Analysis of the eukaryotic prenylome by isoprenoid affinity tagging
Analysis of the eukaryotic prenylome by isoprenoid affinity tagging
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DOI：
10.1038/nchembio.149
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发表时间：
2009-04-01
影响因子：
14.8
通讯作者：
Alexandrov, Kirill
中科院分区：
文献类型：
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作者：
Nguyen, Uyen T. T.;Guo, Zhong;Alexandrov, Kirill
蛋白质异戊烯化是真核细胞中广泛存在的一种现象，它影响许多重要的信号分子。我们描述了工程蛋白异戊二烯基转移酶和它们的通用合成底物，生物素-香叶焦磷酸的结构指导设计。这些新工具使我们能够检测细胞和器官中的飞摩尔量的可异戊二烯基化蛋白质，并鉴定其同源蛋白质异戊二烯基转移酶。使用这种方法，我们分析了蛋白质异戊二烯基转移酶抑制剂的体内作用。尽管一些抑制剂显示出预期的活性，但其他抑制剂缺乏体内活性或靶向比以前认为的更广谱的异戊二烯基转移酶。为了定量异戊烯化抑制剂的体内作用，我们通过质谱分析了蛋白质组中生物素-香叶基-标记的RabGTP酶。我们还表明，网站的积极囊泡运输进行大部分的RabGTP酶。这种方法能够定量蛋白质组范围内的蛋白质异戊二烯化的调节和治疗剂的调制分析。
Protein prenylation is a widespread phenomenon in eukaryotic cells that affects many important signaling molecules. We describe the structure-guided design of engineered protein prenyltransferases and their universal synthetic substrate, biotin-geranylpyrophosphate. These new tools allowed us to detect femtomolar amounts of prenylatable proteins in cells and organs and to identify their cognate protein prenyltransferases. Using this approach, we analyzed the in vivo effects of protein prenyltransferase inhibitors. Whereas some of the inhibitors displayed the expected activities, others lacked in vivo activity or targeted a broader spectrum of prenyltransferases than previously believed. To quantitate the in vivo effect of the prenylation inhibitors, we profiled biotin-geranyl-tagged RabGTPases across the proteome by mass spectrometry. We also demonstrate that sites of active vesicular transport carry most of the RabGTPases. This approach enables a quantitative proteome-wide analysis of the regulation of protein prenylation and its modulation by therapeutic agents.