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: 9262253
• 负责人: ERIC Robert STRIETER
• 金额: $ 30.7万
• 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/9262253
• 关键词: Address; Binding; Biochemical; Biological; Biological Assay; Biology; C-terminal; Cells; Cellular biology; Cervical; Chemicals; Cleaved cell; Collection; Complement; Data; Deubiquitinating Enzyme; Development; Drug Targeting; Enzymes; Esophageal; Eukaryotic Cell; Event; Excision; Family; Foundations; Genetic Transcription; Goals; Heart Diseases; Hepatocyte; Human Genome; Hydrolase; Investigation; Kinetics; Knowledge; Laboratories; Lead; Lysine; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Measures; Methods; Modeling; Modification; Molecular; Molecular Conformation; NMR Spectroscopy; Nature; Neurodegenerative Disorders; Outcome; Pathway interactions; Phase; Physiological; Play; Post-Translational Protein Processing; Process; Property; Proteins; Reagent; Regulation; Research; Roentgen Rays; Role; Science; Signal Pathway; Signal Transduction; Site-Directed Mutagenesis; Structure-Activity Relationship; Techniques; Testing; Therapeutic Intervention; Transforming Growth Factors; Translating; Ubiquitin; Variant; Work; X-Ray Crystallography; adduct; cell motility; design; fighting; human disease; insight; malignant neurologic neoplasms; member; molecular dynamics; nervous system disorder; novel therapeutics; programs; protein function; public health relevance; response; tool; tumor; tumorigenesis; ubiquitin C-terminal hydrolase; ubiquitin ligase

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）。这些酶通过从目标蛋白中去除小蛋白泛素 (Ub) 或修剪 Ub 寡聚物来调节广泛的细胞和生物体生物学。尽管 DUB 很重要，但我们对其工作原理的了解还存在根本性差距。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 催化去除 Ub 中小的 C 末端加合物，而细胞研究数据表明这些酶参与了 Ub 寡聚物的分解。最近，我们的实验室开发了一种简单的化学方法来合成各种泛素寡聚物。使用这些寡聚物来探测 DUB 的功能，我们发现 UCH 家族的两个成员 UCH37 和 UCHL3 选择性水解 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（本文称为支化 Ub 链）。这种活性是前所未有的，因为尚未观察到 UCH37 和 UCHL3 分解其他确定的 Ub 寡聚体的能力，并且支化 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，我们的结果表明支化 Ub 链在生物学中发挥的作用比以往想象的要重要得多。在本申请中，我们建议揭示 UCH 选择性水解支化 Ub 链的机制，并在 UCH37 调控的途径背景下测试这种活性。拟议的工作分为三个具体目标。第一个目标是，我们将扩展化学合成 Ub 链的范围，以研究链分解的动力学和选择性。在第二个目标中，我们将从结构上表征 支化 Ub 链及其与 UCH 的相互作用。与目标 1 中提出的研究一起，这些研究将产生 UCH37 和 UCHL3 功能的工作模型。在目标 3 中，我们将针对 UCH37 对该模型进行测试。许多肿瘤（例如宫颈癌、肝细胞癌和食管癌）显示出异常高水平的 UCH37。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。从我们提出的研究中获得的机制见解具有巨大的潜力，可以转化为抗癌新药的开发。