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ISG15(统称为泛素样蛋白或UBL蛋白)的去泛素化酶(DUBS)的特定抑制剂和底物。我们将这些称为UBL-X试剂小组。这些试剂的特异性是突出的，并已开发出评估DUB同系物和UBL类同系物之间交叉反应的方法。这些试剂使我们能够定义一些单个DUB的可能特异性，导致了关于不同类别DUB表现出的活性光谱的一些令人惊讶的结论，并为开发和表征特定干扰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：我们将在定量标记和动力学方法中使用UBL-X试剂小组来确定来自对照和应激细胞的裂解产物中活性脱苏糖酶的数量和同源特异性。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，即细胞对细胞压力的反应中，苏木糖化蛋白水平的变化是由活性脱苏糖酶的水平控制的。我们将使用UBL-X试剂板来确定SARS病毒木瓜蛋白样蛋白酶DUB活性的酶特异性。这些信息将建议可能的细胞底物，并用于安装高通量药物筛选，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试剂小组来评估已报道的抑制脱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“特定的”抑制剂对脱泛素化酶谱的全球影响。在最广泛的意义上，这些研究将有助于我们了解UBL蛋白的代谢，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.