Mechanism and function of protein prenylation

描述（由申请人提供）：Ras蛋白被法尼基化，抑制法尼基化可阻止细胞转化，这一发现引起了人们对这种翻译后修饰的兴趣；目前，几种靶向法尼基转移酶的候选药物正处于2期试验。然而，尽管取得了这一成功，我们目前对戊烯基转移酶的酶学理解仍然不完整，特别是关于精确的化学机制。此外，关于蛋白质戊酰化的生物学功能的许多方面仍不清楚。在这个项目的前三年，我们对戊烯基转移酶的结构和机制有了重要的了解；我们在理解这种修改的功能方面也取得了进展。本次更新申请的具体目的是：(1)完成我们对蛋白法尼基和香叶基转移酶催化反应的化学机制的研究。将进行立体化学和动力学同位素效应实验。(2)利用我们从类异戊二烯类似物的研究中获得的结构见解，设计一类新的蛋白法尼基转移酶双底物抑制剂。(3)鉴定通过与类异戊二烯相互作用识别戊烯基化蛋白和多肽的其他蛋白。这些将使用光亲和捕获方法进行鉴定，采用生物素化戊酰半胱氨酸和戊酰化肽，结合质谱分析进行目标鉴定。这些研究将确定这种现象有多普遍，并评估存在多少不同的结构类别。(4)采用合成方法和噬菌体展示相结合的方法，获得针对戊烯酰化蛋白与其受体相互作用的抑制剂。这些抑制剂将有助于阐明蛋白质戊酰化的生物学功能。上述目标的完成将为蛋白质戊酰化的机制和功能提供更多的见解。

DESCRIPTION (provided by applicant): The discovery that Ras proteins are farnesylated and that inhibition of farnesylation prevents cellular transformation has generated an explosion of interest in this post-translational modification; currently, several drug candidates that target farnesyl transferase are in Phase 2 trials. However, despite this success, our current understanding of the enzymology of prenyltransferases remains incomplete particularly regarding the precise chemical mechanism. Moreover, many aspects concerning the biological function of protein prenylation remain unclear. In the first three years of this project, we have gained significant insights into the structure and mechanism of prenyltransferases; we have also made progress in understanding functional aspects of this modification. The specific aims of this renewal application are to: (1) Complete our studies of the chemical mechanisms of the reactions catalyzed by protein farnesyl and geranylgeranyl transferase enzymes. Stereochemical and kinetic isotope effect experiments will be performed. (2) Use the structural insights we have gained from our studies with isoprenoid analogues to design a new class of bisubstrate inhibitors of protein farnesyltransferase. (3) Identify additional proteins that recognize prenylated proteins and peptides through interactions with the isoprenoid. These will be identified using a photoaffinity capture method employing biotinylated prenylcysteines and prenylated peptides coupled with mass spectral analysis for target identification. These studies will determine how common this phenomenon is and evaluate how many different structural classes exist. (4) Use a combination of synthetic methods and phage display to obtain inhibitors that target the interaction between prenylated proteins and their receptors. Such inhibitors will be useful for clarifying the biological function of protein prenylation. Completion of the above aims should provide additional insights into the mechanism and function of protein prenylation.