Functions of Ub-like Proteins and Proteases

描述（由申请人提供）：我们已经开发了针对泛素、NeddS、SUMO-1、SUMO-2/3和ISG15的去泛素化酶（DUBs）的特异性抑制剂和底物（统称泛素样蛋白或UBL蛋白）。我们将这些称为UBL-X试剂面板。这些试剂的特异性非常突出，并且已经开发了评估DUB同源物和UBL相似物之间交叉反应性的方法。这些试剂使我们能够确定许多单个dub的可能特异性，并得出关于不同类别dub所表现出的活性谱的一些令人惊讶的结论，并为开发和表征特异性干扰选定UBL蛋白代谢途径的抑制剂提供了新的机会。在接下来的一段时间里：我们将使用UBL-X试剂面板进行定量标记和动力学方法，以确定对照和应激细胞裂解物中活性脱氧酶的数量和平行特异性。将其特异性与纯化的催化结构域进行比较。我们将检验这一假设，即在细胞应激的反应中，细胞中被水解蛋白水平的变化是由活性脱羧酶的水平控制的。我们将使用UBL-X试剂确定SARS病毒木瓜蛋白酶所表现出的DUB活性的酶学特异性。这一信息将提示可能的细胞底物，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药物。我们将使用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“特异性”抑制剂对去泛素化酶谱的整体影响。从广义上讲，这些研究将有助于我们对UBL蛋白代谢的理解，并指导开发用于治疗癌症和微生物感染的抑制剂和药物。

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.