In Vivo Interactome and Dynamics of Cullin-Ring Ligases

描述（由申请人提供）：Cullin-RING连接酶（CRL）代表真核生物中最大的E3泛素连接酶家族，它们参与调节广泛的生物过程，包括发育、细胞周期进程、信号转导和DNA损伤。CRL是泛素-蛋白酶体系统（UPS）中的关键参与者，并通过标记泛素化和随后的蛋白酶体降解的特异性底物来控制蛋白质的命运。与其他RING E3连接酶不同，cullin不直接结合底物;相反，CRL特异性主要依赖于通过蛋白质-蛋白质相互作用与核心CRL复合物动态相互作用的底物受体。在哺乳动物中，存在多个cullin家族成员和特异性衔接子，以及大量底物受体，其可以以模块化方式组装以在体内形成超过400个CRL。据估计，CRL靶向近20%的蛋白酶体降解底物，包括许多癌基因和肿瘤抑制基因。鉴于其在UPS和细胞生物学中的至关重要性，CRL功能的失调可能导致许多不同的疾病，包括癌症。因此，对CRL生物学的详细了解不仅可以进一步了解它们在癌症发展过程中的调节方式，还可以为未来的癌症治疗提供新的分子靶点，靶向选择性蛋白质降解。与目前通过一般蛋白酶体抑制靶向UPS的药物相比，这将是开发更有效和毒性更小的癌症治疗的更具吸引力的策略。蛋白质-蛋白质相互作用对于建立CRL的多样性组装和活性是必不可少的。我们假设，映射的CRL相互作用网络在体内不仅可以让我们获得一个完整的描述CRL系统在活细胞中，但也有重要的影响，影响特定的CRL途径的药理学试剂的鉴定。为了获得在活细胞中的CRL景观，我们建议采用QTAX策略来定义体内蛋白质相互作用组和动态的CRL，旨在确定新的调节剂的CRL复合物。以下是我们的具体目标：1）确定CRL复合物在活细胞中的体内相互作用组; 2）阐明CRL 4复合物在DNA损伤反应中的体内相互作用动力学，以阐明其功能和调控。

DESCRIPTION (provided by applicant): Cullin-RING ligases (CRLs) represent the largest family of E3 ubiquitin ligases in eukaryotes and they are involved in regulating a wide array of biological processes including development, cell cycle progression, signal transduction, and DNA damage. CRLs are key players in the ubiquitin-proteasome system (UPS) and control protein fate by marking specific substrates for ubiquitination and subsequent proteasomal degradation. Unlike other RING E3 ligases, cullins do not bind substrates directly; instead, CRL specificity relies mainly on substrate receptors that dynamically interact with core CRL complexes through protein-protein interactions. In mammals, there are multiple cullin family members and specific adaptors, as well as a large number of substrate receptors, which can assemble in a modular manner to form over 400 CRLs in vivo. CRLs are estimated to target nearly 20% of proteasomal degradation substrates including many oncogenes and tumor suppressor genes. Given its critical importance in the UPS and cell biology, dis-regulation of CRLs' function can lead to many different diseases including cancer. Therefore, detailed understanding of the CRL biology would not only further our understanding of how they are regulated during cancer development but also provide novel molecular targets for future cancer therapeutics targeting at selective protein degradation. This would be a much more attractive strategy for developing more effective and less toxic cancer treatment in comparison to current drugs targeting the UPS through general proteasome inhibition. Protein-protein interactions are essential for establishing CRLs' diverse assemblies and activities. We hypothesize that mapping the CRL interaction networks in vivo not only would allow us to obtain a complete description of CRL system in living cells but also has important implications for the identification of pharmacological agents that affect particular CRL pathways. To obtain the CRL landscape in living cells, we propose to employ the QTAX strategy to define in vivo protein interactome and dynamics of CRLs, aiming to identify new regulators of the CRL complexes. Here are our specific aims: 1) To define the in vivo interactome of CRL complexes as they occur in living cells; 2) To unravel the in vivo interaction dynamics of CRL4 complexes during DNA damage response to elucidate their function and regulation.