Role of RAG1 Ubiquitin Ligase Activity in Recombination

RAG1 泛素连接酶活性在重组中的作用

基本信息

批准号：
7447622
负责人：
JESSICA Morgan JONES
金额：
$ 0.37万
依托单位：
GEORGETOWN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-06-20 至 2010-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7447622
关键词：
Affect Affinity Alleles B-Lymphocytes Binding Biochemical Biological Assay Biological Models Cell-Free System Cells Complex DNA DNA Binding Development Dominant-Negative Mutation Elements Enzymes Fingers Gene Rearrangement Genetic Recombination Human Immune Immune system Immunoprecipitation In Vitro Kinetics Laboratories Lead Length Lymphocyte Lysine Mass Spectrum Analysis Measures Metabolic Methods Mutagenesis Mutate Mutation Nuclear Magnetic Resonance Patients Physiological Positioning Attribute Protein Binding Proteins Rate Research Personnel Role Sequence Alignment Signal Transduction Specificity Structure Surface Plasmon Resonance Techniques Testing Ubiquitin Ubiquitin-Conjugating Enzymes Ubiquitination V(D)J Recombination Work Yeasts Zinc base in vivo molecular modeling mutant novel programs protein expression recombinase research study ubiquitin ligase ubiquitin-protein ligase yeast two hybrid system

项目摘要

The RAG1 protein is part of a multi-functional complex that carries out V(D)J recombination, a programmed DNA rearrangement that is required for development of the immune system in humans. Either alone or in the context of its recombinase partner RAG2, RAG1 encompasses multiple biochemical activities, including DNA binding, DNA cleavage, and ubiquitin ligation.The working hypothesis of this laboratory is that RAGl's integral ubiquitin ligase (E3) activity works with the ubiquitin conjugation machinery to regulate V(D)J recombination and lymphocyte development. This is based on the observations that 1) the zinc binding RING finger domain of RAG1 acts as a ubiquitin ligase (E3) in vitro and promotes covalent attachment of the small ubiquitin protein to a specific RAG1 lysine residue, 2) RAG1 is ubiquitylated in intact cells, 3) mutation or deletion of the RING finger in model systems leads to altered protein expression and decreased recombination, and 4) mutation of the RING finger or deletion of the region that is ubiquitylated can lead to severe immune deficiency in human patients. The specific hypothesis of this proposal is that RAGl's RING domain interacts with ubiquitin conjugating (E2) enzymes to promote V(D)J recombination. This hypothesis will be tested through a detailed functional and physical analysis of the interaction between the RAG1 RING finger and various E2 enzymes, followed by functional analysis of V(D)J recombination. 1.Test the prediction that the RAG1-E2 binding interface uses structural elements analogous to other RING-E2 pairs. Two independent methods will be used to identify the binding interface. 2. Test the hypothesis that alterations to the zinc binding RING finger domain change the affinity and specificity of RAG1-E2 binding and E3 activity. Multiple independent methods will be used to analyze mutants at the binding interface. 3. Test the hypothesis that RAG1 ubiquitin ligase activity/ubiquitylation promotes V(D)J recombination in cells and that ubiquitylation modulates RAGl's other biochemical activities in vitro. Mutations that diminish E3 activity will be examined in V(D)J recombination and cleavage asays.

RAG1蛋白是进行V（d）J重组的多功能复合物的一部分在人类中开发免疫系统所需的程序性DNA重排。任何一个 RAG1单独或在其重组酶伴侣RAG2的背景下包括多种生化活动，包括DNA结合，DNA裂解和泛素连接。该实验室的工作假设为 RAGL的整体泛素连接酶（E3）活性与泛素结合机械作用，可调节 V（d）J重组和淋巴细胞发育。这是基于1）锌结合的观察结果 RAG1的环指结构域在体外充当泛素连接酶（E3），并促进了共价附着针对特定RAG1赖氨酸残基的小泛素蛋白，2）Rag1在完整细胞中被泛素化，3）模型系统中环手指的突变或缺失导致蛋白质表达改变并减少重组和4）环手指的突变或被泛素化区域的缺失可能导致人类患者的严重免疫缺陷。该提议的具体假设是Ragl的环域与泛素共轭（E2）酶相互作用，以促进V（d）J重组。该假设将通过详细的功能和物理分析来检验 RAG1环手指和各种E2酶，然后进行V（d）J重组的功能分析。 1.检验预测，RAG1-E2结合界面使用类似于其他的结构元素环-E2对。将使用两种独立的方法来识别绑定界面。 2。检验以下假设：锌结合环指域的改变改变了亲和力 RAG1-E2结合和E3活性的特异性。多种独立方法将用于分析结合界面处的突变体。 3。测试Rag1泛素连接酶活性/泛素化的假设促进V（d）J 细胞中的重组，并在体外调节RAGL的其他生化活性。在V（d）J重组和裂解Asay中将检查减少E3活性的突变。