UNDERSTANDING THE FUNCTION OF DEUBIQUITINASES USING CHEMICAL TOOLS

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中去除小蛋白泛素（Ub）或修剪Ub低聚物来调节广泛的细胞和有机体生物学。尽管dub很重要，但我们对它们如何工作的了解还存在根本性的差距。被称为Ub c端水解酶（UCHs）的dub家族体现了这种情况。生化数据表明，uch可以催化Ub的小c端加合物的去除，而细胞研究数据表明，这些酶参与Ub低聚物的分解。最近，我们的实验室开发了一种直接的化学方法来合成广泛的泛素低聚物。利用这些低聚物来探测DUBs的功能，我们发现了UCH家族的两个成员UCH37和UCHL3，它们选择性地水解Ub链，其中单个Ub亚基通过两个赖氨酸残基被两个Ub分子修饰（这里称为支链Ub链）。这种活性是前所未有的，因为尚未观察到UCH37和UCHL3拆除其他已定义的Ub低聚物的能力，并且支链Ub链的功能完全未知。考虑到UCHL3和UCH37在细胞分化、发育和运动中的重要性，我们的研究结果表明支链Ub链在生物学中发挥的作用比以往所认识的要重要得多。在这项应用中，我们提出揭示UCHs选择性水解支链Ub的机制，并在UCH37调控的途径中测试这种活性。拟议的工作分为三个具体目标。在第一个目标中，我们将扩展化学合成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，我们将从结构上描述

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.