Determining the scope of prenylatable protein sequences

项目摘要 本研究调查了异戊烯化CaaX蛋白的法呢基转移酶（FTase）的特异性。 探索FTase的体内活性的研究历史上使用了报告基因（例如Ras GTP酶），其 经历复杂的多步翻译后修饰（PTM），包括初始法尼基化，然后是 CaaX蛋白水解和羧基甲基化。本研究利用了Hsp40 Ydj1，一种仅法尼基化的 我们已经开发了一系列方法来监测其PTM状态。它的使用表明， 法尼基化不一定与随后的PTM偶联，如CaaX中普遍接受的那样 蛋白质，并且FTase特异性比预期的要严重得多。这些发现 挑战了法尼基化蛋白质如何被修饰以及哪些蛋白质 以FTase为目标我们将扩展我们的研究，使用以下组合来完全解决FTase的特异性： 遗传学、生物化学、生物信息学和生物物理学研究。我们在调查中， 一套非常强大的初步研究结果，几个研究小组的互补专业知识，以及一个 全面的分子工具箱，用于研究法尼基化蛋白质和其他与此相关的酶。 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.