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种脱泛素酶(也称为DUBS)。这些酶通过从目标蛋白中去除小蛋白泛素(Ub)或修剪Ub寡聚体来调节广泛的细胞和生物生物学。尽管配音很重要，但我们对配音如何工作的了解存在根本差距。被称为Ub C-末端水解酶(UCH)的DUB家族就体现了这种情况。生化数据表明，UCH催化从Ub中移除小的C-末端加合物，而来自细胞研究的数据表明，这些酶参与了Ub寡聚体的分解。最近，我们的实验室开发了一种直接的化学方法来合成一系列泛素低聚体。利用这些寡聚体来研究DUB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，其中一个Ub亚单位通过两个赖氨酸残基被两个Ub分子修饰(这里称为分支Ub链)。这种活动是史无前例的，因为还没有观察到UCH37和UCHL3分解其他定义的Ub寡聚体的能力，而且支化的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，我们的结果表明分支Ub链在生物学中发挥着比人们所认识的更重要的作用。在本申请中，我们建议揭示UCHs选择性水解支Ub链的机制，并在UCH37调控的途径的背景下测试这一活性。拟议的工作分为三个具体目标。在第一个目标中，我们将扩大化学合成的Ub链的库，以研究链拆解的动力学和选择性。在第二个目标中，我们将从结构上描述 分支Ub链及其与UCH的相互作用。与目标1中建议的研究一起，这些研究将导致UCH37和UCHL3职能的工作模式。在目标3中，我们将针对UCH37对此模型进行测试。许多肿瘤(如宫颈、肝细胞和食道)显示出异常高的UCH37水平。我们假设UCH37通过破坏细胞迁移的关键调节因子，即分支Ub链来促进肿瘤发生。从我们提议的研究中获得的机制洞察力具有很好的潜力转化为抗癌新药的开发。

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.