Understanding the Function of Deubiquitinases Using Chemical Tools: Administrative Supplement

使用化学工具了解去泛素酶的功能：行政补充

• 批准号: 9895355
• 负责人: ERIC Robert STRIETER
• 金额: $ 3.8万
• 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 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9895355
• 关键词: Administrative Supplement; Affect; Biochemical; Biological; Biology; C-terminal; Cell physiology; Cells; Chemicals; Data; Deubiquitinating Enzyme; Development; Drug Targeting; Enzymes; Excision; Family; Human Genome; Hydrolase; Investigation; Kinetics; Knowledge; Laboratories; Lysine; Malignant Neoplasms; Mass Spectrum Analysis; Methods; Modeling; Modification; Neurodegenerative Disorders; Pathway interactions; Physiological; Play; Proteins; Role; Structure; Testing; Therapeutic Intervention; Ubiquitin; Work; adduct; base; cell motility; member; tool; ubiquitin C-terminal hydrolase

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.

项目摘要 人类基因组编码大约100种去泛素化酶（也称为去泛素化酶）。 作为DUBs）。这些酶通过以下方式调节细胞和生物体的广泛生物学 从靶蛋白中去除小蛋白泛素（Ub）或修整Ub寡聚体。 尽管DUBs很重要，但我们的知识存在根本性的空白 关于他们如何工作。DUB家族被称为Ub C-末端水解酶 （UCHs）体现了这种情况。生化数据表明，UCH催化去除 来自Ub的小C-末端加合物，而细胞研究的数据表明这些 酶解Ub寡聚体。最近，我们的实验室开发了一种 一种直接的化学方法来合成广泛的泛素 低聚物使用这些寡聚体来探测DUBs的功能，我们发现了两个 UCH家族的成员UCH 37和UCHL 3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， （本文称为支链Ub链）。这是史无前例的，因为 UCH 37和UCHL 3拆除其他定义的Ub寡聚体的能力尚未被证实。 观察和分支的Ub链的功能是完全未知的。考虑 UCHL 3和UCH 37在细胞分化、发育和运动中的重要性， 我们的研究结果表明，分支的Ub链在生物学中发挥的作用远比 一直感激。在本申请中，我们提出揭示的机制， UCH选择性地水解支链Ub链，并在一个实施方案的背景下测试这种活性。 由UCH 37调节的通路。拟议的工作分为三个具体目标。在 第一个目标，我们将扩大化学合成的Ub链的库， 研究链分解的动力学和选择性。第二个目标，我们将 结构表征分支Ub链及其与UCH相互作用。一起 通过目标1中提出的研究，这些调查将导致为 UCH 37和UCHL 3的功能。在aim 3中，我们将通过开发 基于质谱的方法检测细胞中的分支Ub缀合物 萃取物我们假设异常的细胞内UCH浓度影响了细胞内的细胞毒性。 分支Ub链的稳态水平，这反过来又影响细胞功能。