Understanding the Function of Deubiquitinases Using Chemical Tools: Administrative Supplement

项目总结 人类基因组编码大约100种脱泛素酶(也称为 作为配音)。这些酶通过以下方式调节广泛的细胞和生物生物学 从靶蛋白中去除小蛋白泛素(Ub)或修剪Ub寡聚体。 尽管配音很重要，但我们的知识中存在着根本的差距 关于它们是如何工作的。被称为Ub C-末端水解酶的DUB家族 (UCHs)就体现了这种情况。生化数据表明，UCHs催化了这种去除 来自Ub的小C-末端加合物，而来自细胞研究的数据表明这些 Ub寡聚体分解中的酶。最近，我们实验室研制出了一种 一种合成多种泛素的直接化学方法 齐聚物。利用这些低聚物来探索DUBS的功能，我们发现了两个 UCH家族的成员，UCH37和UCHL3，选择性地在 其中单个Ub亚基通过两个赖氨酸残基被两个Ub分子修饰 (这里称为分支Ub链)。这次活动是史无前例的，因为 UCH37和UCHL3分解其他已定义的Ub低聚体的能力尚未达到 观察到了分支的Ub链，其功能完全未知。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链在生物学中的作用远远超过 从未被赏识过。在本申请中，我们建议揭示 UCH选择性地水解分支的Ub链，并在 受UCH37调控的通路。拟议的工作分为三个具体目标。在……里面 第一个目标是，我们将把化学合成的Ub链的库扩大到 考察了解链反应的动力学和选择性。在第二个目标中，我们将 从结构上表征支化的Ub链及其与UCH的相互作用。同舟共济 根据目标1中建议的研究，这些调查将导致 UCH37和UCHL3的功能。在目标3中，我们将通过开发 基于质谱学的细胞内分支Ub结合物的检测方法 萃取物。我们假设UCH细胞内的异常浓度会影响 分支Ub链的动态平衡水平，进而影响细胞功能。

PROJECT SUMMARY 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 by developing mass spectrometry based methods to detect branched Ub conjugates in cellular extracts. We hypothesize that aberrant intracellular concentrations of UCHs affect the homeostatic levels of branched Ub chains, which in turn affects cellular function.