Role of Ubiquitin Like Protein ISG15 Conjugation

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(泛素化)的了解迅速扩大，因此蛋白质泛素化被发现在细胞功能的各个方面发挥着重要作用，包括细胞周期、膜受体信号转导、内吞、蛋白质质量控制、转录和DNA修复。相比之下，人们对ISG15蛋白修饰的作用知之甚少。ISG15由干扰素刺激基因编码。它的表达被I型干扰素以及细菌和病毒感染高度上调。由于ISG15不存在于酵母和线虫等简单的真核生物中，因此它不太可能是一个看家基因。相反，它应该参与复杂生物体的特殊功能，如人类和老鼠。在过去的四年资助期间，我们已经确定了一组ISG15靶点，表征了ISG15修饰对其中一些靶点的影响，鉴定了ISG15 E2和一些E3酶，并建立了ISG15 E1基因敲除小鼠。这项关于下一个两年资助期的提案将检验这样一个假设，即蛋白质ISG15修饰在免疫反应期间调节细胞功能方面发挥着重要作用。在特殊目的#1中提出的研究将通过高通量筛选ISGylated蛋白来识别ISG15修饰位点。在特殊目的#3中提出的研究将描述ISG15激活酶UBE1L基因敲除小鼠的特征，以检验蛋白质ISG化功能的分子机制。这些拟议的研究将解决有关蛋白质ISG化的重要问题，并可能为人类疾病的预防和治疗提供有价值的见解，如病原体感染和免疫缺陷。

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 (ubiquitylation) has expanded rapidly and as a consequence protein ubiquitylation has been found to play important roles in various aspects of cellular function, including the cell cycle, membrane receptor signal transduction, endocytosis, protein quality control, transcription, and DNA repair. In contrast, little is known about the role of protein modification by ISG15. ISG15 is encoded by an interferon stimulated gene. Its expression is highly upregulated by Type I interferon and by bacterial and viral infections. Since ISG15 is not found in simple eukaryotic organisms, such as yeast and nematodes, it is unlikely to be a house keeping gene. Instead, it should be involved in specialized functions in complex organisms, such as human and mouse. In the past four year funding period, we have identified a group of ISG15 targets, characterized the effect of ISG15 modification on some of these targets, identified the ISG15 E2 and a few of the E3 enzymes, and established ISG15 E1 knockout mice. This proposal for the next two-year funding period will test the hypothesis that protein ISG15 modification plays an important role in modulating cellular function during immune responses. The studies proposed in Specific Aim #1 will identify ISG15 modification sites via high throughput screening of ISGylated proteins. The studies proposed in Specific Aim #3 will characterize ISG15 activating enzyme UBE1L knockout mice to examine molecular mechanisms of the function of protein ISGylation. These proposed studies will address important questions about protein ISGylation and may provide valuable insights into the prevention and therapeutic treatment of human diseases, such as pathogen infections and immune defects.