Understanding the Function of Deubiquitinases Using Chemical Tools

使用化学工具了解去泛素酶的功能

• 批准号: 9381472
• 负责人: ERIC Robert STRIETER
• 金额: $ 3.21万
• 依托单位: UNIVERSITY OF MASSACHUSETTS AMHERST
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9381472
• 关键词: Understanding; Function; Deubiquitinases; Using; Chemical

DESCRIPTION (provided by applicant): The human genome encodes approximately 100 deubiquitinating enzymes (also known as DUBs). These enzymes regulate a broad swath of cell and organismal biology by removing the small protein ubiquitin (Ub) from target proteins or trimming Ub oligomers. Despite the importance of DUBs, there are fundamental gaps in our knowledge regarding how they work. The family of DUBs known as the Ub C-terminal hydrolases (UCHs) embodies this situation. Biochemical data suggests UCHs catalyze the removal of small C-terminal adducts from Ub, whereas data from cellular studies implicates these enzymes in the disassembly of Ub oligomers. Recently, our laboratory developed a straightforward chemical approach towards the synthesis of a wide array of ubiquitin oligomers. Using these oligomers to probe the function of DUBs, we discovered two members of the UCH family, UCH37 and UCHL3, selectively hydrolyze Ub chains in which a single Ub subunit is modified with two Ub molecules through two lysine residues (herein referred to as branched Ub chains). This activity is unprecedented, as the capacity of UCH37 and UCHL3 to dismantle other defined Ub oligomers has not been observed and the function of branched Ub chains is entirely unknown. Considering the importance of UCHL3 and UCH37 in cellular differentiation, development, and motility, our results suggest branched Ub chains play far more important roles in biology than ever appreciated. In this application, we propose to uncover the mechanism by which UCHs selectively hydrolyze branched Ub chains and test this activity in the context of a pathway regulated by UCH37. The proposed work is divided into three specific aims. In the first aim, we will expand the repertoire of chemically synthesized Ub chains to investigate the kinetics and selectivity of chain disassembly. In the second aim, we will structurally characterize branched Ub chains and their interactions with UCHs. Together with the studies proposed in aim 1, these investigations will lead to working model for the function of UCH37 and UCHL3. In aim 3, we will put this model to the test with regards to UCH37. A number of tumors (e.g., cervical, hepatocellular, and esophageal) display abnormally high levels of UCH37. We hypothesize that UCH37 promotes tumorigenesis by disrupting a critical regulator of cellular migration, i.e., branched Ub chains. The mechanistic insights gained from our proposed studies have excellent potential to be translated into the development of new drugs to fight cancer.

描述（由申请人提供）：人类基因组编码大约100种去泛素化酶（也称为DUB）。这些酶通过从靶蛋白或修剪UB的低聚物中去除小蛋白泛素（UB）来调节细胞和生物生物学的广泛细胞。尽管配音很重要，但我们关于它们的工作方式仍然存在根本的差距。称为UB C末端水解酶（UCHS）的配音家族体现了这种情况。生化数据表明，UCHS催化了UB中的小C末端加合物的去除，而来自细胞研究的数据则暗示了这些酶在UB低聚物的拆卸中。最近，我们的实验室开发了一种直接的化学方法，用于合成各种泛素低聚物。使用这些低聚物来探测配音的功能，我们发现了UCH家族的两个成员UCH37和UCHL3，有选择地水解UB UB链，其中一个单个UB亚基通过两个UB分子通过两个赖氨酸残基修饰（以下称为分支UB链链）。这项活动是前所未有的，因为尚未观察到UCH37和UCHL3拆除其他定义的UB低聚物的能力，并且分支UB链的功能完全未知。考虑到UCHL3和UCH37在细胞分化，发育和运动性中的重要性，我们的结果表明，分支UB链在生物学中起着比以往任何时候所欣赏的重要作用。在此应用中，我们建议揭示UCHS选择性水解分支UB链的机制，并在UCH37调节的途径中测试该活动。拟议的工作分为三个特定目标。在第一个目标中，我们将扩大化学合成的UB链的曲目，以研究链拆卸的动力学和选择性。在第二个目标中，我们将在结构上表征 分支UB链及其与UCH的相互作用。与AIM 1中提出的研究一起，这些研究将导致UCH37和UCHL3功能的工作模型。在AIM 3中，我们将对UCH37进行测试。许多肿瘤（例如宫颈，肝细胞和食道）表现出异常高的UCH37。我们假设UCH37通过破坏细胞迁移的关键调节剂，即分支UB链来促进肿瘤发生。我们提出的研究从我们提出的研究中获得的机械见解具有很大的潜力，可以转化为与癌症作斗争的新药的发展。