In Vivo Interactome and Dynamics of Cullin-Ring Ligases

描述(申请人提供)：库林-环连接酶(CRL)是真核生物中最大的E3泛素连接酶家族，参与调控广泛的生物过程，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)的关键成员，通过标记泛素化和随后的蛋白酶体降解的特定底物来控制蛋白质的命运。与其他环E3连接酶不同，库林斯不直接结合底物；相反，CRL的特异性主要依赖于底物受体，底物受体通过蛋白质-蛋白质相互作用与核心CRL复合体动态相互作用。在哺乳动物中，有多个cullin家族成员和特定的接头，以及大量的底物受体，它们可以在体内以模块化的方式组装成400多个CRL。据估计，CRL靶向于近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和细胞生物学中的关键作用，CRLS功能的失调可能导致包括癌症在内的许多不同的疾病。因此，对CRL生物学的详细了解不仅将进一步加深我们对它们在癌症发生过程中如何调控的理解，而且还将为未来针对选择性蛋白质降解的癌症治疗提供新的分子靶点。与目前通过抑制蛋白酶体抑制UPS的药物相比，这将是一个更具吸引力的策略，用于开发更有效和毒性更低的癌症治疗方法。蛋白质之间的相互作用对于建立CRL的不同组装和活性是必不可少的。我们推测，绘制体内CRL相互作用网络的图谱不仅可以让我们对活细胞中的CRL系统进行完整的描述，而且对于识别影响特定CRL途径的药物也有重要意义。为了获得活细胞中CRL的图景，我们建议使用QTAX策略来定义体内CRL的蛋白质相互作用组和动力学，目的是识别CRL复合体的新的调节因子。我们的具体目标是：1)确定活细胞中CRL复合体的体内相互作用组；2)揭示CRL4复合体在DNA损伤反应中的体内相互作用动力学，以阐明它们的功能和调节。

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.