Analysis of Lymphocyte Development in Mice Expressing E3-Deficient RAG1 Alleles

表达 E3 缺陷 RAG1 等位基因的小鼠淋巴细胞发育分析

• 批准号: 8417642
• 负责人: JESSICA Morgan JONES
• 金额: $ 7.46万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-15 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8417642
• 关键词: 26S proteasome; Alleles; Amino Acid Substitution; Biochemical; Biological; Bone Marrow; Bromodeoxyuridine; Cell Separation; Cell physiology; Cell-Free System; Cells; Chromosomal translocation; Complex; DNA; DNA Binding; DNA Repair; Development; Epigenetic Process; Eukaryota; Gene Rearrangement; Genetic Recombination; Histones; Human; Immune; Immune system; In Vitro; Kinetics; Knock-in Mouse; Label; Lead; Left; Life; Ligation; Link; Lymphocyte; Lymphoma; Measures; Modeling; Mus; Mutation; Patients; Physiological; Property; Proteins; Proteomics; Regulation; Research; Role; SCID Mice; Serine; Shotguns; Spleen; Staging; System; Testing; Thymus Gland; Ubiquitin; Ubiquitination; V(D)J Recombination; Variant; alpha Karyopherins; gel electrophoresis; mouse model; programs; protein degradation; recombinase; trafficking; ubiquitin ligase; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): 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. Ubiquitin conjugation is involved in regulation of nearly every biochemical system in eukaryotes, and can mark proteins for degradation by the 26S proteasome or alter their biological properties. Ubiquitin ligases such as RAG1 choose the targets of ubiquitylation, suggesting that RAG1 may exert a regulatory influence over recombination and lymphocyte development. It has been shown previously that mutations in the RAG1 ubiquitin ligase domain are associated with severe immune deficiency in human patients. Consistent with this, we have found that disruption of ubiquitin ligase activity leads to a reduction in recombination in vitro under conditions where RAG1 is limiting, and the isolated RAG1 E3 domain can promote ubiquitylation of several substrates in cell-free systems. The specific hypothesis of this proposal is that RAG1 ubiquitin ligase activity promotes V(D)J recombination during lymphocyte development. This hypothesis will be tested through a detailed examination of the physiological role(s) of RAG1 E3 activity in lymphocyte development in the mouse, including an examination of ubiquitylation of putative RAG1 substrates. We will develop three separate strains of knock-in mice expressing E3-deficient RAG1 alleles, each of which has been extensively characterized in cell-free and in vitro systems, and two of which correspond to alleles identified in immunodeficient human patients. Analysis of these murine models will be absolutely critical in establishing the role of RAG1 ubiquitin ligase activity in lymphocyte development.

描述（由申请人提供）：RAG1蛋白是一种多功能复合物的一部分，该复合物进行了V（d）J重组，这是一种编程的DNA重排，是人类免疫系统开发所需的。 RAG1单独或在其重组酶伴侣RAG2的背景下，包括多种生化活性，包括DNA结合，DNA裂解和泛素连接。泛素结合涉及真核生物中几乎每个生化系统的调节，并且可以标记26S蛋白酶体降解或改变其生物学特性的蛋白质。泛素连接酶（例如RAG1）选择泛素化靶标，表明RAG1可能对重组和淋巴细胞发育产生调节作用。先前已经显示，RAG1泛素连接酶结构域中的突变与人类患者的严重免疫缺陷有关。与此一致，我们发现在RAG1受到限制的条件下，泛素连接酶活性的破坏会导致体外重组的减少，并且分离的RAG1 E3结构域可以促进无细胞系统中几种底物的泛素化。该提议的具体假设是Rag1泛素连接酶活性在淋巴细胞发育过程中促进V（d）J重组。该假设将通过对小鼠淋巴细胞发育中RAG1 E3活性的生理作用进行详细研究，包括对推定RAG1底物的泛素化的检查。我们将开发三种表达E3缺陷rag1等位基因的敲入小鼠的菌株，它们在无细胞和体外系统中都广泛表征，其中两只对应于免疫缺陷的人类患者中鉴定出的等位基因。对这些鼠模型的分析对于确定Rag1泛素连接酶活性在淋巴细胞发育中的作用至关重要。