Global Profiling of Huntingtin-associated protein E (HYPE)-Mediated AMPylation through a Chemical Proteomic Approach
Global Profiling of Huntingtin-associated protein E (HYPE)-Mediated AMPylation through a Chemical Proteomic Approach
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DOI：
10.1074/mcp.o115.054429
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发表时间：
2016-02-01
影响因子：
7
通讯作者：
Tate, Edward W.
中科院分区：
文献类型：
--
作者：
Broncel, Malgorzata;Serwa, Remigiusz A.;Tate, Edward W.
细菌毒力因子对哺乳动物小 GTP 酶的 AMP 化可能是细菌感染宿主细胞的关键步骤，并构成潜在的药物靶点。这种翻译后修饰也存在于真核生物中，AMP 转移酶活性最近被认为是由含有环状 AMP 结构域的蛋白 (FICD) 蛋白诱导的 HYPE 丝，该蛋白从秀丽隐杆线虫到人类都是保守的。与细菌 AMP 转移酶相反，通过免疫沉淀和质谱方法仅鉴定了少量 HYPE 底物，并且哺乳动物细胞中的全部靶标尚未确定。我们在这里描述了哺乳动物细胞裂解物中 HYPE 介导的 AMPylation 的全局化学蛋白质组筛选和底物验证的第一个例子。通过基于定量质谱的蛋白质组学与提供 AMP 修饰的 MS/MS 证据的新型化学蛋白质组学工具相结合，我们总共鉴定了 25 种 AMP 化蛋白质，包括先前验证的底物内质网 (ER) 伴侣 BiP (HSPA5)，以及参与基因表达、ATP 生物合成和细胞骨架维持途径的新型底物。该数据集代表了迄今为止报道的最大的 AMPylated 人类蛋白质库，也是底物特异性研究的基础，最终可以破译真核 AMPylation 中涉及的复杂生物网络。
AMPylation of mammalian small GTPases by bacterial virulence factors can be a key step in bacterial infection of host cells, and constitutes a potential drug target. This posttranslational modification also exists in eukaryotes, and AMP transferase activity was recently assigned to HYPE Filamentation induced by cyclic AMP domain containing protein (FICD) protein, which is conserved from Caenorhabditis elegans to humans. In contrast to bacterial AMP transferases, only a small number of HYPE substrates have been identified by immunoprecipitation and mass spectrometry approaches, and the full range of targets is yet to be determined in mammalian cells. We describe here the first example of global chemoproteomic screening and substrate validation for HYPE-mediated AMPylation in mammalian cell lysate. Through quantitative mass-spectrometry-based proteomics coupled with novel chemoproteomic tools providing MS/MS evidence of AMP modification, we identified a total of 25 AMPylated proteins, including the previously validated substrate endoplasmic reticulum (ER) chaperone BiP (HSPA5), and also novel substrates involved in pathways of gene expression, ATP biosynthesis, and maintenance of the cytoskeleton. This dataset represents the largest library of AMPylated human proteins reported to date and a foundation for substrate-specific investigations that can ultimately decipher the complex biological networks involved in eukaryotic AMPylation.