Ubiquitin Like Protein ISG15 Conjugation

泛素样蛋白 ISG15 缀合

• 批准号: 6687384
• 负责人: DONG-ER ZHANG
• 金额: $ 40.36万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6687384
• 关键词: cell line; clinical research; cytokine; gene targeting; human tissue; immunoaffinity chromatography; laboratory mouse; ligase; liquid chromatography mass spectrometry; matrix assisted laser desorption ionization; phosphorylation; posttranslational modifications; protein purification; protein structure function; ubiquitin

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.

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，我们对蛋白质泛素化的认识迅速扩大，因此蛋白质泛素化被发现在细胞周期、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发挥着重要作用。相比之下，人们对ISG15蛋白修饰的作用知之甚少。我们克隆了去泛素酶家族的一个新成员，命名为UBP43。进一步的研究表明，UBP43是一种能特异性地将ISG15从其结合物中移除的蛋白酶。I型干扰素(INFA/b)和脂多糖强烈上调UBP43的表达和蛋白ISG化。我们已经产生了UBP43基因敲除小鼠。UBP43缺陷小鼠在发育和对干扰素和内毒素治疗的反应中表现出根本的缺陷。这一提议将检验这样一种假设，即蛋白质ISG15修饰在维持应激条件下细胞新陈代谢方面发挥着基础性作用。在特殊目标#1中提出的研究将确定ISG15的分子靶标。我们已经为这种方法建立了一种免疫亲和纯化方法。在特殊目的#2中提出的研究将通过定义目标蛋白中的ISG15修饰位点并研究ISG化对目标蛋白的影响来表征蛋白质ISG化的功能。我们已经鉴定了几种ISGylated蛋白。在特殊目的#3中提出的研究将产生条件性ISG15激活酶UBE1L基因敲除小鼠，以分析蛋白质ISG化的功能。提出的实验将解决有关蛋白质ISG化的重要问题，并可能为病毒感染和癌症等人类疾病的治疗提供有价值的见解。