Role of RACK1 in RAG1 degradation and B cell development

RACK1 在 RAG1 降解和 B 细胞发育中的作用

• 批准号: 10302865
• 负责人: Patrick C. Swanson
• 金额: $ 21.83万
• 依托单位: CREIGHTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-16 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10302865
• 关键词: Adaptive Immune System; Affect; Antigen Receptors; B-Cell Development; B-Lymphocytes; Benign; Binding Proteins; Cell Line; Cell model; Cells; Code; Complex; Cullin Proteins; DNA Double Strand Break; Data; Development; Electron Transport Complex III; Ephrin-A5; Epitopes; Exons; Family; Foundations; Gene Rearrangement; Genetic Transcription; Genomic Instability; HSP 90 inhibition; Half-Life; Heat-Shock Proteins 90; Immune; Immunity; Immunologic Deficiency Syndromes; Impairment; In Vitro; Knockout Mice; Laboratories; Lead; Length; Lymphocyte; Malignant Neoplasms; Mediating; Molecular; Mutation; Nonhomologous DNA End Joining; Organism; Pathogenicity; Physiological; Population Heterogeneity; Process; Protein Kinase C; Proteins; Proteomics; Publishing; Receptor Gene; Receptor Signaling; Regulation; Research; Role; Signal Pathway; Site; Site-Directed Mutagenesis; Source; Structure; Surface Antigens; System; T-Lymphocyte; Testing; Therapeutic; Time; Ubiquitin; V(D)J Recombination; Work; adaptive immunity; base; insight; knock-down; member; microbial; microorganism; multicatalytic endopeptidase complex; novel; overexpression; protein degradation; protein protein interaction; receptor; receptors for activated C kinase; reconstitution; repaired; small molecule inhibitor; ubiquitin ligase; ubiquitin-protein ligase

PROJECT SUMMARY B and T lymphocytes form the foundation of our adaptive immune system, which is based on specific recognition of foreign molecules by structurally diverse surface antigen receptors. Structural diversity in these receptors originates through site-specific rearrangement of the antigen receptor genes during lymphocyte development. This rearrangement process, called V(D)J recombination, is initiated when the RAG1/2 proteins introduce DNA double-strand breaks (DSBs) at antigen receptor gene segments, and is completed when the DSBs are sensed and repaired by non-homologous end-joining. This process is subjected to many layers of regulation, but an elementary means to constrain V(D)J recombination is to control the level of the RAG proteins themselves. Work in this laboratory suggests that RAG1 levels are controlled by a RAG1 interacting protein we identified called Vpr binding protein (VprBP; also called DCAF1), which exerts its control through two different mechanisms: first, by promoting timely proteasome-dependent degradation of RAG1 through VprBP’s association with a Cul4-DDB1 E3 ubiquitin ligase complex; and second, by regulating Rag transcriptional induction under conditions that stimulate V(D)J recombination. The molecular details underlying these mechanisms remain unclear. We have recently performed more detailed interactome studies, confirming VprBP and DDB1 association with full-length RAG1 and identifying Receptor of Activated C Kinase 1 (RACK1) as a promising co-factor in mediating RAG1 degradation based on previous work establishing it as an adaptor for other modular cullin E3 ubiquitin ligases. These findings lead us to hypothesize that RACK1 regulates RAG1 degradation and V(D)J recombination. To test this hypothesis, we will (i) establish the role of RACK1 in regulating full-length RAG1 degradation in vitro and in cell models and, based on previous studies, its potential for competitive inhibition by HSP90 and modulation by signaling pathways involved in activating Protein Kinase C; and (ii) establish how loss of RACK1 in B cells affects B cell development, RAG1 protein levels, and V(D)J recombination. Establishing the molecular basis for RACK1 interactions with full-length RAG1 and the Cul4- DDB1 ubiquitin ligase complex, the role of RACK1 (and potentially HSP90 and receptor signaling pathways) in regulating RAG1 degradation, and the consequence of losing RACK1 expression in B cells on B cell development, RAG1 protein levels, and V(D)J recombination, would define a new physiological role for RACK1 (and HSP90) in V(D)J recombination, and provide new insights into sources for altered immune repertoire and genomic instability caused by dysregulated RAG1 expression. This work would also extend the paradigm for RACK1 as a multi-functional adaptor to E3 ubiquitin ligases and potentially reveal new avenues to therapeutically regulate RACK1-dependent protein turnover.

项目摘要 B和T淋巴细胞构成了我们自适应免疫系统的基础，该系统基于特定 通过结构潜水表面抗原受体对外国分子的识别。这些结构多样性 受体通过淋巴细胞期间抗原受体基因的位点特异性重排 发展。当RAG1/2蛋白质时，启动了称为V（d）J重组的重排过程 在抗原受体基因段中引入DNA双链断裂（DSB），并在 通过非同理学端连接来感知和修复DSB。此过程受到许多层次 调节，但基本的方法来限制V（d）J重组是控制抹布的水平 蛋白质本身。该实验室的工作表明RAG1水平由RAG1相互作用控制 我们确定称为VPR结合蛋白（VPRBP；也称为DCAF1）的蛋白质，该蛋白通过 两种不同的机制：首先，通过促进RAG1的及时蛋白酶体依赖性降解 VPRBP与CUL4-DDB1 E3泛素连接酶复合物的关联；其次，通过调节抹布 在刺激V（d）J重组的条件下转录诱导。分子细节的基础 这些机制尚不清楚。我们最近进行了更详细的Interactome研究，确认 VPRBP和DDB1与全长RAG1的关联，并识别激活C激酶1（RACK1）的受体 作为基于以前的工作，将其作为适配器的介导RAG1降解的有前途的联合因素 用于其他模块化Cullin E3泛素连接酶。这些发现使我们假设RACK1调节 RAG1降解和V（D）J重组。为了检验这一假设，我们将（i）确定RACK1的作用 调节体外和细胞模型中的全长rag1降解，并基于先前的研究 HSP90的竞争抑制和通过激活蛋白激酶的信号通路进行调节 c; （ii）确定B细胞中RACK1的损失如何影响B细胞的发展，RAG1蛋白水平和V（D）J 重组。建立与全长rag1和cul4-相互作用的分子基础 DDB1泛素连接酶复合物，RACK1（以及潜在的HSP90和接收器信号通路）的作用 调节rag1降解，以及在B细胞上B细胞中RACK1表达失去RACK1的结果 开发，RAG1蛋白水平和V（D）J重组将定义RACK的新生理作用1 （和HSP90）在V（d）J重组中，并为改变免疫库的来源提供了新的见解。 由RAG1表达失调引起的基因组不稳定性。这项工作还将扩展 RACK1作为E3泛素连接酶的多功能适配器，并有可能揭示新的途径 治疗调节RACK1依赖性蛋白质周转。