Regulation and specificity of deubiquitylating enzyme complex

描述（由申请人提供）：人去泛素化酶（DUB）的异常调节与许多人类疾病（包括癌症、神经退行性疾病和病毒感染）的发病机制有关。泛素特异性蛋白酶（USP）构成已知的五个DUB家族中最大的家族。人USP正在成为药理学干预的有希望的靶点。然而，缺乏对USP的特异性和调节的理解阻碍了开发新疗法的进展。值得注意的是，最近对人类DUB的全球蛋白质组学分析显示，超过30种人类USP与WD 40重复蛋白特异性相关。鉴于其广泛存在，WD 40重复蛋白和USP之间的相互作用可能代表了调节USP活性的根本重要方式。本申请的重点是一个典型的USP WD 40重复蛋白复合物，人USP 1/UAF 1，及其密切的同源物。本申请的首要目标是深入了解人USP和USP WD 40-重复蛋白复合物的特异性和调控。有三个具体的目标：1）确定USP 1和USP 2（USP 1的结构同系物）中的二分USP-Ub相互作用的能量。通过双突变分析评估双链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USP 1/UAF 1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UAF 1上调USP 1和密切相关的人USP 46活性的分子机制。我们将表征USP 1/UAF 1和USP 46/UAF 1复合物，并绘制UAF 1与两种USP之间的相互作用位点。将通过酶学方法研究UAF 1刺激USP酶活性的分子基础。综上所述，这些研究有可能揭示流行的USP WD 40-重复蛋白复合物的特异性和调节的重要见解。此外，我们的研究将为这类重要的人类USP的药理学干预提出新的方向。 公共卫生相关性：人泛素特异性蛋白酶（USP）与许多人类疾病有关，包括神经退行性疾病、癌症、共济失调和病毒感染。为了将USP用于治疗目的，需要更好地理解USP的特异性和法规。这些知识对于开发能够特异性抑制人泛素特异性蛋白酶的治疗剂是必不可少的。

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. PUBLIC HEALTH RELEVANCE: Human ubiquitin-specific proteases (USPs) have been linked to a number of human diseases, including neurodegenerative disease, cancer, ataxia and viral infection. In order to exploit USP for therapeutic purposes, a better understanding of USP's specificity and regulation is required. Such knowledge is essential for developing therapeutics that can specifically inhibit the human ubiquitin-specific protease.