In Vivo Interactome and Dynamics of Cullin-Ring Ligases

描述（由申请人提供）：Cullin-RING连接酶（CRLs）是真核生物中最大的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）的关键参与者，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RING E3连接酶不同，cullins不直接结合底物；相反，CRL特异性主要依赖于底物受体通过蛋白-蛋白相互作用与核心CRL复合物动态相互作用。在哺乳动物中，cullin家族有多个成员和特定的接头，以及大量的底物受体，它们可以模块化地组装在一起，在体内形成400多个crl。据估计，crl靶向近20%的蛋白酶体降解底物，包括许多致癌基因和肿瘤抑制基因。鉴于其在UPS和细胞生物学中的重要作用，crl功能的失调可导致包括癌症在内的许多不同疾病。因此，对CRL生物学的详细了解不仅有助于我们进一步了解它们在癌症发展过程中是如何被调控的，而且还为未来针对选择性蛋白质降解的癌症治疗提供了新的分子靶点。与目前通过一般蛋白酶体抑制靶向UPS的药物相比，这将是一个更有吸引力的策略，可以开发更有效、毒性更低的癌症治疗方法。蛋白质-蛋白质相互作用是建立crl多样化组装和活性的必要条件。我们假设，绘制体内的CRL相互作用网络不仅可以让我们获得活细胞中CRL系统的完整描述，而且对识别影响特定CRL途径的药物也具有重要意义。为了获得CRL在活细胞中的景观，我们建议采用QTAX策略来定义体内蛋白质相互作用组和CRL动力学，旨在识别CRL复合物的新调控因子。以下是我们的具体目标：1)定义CRL复合物在活细胞中发生的体内相互作用；2)揭示CRL4复合物在DNA损伤应答过程中的体内相互作用动态，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.