Role of RACK1 in RAG1 degradation and B cell development

项目总结 B和T淋巴细胞构成了我们适应性免疫系统的基础，它基于特定的 通过结构不同的表面抗原受体识别外源分子。这些地区的结构多样性 受体起源于淋巴细胞中抗原受体基因的特异性重排 发展。这种重排过程被称为V(D)J重组，当RAG1/2蛋白启动时 在抗原受体基因片段引入DNA双链断裂(DSB)，并在 双链断裂是通过非同源末端连接来感知和修复的。这一过程经历了许多层次的 但限制V(D)J重组的一个基本手段是控制RAG的水平 蛋白质本身。该实验室的工作表明，RAG1水平受RAG1相互作用的控制 我们确定的一种蛋白质称为VPR结合蛋白(VprBP；也称为DCAF1)，它通过 两种不同的机制：第一，通过促进蛋白酶体依赖的RAG1的及时降解 VprBP与CUL4-DDB1 E3泛素连接酶复合体的结合；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。潜在的分子细节 这些机制仍不清楚。我们最近进行了更详细的互动组研究，证实了 VprBP和DDB1与全长RAG1的关系及活化C-激酶受体RACK1的识别 作为一种有前景的辅助因子，在调节RAG1降解方面，基于先前的工作，将其建立为一个适配器 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复合。为了检验这一假设，我们将(I)确定RACK1在 在体外和细胞模型中调节全长RAG1的降解以及基于先前的研究，其潜力 对HSP90的竞争性抑制和参与蛋白激酶激活的信号通路的调节 C；和(Ii)确定B细胞中RACK1的缺失如何影响B细胞发育、RAG1蛋白水平和V(D)J 重组。RACK1与全长RAG1和CUL4-相互作用的分子基础 DDB1泛素连接酶复合体，RACK1(以及潜在的HSP90和受体信号通路)在 调节RAG1的降解，以及B细胞上RACK1表达缺失的后果 RACK1的发育、RAG1蛋白水平和V(D)J重组将定义一个新的生理角色 (和HSP90)在V(D)J重组中的表达，并为改变免疫谱系的来源和 RAG1表达异常引起的基因组不稳定性。这项工作还将把范例扩展到 RACK1作为E3泛素连接酶的多功能适配子，并可能揭示新的途径 从治疗上调节RACK1依赖的蛋白质周转。

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.