Determining the scope of prenylatable protein sequences

项目总结 本研究探讨了异戊二烯基化CAAX蛋白的法尼基转移酶(FTase)的特异性。 探索FTase体内活性的研究历史上使用了报告(例如Ras GTP酶)， 经历复杂的多步骤翻译后修饰(PTM)，包括最初的法尼化，然后是 CAAX蛋白降解和羧甲基化。这项研究利用了Hsp40Ydj1，一种仅有法尼化的基因 我们已经为其开发了一系列方法来监控其PTM状态。它的使用揭示了 法尼化不一定与随后的PTM偶联，这是CAAX普遍接受的 蛋白质，而且FT酶的专一性比预期的要混杂得多。这些发现 挑战关于法尼化蛋白质是如何修饰的以及哪些蛋白质是 被FTase锁定。我们将扩展我们的研究，以充分解决FTase的特异性使用组合 遗传学、生物化学、生物信息学和生物物理学研究。我们利用我们的调查和 一组异常强大的初步发现，几个研究小组的互补专业知识，以及 用于研究法尼化蛋白质和与此相关的其他酶的综合分子工具箱 翻译后修饰途径。

PROJECT SUMMARY This study investigates the specificity of farnesyl transferase (FTase) that isoprenylates CaaX proteins. Studies probing the in vivo activity of the FTase have historically used reporters (e.g. Ras GTPases) that undergo complex multi-step post-translational modification (PTM), involving initial farnesylation followed by CaaX proteolysis and carboxyl methylation. This study takes advantage of Hsp40 Ydj1, a farnesylation-only reporter for which we have developed a range of methods to monitor its PTM status. Its use reveals that farnesylation is not necessarily coupled to subsequent PTMs as has been generally accepted for CaaX proteins, and that FTase specificity is significantly more promiscuous than anticipated. These findings challenge the conventional paradigm for how farnesylated proteins are modified and which proteins are targeted by FTase. We will extend our studies to fully resolve the specificity of FTase using a combination of genetic, biochemical, bioinformatic, and biophysical studies. We bring to bear on our investigations an exceptionally strong set of preliminary findings, the complementary expertise of several research groups, and a comprehensive molecular toolbox for the study of farnesylated proteins and other enzymes associated with this post-translational modification pathway.