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: 10430247
• 负责人: Patrick C. Swanson
• 金额: $ 18.19万
• 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 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10430247
• 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; 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淋巴细胞形成我们的适应性免疫系统的基础，这是基于特定的 通过结构多样的表面抗原受体识别外来分子。结构多样性在这些 受体起源于淋巴细胞增殖过程中抗原受体基因的特异性重排 发展这种重排过程称为V（D）J重组，当RAG 1/2蛋白 在抗原受体基因片段处引入DNA双链断裂（DSB），并且当 DSB通过非同源末端连接被感测和修复。这一过程受到许多层的影响， 调节，但限制V（D）J重组的基本手段是控制RAG的水平 蛋白质本身该实验室的工作表明，RAG 1水平是由RAG 1相互作用控制的。 我们鉴定的蛋白质称为Vpr结合蛋白（VprBP;也称为DCAF 1），它通过以下途径发挥其控制作用： 两种不同的机制：第一，通过促进RAG 1的及时蛋白酶体依赖性降解， VprBP与Cul 4-DDB 1 E3泛素连接酶复合物的结合;第二，通过调节Rag 在刺激V（D）J重组的条件下进行转录诱导。这些分子细节 这些机制仍不清楚。我们最近进行了更详细的相互作用组研究，证实 VprBP和DDB 1与全长RAG 1相关并鉴定活化C激酶1（RACK 1）的受体 作为一个有前途的辅因子介导RAG 1降解的基础上，以前的工作建立它作为一个适配器 其他模块化cullin E3泛素连接酶。这些发现使我们假设RACK 1调节 RAG 1降解和V（D）J重组。为了验证这一假设，我们将（i）确定RACK 1在以下方面的作用： 在体外和细胞模型中调节全长RAG 1降解，并根据先前的研究， 通过HSP 90竞争性抑制和通过参与激活蛋白激酶的信号通路调节 C;和（ii）确定B细胞中RACK 1的缺失如何影响B细胞发育、RAG 1蛋白水平和V（D）J 重组建立RACK 1与全长RAG 1和Cul 4-相互作用的分子基础。 DDB 1泛素连接酶复合物，RACK 1（以及潜在的HSP 90和受体信号通路）在 调节RAG 1降解，以及B细胞中RACK 1表达缺失对B细胞的影响 RACK 1的发育、RAG 1蛋白水平和V（D）J重组将定义RACK 1的新生理作用 (and HSP 90）在V（D）J重组中的作用，并为改变免疫库的来源提供新的见解， RAG 1表达失调引起的基因组不稳定性。这项工作也将扩展范例， RACK 1作为E3泛素连接酶的多功能接头，并可能揭示新的途径， 治疗性调节RACK 1依赖性蛋白质周转。