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理有关，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）构成了五个已知配音家庭中最大的家庭。人类USP正在成为药理干预的有希望的目标。但是，缺乏对USP的特异性和调节的理解阻碍了发展新型治疗剂的进步。值得注意的是，最近对人配音的全球蛋白质组学分析表明，超过三十多名人类USP与WD40重复蛋白特别相关。鉴于其广泛的发生，WD40重复蛋白和USP之间的相互作用可能代表了调节USP活性的根本重要方法。该应用集中在典型的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对人类USP和USP WD40重复蛋白复合物的特异性和调控有深入的了解。有三个特定的目的：1）确定USP1和USP2（USP1的结构同源物）中两部分USP-UB相互作用的能量学。通过双重突变分析评估双方相互作用对泛素识别和催化的贡献； 2）使用生理底物及其接近模拟物探测USP1/UAF1的特异性。将开发一种新的基于α的测定法，以提高去泛素化测定法的吞吐量和敏感性； 3）确定UAF1上调USP1和密切相关的人USP46的活性的分子机制。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。通过酶学方法询问UAF1刺激USP酶活性的分子基础。综上所述，这些研究有可能发现对普遍的USP WD40重复蛋白复合物的特异性和调节的重要见解。此外，我们的研究将提出对这类人类USP的药理干预的新方向。