Regulation and specificity of deubiquitylating enzyme complex

去泛素化酶复合物的调节和特异性

• 批准号: 8464161
• 负责人: Zhihao Zhuang
• 金额: $ 24.38万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2017-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8464161
• 关键词: Active Sites; Ataxia; Binding; Binding Sites; Biological Assay; C-terminal; Catalysis; Chemicals; Complex; Coupled; Crosslinker; Enzymes; Face; Family; Free Energy; Gel; Goals; Homologous Gene; Human; Human Ubiquitin; Intervention; Iodoacetamide; Kinetics; Knowledge; Link; Lysine; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Modeling; Molecular; Molecular Conformation; Mono-S; Multienzyme Complexes; Mutation Analysis; N-terminal; Neurodegenerative Disorders; Pathogenesis; Phosphorylation; Physiological; Polyubiquitin; Positioning Attribute; Process; Proliferating Cell Nuclear Antigen; Protease Inhibitor; Protein Family; Proteins; Proteomics; Regulation; Serine; Site; Site-Directed Mutagenesis; Specificity; Structure; System; Tail; Testing; Therapeutic; Therapeutic Agents; Ubiquitin; Ubiquitin C; Virus Diseases; base; crosslink; design; gel electrophoresis; human disease; improved; insight; novel therapeutics; protein complex; protein function; ubiquitin ligase; ubiquitin-aldehyde; ubiquitin-specific protease

DESCRIPTION (provided by applicant): Abnormal regulation of human deubiquitylating enzymes (DUBs) has been implicated in the pathogenesis of a number of human diseases including cancer, neurodegenerative disorders and viral infection. Ubiquitin-specific proteases (USPs) constitute the largest family among the five known DUB families. Human USPs are emerging as promising targets for pharmacological intervention. However, the lack of understanding of the specificity and regulation of USPs has hindered the progress in developing novel therapeutics. Remarkably, a recent global proteomic analysis of human DUBs revealed that over thirty human USPs are associated specifically with WD40-repeat proteins. Given its widespread occurrence, the interaction between WD40- repeat proteins and USPs likely represents a fundamentally important way of regulating USP activity. This application focuses on a prototypical USP WD40-repeat protein complex, human USP1/UAF1, and its close homologs. The overarching goal of this application is to gain an in-depth understanding of the specificity and regulation of human USPs and the USP WD40-repeat protein complexes. There are three specific aims: 1) Determine the energetics of the bipartite USP-Ub interaction in USP1 and USP2 (a structural homolog of USP1). Assess the contribution of the bipartite interaction to ubiquitin recognition and catalysis by double mutation analysis; 2) Probe the specificity of USP1/UAF1 using a physiological substrate and its close mimics. A new AlphaScreen-based assay will be developed to improve both the throughput and sensitivity of the deubiquitylation assay; 3) Determine the molecular mechanism by which UAF1 upregulates the activity of USP1 and the closely related human USP46. We will characterize the USP1/UAF1 and USP46/UAF1 complexes and map the interaction sites between UAF1 and the two USPs. The molecular basis for the stimulation of the USP enzymatic activity by UAF1 will be interrogated by enzymological approaches. Taken together, these studies have the potential to uncover important insights into the specificity and regulation of the prevalent USP WD40-repeat protein complexes. Further, our study will suggest new directions for pharmacologic intervention of this important class of human USPs.

描述（由申请人提供）：人类去泛素化酶（DUB）的异常调节与许多人类疾病的发病机制有关，包括癌症、神经退行性疾病和病毒感染。泛素特异性蛋白酶 (USP) 是五个已知 DUB 家族中最大的家族。人类 USP 正在成为药物干预的有希望的目标。然而，对 USP 的特异性和监管缺乏了解阻碍了新疗法的开发进展。值得注意的是，最近对人类 DUB 进行的全球蛋白质组学分析表明，超过 30 个人类 USP 与 WD40 重复蛋白特异性相关。鉴于其广泛存在，WD40-重复蛋白和USP之间的相互作用可能代表了调节USP活性的一种根本上重要的方式。该应用重点关注原型 USP WD40 重复蛋白复合物、人 USP1/UAF1 及其密切同源物。该应用的总体目标是深入了解人类 USP 和 USP WD40 重复蛋白复合物的特异性和调节。共有三个具体目标：1) 确定 USP1 和 USP2（USP1 的结构同系物）中 USP-Ub 双向相互作用的能量学。通过双突变分析评估二分相互作用对泛素识别和催化的贡献； 2) 使用生理底物及其近似模拟物探测 USP1/UAF1 的特异性。将开发一种新的基于 AlphaScreen 的检测方法，以提高去泛素化检测的通量和灵敏度； 3)确定UAF1上调USP1及其密切相关的人类USP46活性的分子机制。我们将表征 USP1/UAF1 和 USP46/UAF1 复合物，并绘制 UAF1 和两个 USP 之间的相互作用位点图。 UAF1 刺激 USP 酶活性的分子基础将通过酶学方法进行探究。总而言之，这些研究有可能揭示关于流行的 USP WD40 重复蛋白复合物的特异性和调节的重要见解。此外，我们的研究将为这一类重要的人类 USP 的药理干预提出新的方向。