Functions of Ub-like Proteins and Proteases

描述（由申请人提供）：我们已经开发了作用于泛素、NeddS、SUMO-1、SUMO-2/3和ISG 15（统称为泛素样或UBL蛋白）的去泛素化酶（DUB）的特异性抑制剂和底物。我们将这些称为UBL-X试剂组。这些试剂的特异性是突出的，并且已经开发了评估DUB同源物和UBL旁系同源物之间的交叉反应性的方法。这些试剂使我们能够定义许多单个DUB的可能特异性，导致关于不同类别DUB所表现出的活性谱的一些令人惊讶的结论，并提出了开发和表征特异性干扰UBL蛋白质代谢的选定途径的抑制剂的新机会。在今后一段时期内：我们将在定量标记和动力学方法中使用UBL-X试剂组，以确定来自对照和应激细胞的裂解物中活性去小泛素化酶的量和特异性。将特异性与纯化的催化结构域进行比较。我们将测试的假设，在细胞水平的sumoylated蛋白在细胞应激反应的变化是由活性desumoylating酶的水平控制。我们将使用UBL-X试剂组确定SARS病毒木瓜蛋白酶样蛋白酶所表现出的DUB活性的酶特异性。这些信息将提示可能的细胞基质，并用于进行高通量药物筛选，以确定可能有助于限制冠状病毒感染传播的抗病毒剂。我们将使用一组UBL-X试剂来评估已报道的抑制去泛素化酶的抑制剂的特异性。这将提供一种快速和选择性的方法来评估“特定”抑制剂对去泛素化酶谱的全局影响。从最广泛的意义上说，这些研究将有助于我们理解UBL蛋白质代谢，并指导用于治疗癌症和微生物感染的抑制剂和药物的开发。

DESCRIPTION (provided by applicant): We have developed specific inhibitors of, and substrates for, deubiquitinating enzymes (DUBs) acting upon ubiquitin, NeddS, SUMO-1, SUMO-2/3, and ISG15 (collectively referred to as ubiquitin-like or UBL proteins). We refer to these as the UBL-X panel of reagents. The specificity of these reagents is outstanding and methods to assess cross reactivity between DUB homologs and UBL paralogs have been developed. These reagents have allowed us to define the likely specificity of a number of individual DUBs, led to some surprising conclusions regarding the spectrum of activities exhibited by different classes of DUBs, and suggested new opportunities for developing and characterizing inhibitors that specifically interfere with selected pathways of UBL protein metabolism. In the coming period: We will use the UBL-X panel of reagents in quantitative labeling and kinetic approaches to determine the amounts and paralog specificity of active desumoylating enzymes in lysates from control and stressed cells. The specificity will be compared to that of the purified catalytic domains. We will test the hypothesis that changes in cellular levels of sumoylated proteins in response to cellular stress are controlled by levels of active desumoylating enzymes. We will determine the enzymatic specificity of the DUB activity exhibited by the SARS virus papain-like protease using the panel of UBL-X reagents. This information will suggest possible cellular substrates and be used to mount a high throughput drug screen to identify antiviral agents that may be useful in limiting the spread of coronavirus infections. We will use the panel of UBL-X reagents to evaluate the specificity of inhibitors that have been reported to inhibit deubiquitinating enzymes. This will provide a rapid and selective method to evaluate the global effects of "specific" inhibitors on the spectrum of deubiquitinating enzymes. In the broadest sense, these studies will contribute to our understanding of UBL protein metabolism and guide development of inhibitors and drugs for the treatment of cancers and microbial infections.