Role of Ubiquitin Like Protein ISG15 Conjugation

描述（由申请人提供）： 本研究的长期目标是了解泛素样蛋白ISG15修饰蛋白质的生物学功能。近年来，人们对蛋白质泛素化的认识迅速扩展，发现蛋白质泛素化在细胞周期、膜受体信号转导、内吞作用和DNA修复等细胞功能的各个方面都发挥着重要作用。相比之下，关于ISG15对蛋白质修饰的作用知之甚少。我们克隆了去泛素化酶家族的一个新成员，称为UBP 43。进一步的研究表明，UBP 43是一种蛋白酶，可以特异性地从其缀合物中去除ISG15。I型干扰素（INFa/B）和LPS强烈上调UBP 43表达和蛋白质ISG化。我们已经产生了UBP43敲除小鼠。UBP43缺陷型小鼠在发育和对干扰素和LPS治疗的反应中显示出根本性缺陷。该提案将测试蛋白ISG15修饰在应激条件下维持细胞代谢中起重要作用的假设。具体目标#1中提出的研究将确定ISG15的分子靶点。我们已经建立了一种免疫亲和纯化方法，这样的方法。具体目标#2中提出的研究将通过定义靶蛋白中的ISG 15修饰位点并研究ISG化对靶蛋白的影响来表征蛋白ISG化的功能。我们已经鉴定了几种ISGylated蛋白。具体目标#3中提出的研究将产生条件性ISG15活化酶UBE1L敲除小鼠，以分析蛋白ISG化的功能。提出的实验将解决有关蛋白质ISGylation的重要问题，并可能为人类疾病（如病毒感染和癌症）的治疗提供有价值的见解。

DESCRIPTION (provided by applicant): The long-term goal of this study is to understand the biological function of protein modification by the ubiquitin like protein ISG15. In recent years our knowledge of protein ubiquitination has expanded rapidly and as a consequence protein ubiquitination has been found to play important roles in various aspects of cellular functions, including cell cycle, membrane receptor signal transduction, endocytosis, and DNA repair. In contrast, little is known about the role of protein modification by ISG15. We cloned a novel member of the deubiquitinating enzyme family, termed UBP43. Further studies have demonstrated that UBP43 is a protease that specifically removes ISG15 from its conjugates. Type I interferon (INFa/b) and LPS strongly upregulate both UBP43 expression and protein ISGylation. We have generated UBP43 knockout mice. UBP43 deficient mice show fundamental defects in development and in response to interferon and LPS treatment. This proposal will test the hypothesis that protein ISG15 modification plays a fundamental role in maintaining cellular metabolism under conditions of stress. The studies proposed in Specific Aim#1 will identify the molecular targets of ISG15. We have established an immuno-affinity purification method for such an approach. The studies proposed in Specific Aim #2 will characterize the function of protein ISGylation by defining ISG15 modification sites in target proteins and studying the effect of ISGylation on target proteins. We have identified several ISGylated proteins. The studies proposed in Specific Aim #3 will generate conditional ISG15 activating enzyme UBE1L knockout mice to analyze the function of protein ISGylation. The experiments proposed will address important questions about protein ISGylation and may provide valuable insights into the therapeutic treatment of human diseases, such as viral infection and cancer.