Mechanism and function of protein prenylation

蛋白质异戊二烯化的机制和功能

• 批准号: 7035772
• 负责人: MARK D DISTEFANO
• 金额: $ 24.96万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-06-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7035772
• 关键词: affinity labeling; alkyltransferase; enzyme inhibitors; enzyme mechanism; farnesyl compound; geranyl compound; intermolecular interaction; isoprenoid; mass spectrometry; peptide library; posttranslational modifications; protein structure function; stereochemistry

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.

描述（由申请人提供）：Ras 蛋白被法尼基化以及抑制法尼基化可防止细胞转化的发现引起了人们对这种翻译后修饰的极大兴趣；目前，几种针对法呢基转移酶的候选药物正在进行二期试验。然而，尽管取得了这一成功，我们目前对异戊烯基转移酶酶学的理解仍然不完整，特别是在精确的化学机制方面。此外，有关蛋白质异戊二烯化生物学功能的许多方面仍不清楚。在这个项目的前三年，我们对异戊二烯基转移酶的结构和机制有了重要的了解；我们在理解这一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）完成对蛋白质法呢基和香叶基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研究中获得的结构见解来设计一类新的蛋白质法呢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异戊二烯化蛋白质和肽的其他蛋白质。这些将使用光亲和捕获方法进行鉴定，该方法采用生物素化异戊二烯半胱氨酸和异戊二烯化肽，并结合质谱分析进行目标鉴定。这些研究将确定这种现象的普遍性，并评估存在多少种不同的结构类别。 (4) 结合合成方法和噬菌体展示来获得针对异戊二烯化蛋白与其受体之间相互作用的抑制剂。此类抑制剂将有助于阐明蛋白质异戊二烯化的生物学功能。上述目标的完成应该为蛋白质异戊二烯化的机制和功能提供更多的见解。