质组学分析确定了所有的蛋白酶体亚基、已知的调节因子和最近指定的合作伙伴。此外，在不同水平的泛素-蛋白酶体系统中涉及的其他蛋白质也首次被鉴定为pip。其中之一，泛素特异性蛋白酶USP7，也被称为HAUSP，是p53-HDM2通路的重要参与者。利用以HAUSP为诱饵的反向免疫沉淀的互补方法进一步证实了相互作用的特异性。总之，我们提供了一个有价值的工具，通过识别可能影响蛋白酶体功能的伴侣，有助于更好地理解任何活的人类细胞和/或器官/组织以及不同细胞生理状态下的复杂蛋白质水解机制。中国生物医学工程学报（英文版），2009。
An affinity purification strategy was developed to characterize human proteasome complexes diversity as well as endogenous proteasome-interacting proteins (PIPs). This single step procedure, initially used for 20 S proteasome purification, was adapted to purify all existing physiological proteasome complexes associated to their various regulatory complexes and to their interacting partners. The method was applied to the purification of proteasome complexes and their PIPs from human erythrocytes but can be used to purify proteasomes from any human sample as starting material. The benefit of in vivo formaldehyde cross-linking as a stabilizer of protein-protein interactions was studied by comparing the status of purified proteasomes and the identified proteins in both protocols (with or without formaldehyde cross-linking). Subsequent proteomics analyses identified all proteasomal subunits, known regulators, and recently assigned partners. Moreover other proteins implicated at different levels of the ubiquitin-proteasome system were also identified for the first time as PIPs. One of them, the ubiquitin-specific protease USP7, also known as HAUSP, is an important player in the p53-HDM2 pathway. The specificity of the interaction was further confirmed using a complementary approach that consisted of the reverse immunoprecipitation with HAUSP as a bait. Altogether we provide a valuable tool that should contribute, through the identification of partners likely to affect proteasomal function, to a better understanding of this complex proteolytic machinery in any living human cell and/or organ/tissue and in different cell physiological states. Molecular & Cellular Proteomics 8: 1150-1164, 2009.