CRL泛素连接酶受拟素化修饰(Neddylation)动态调控的机制与功能研究

Cullin-RING ligase（CRL）泛素连接酶在癌症中活性多上调，介导了细胞生长、存活、自噬、迁移和免疫逃逸等癌变特质相关蛋白的降解。 拟素化修饰激活CRL，其抑制剂MLN4924是临床二期药物。去拟素化酶CSN抑制CRL,但相关互作机制，动态调控规律和生物学效应尚不清楚。肌醇代谢通路里的六磷酸肌醇（IP6）能促进CRL和CSN的结合，但生化机理和生理意义未知。本项目拟一，解析IP6结合CSN调控CRL动态拟泛素化的结构和机制；二，制备IP6结合缺失的CSN突变体小鼠并结合肿瘤模型表征IP6-CSN-CRL复合物如何参与调控肿瘤发生；三，筛选IP6合成酶IP5K的抑制剂并鉴定其与MLN4924或CSN抑制剂CSN5i-3联合进行肿瘤干预的效果。四，明确CSN是否参与基于CRL的PROTAC小分子药物的抵抗并探索IP5Ki诱发的CRL-CSN解离是否可与PROTAC小分子协同作用。

Cullin Ring E3 ligases(CRLs) are a major family of protein ubiquitination.machineries that are aberrantly active in cancer and mediate the degradation of many proteins involved in carcinogenic process such as cell survival, growth, metabolism, autophagy, migration and immune evasion. Neddylation activates CRL, and the neddylation inhibitor MLN4924 is currently in phase II clinical trials. The COP9 signalosome (CSN) binds, deneddylates, and inactivates CRL, but mechanisms underlying dynamic CRL-CSN interactions and their biological consequences remain incompletely understood. Our group recently discovered a role for the small metabolite inositol hexakisphosphate (IP6) in promoting CRL-CSN complex assembly （PNAS 2016）. However, key elements required to establish IP6 as a CRL-CSN regulator, such as the biochemical/structural basis and physiological importance of the IP6-CRL-CSN complexes, remain unknown. In this project, we will employ a cross-disciplinary approach to: first, determine structural basis of IP6 acing as an CRL-CSN intermolecular glue and dissect the biochemical impact of IP6 at the CRL-CSN interface: as a regulator of CSN’s deneddylase activity or a modulator of CSN-E2 ligase competition for CRL binding; second, probe the physiological and pathological functions of IP6-CRL-CSN ternary complex in genetic and chemically-induced tumor models by using knockin mice with IP6-binding deficient CSN mutant alleles; third, screen for lead compounds that inhibits the IP6 synthase IP5K(IP5Ki) for tumor chemical prevention and explore potential synergistic effect of IP5Ki in combination with MLN4924 or the CSN deneddylase inhibitor CSN5i-3; fourth, identify potential role of CSN in mediating CRL-based PROTAC drug resistance and explore whether IP5Ki can promote therapeutic effects of CRL-based PROTAC molecules. Successful execution of this project will set a classical example on studying dynamic regulation of CRL neddylation modification and provide new targeting strategy to meet the urgent need for anti-cancer therapy.