Regulation and specificity of deubiquitylating enzyme complex

描述(由申请人提供)：人类脱泛素酶(DUBS)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。泛素特异性蛋白水解酶(USPS)是已知的5个DuB家族中最大的家族。人类USPS正在成为药物干预的有希望的靶点。然而，对USPS的特异性和调控缺乏了解，阻碍了新疗法的发展。值得注意的是，最近一项对人类DUB的全球蛋白质组分析显示，超过30个人类USP与WD40-Repeat蛋白特异相关。鉴于其广泛存在，WD40重复蛋白与USP之间的相互作用可能是调节USP活性的一种根本重要方式。本应用的重点是一个典型的USP WD40-重复蛋白复合体，人的USP1/UAF1，及其相近的同源物。这项应用的首要目标是深入了解人类USPS和USP WD40-重复蛋白复合体的特异性和调节。具体目标有三个：1)确定USP1和USP2(USP1的结构同系物)中USP-Ub相互作用的能量学。通过双突变分析评估二聚体相互作用对泛素识别和催化的贡献；2)利用一种生理底物及其紧密的模拟物来探索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吞吐量和灵敏度；3)确定UAF1上调USP1和密切相关的人类USP46活性的分子机制。我们将表征USP1/UAF1和USP46/UAF1络合物，并绘制UAF1与两个USPP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讨。综上所述，这些研究有可能揭示出对流行的USP WD40-重复蛋白复合体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USPS的药物干预提供新的方向。 公共卫生相关性：人类泛素特定蛋白水解酶(USPS)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，需要更好地了解USP的特异性和调节。这些知识对于开发能够特异性抑制人类泛素特异性蛋白水解酶的疗法是必不可少的。

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.