六磷酸肌醇-CRL-CSN复合物调控胰岛素分泌和糖尿病发生的功能与机制研究

Cullin Ring E3 ligases(CRLs) are a major family of protein ubiquitination machineries that together mediate the turnover of 20% of the proteome. CRL activities are dysregulated in many disease, including diabetes, and they are therefore increasingly appreciated as potential drug targets. CRLs are activated by neddylation, a ubiquitin-like modification, and are tightly regulated by dynamic complex formation with the deneddylase COP9 Signalosome (CSN). CSN sequesters yet protects CRL, enabling stimuli-dependent CRL activation, but how they interact remains poorly understood. The GPCR messenger inositol triphosphate (IP3) can be step-wise phosphorylated by a series of inositol phosphate kinases to generate IP4, IP5, IP6 and the inositol pyrophosphate IP7. Among them, IP6 is the most abundant and is essential for cell viability, but underlying reasons remain mysterious. Our group recently reported a role for IP6 in promoting CRL-CSN interaction （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 whether IP6 acts as an CRL-CSN intermolecular glue; second, 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; third, probe the physiological and pathological function of IP6-CRL-CSN ternary complex in regulating insulin secretion and obesity/diabetes development by using knockin mice with IP6-binding deficient CSN mutant alleles; fourth, explore the therapeutic potential of the neddylation inhibitor MLN4924, or the membrane permeable IP6 analog inositol trispyrophosphate, in modulating obesity/diabetes development via controlled insulin secretion. Successful execution of this project will set a classical example on studying regulatory mechanisms of inositol phosphates in general and provide new targeting strategy to meet the urgent need for anti-obesity/diabetes therapy.

Cullin-RING ligase（CRL）家族泛素连接酶介导了总泛素化的20%，在糖尿病等多种疾病中失调。CRL由类泛素化(Neddylation)激活，被去类泛素化酶CSN动态调控，二者互作的机制了解不多。GPCR信使三磷酸肌醇(IP3)被多个肌醇激酶逐步磷酸化，生成的多磷酸肌醇分子(IP4，IP5, IP6, IP7)较少被研究。我们前期发现IP6促进CRL和CSN的互作，但IP6螯合CRL-CSN复合物的生化机理和生理意义不清楚。本项目拟解析IP6调节CSN酶活或辅助CSN与E2连接酶竞争而抑制CRL类泛素化等机制；并根据前期结果，利用IP6结合缺失的CSN突变体小鼠研究该复合物调控胰岛素分泌、肥胖和糖尿病发生的功能；研究成果将成阐明 IP6调控CRL-CSN泛素连接酶复合体的分子机制，同时拓展六磷酸肌醇与糖尿病这一新领域，为靶向IP6的合成酶IP5K的转化研究奠定基础。

Cullin RING Ligase (CRL)是作用于蛋白泛素化降解的一类最重要的E3泛素连接酶。CRL降解底物时需要被拟素化修饰（Neddylation）激活，COP1是CRL的底物受体，经此研究发现COP1-CSN在葡萄糖诱导的胰岛素分泌过程中的拮抗作用。该研究通过表征IP6结合缺失而导致CSN功能受损的CSN2-K70E突变体小鼠，发现纯和突变会导致小鼠胚胎致死，而杂合突变体小鼠具有高胰岛素血症（ Hyperinsulinemia）和肥胖( obesity)表型。据此我们探究出 CRL-CSN 复合物互作的界面，并解析得到 IP6- CRL-CSN2 复合物的晶体结构，证实IP6 结合口袋可作为调控CRL 拟素化修饰及活化的新靶点。经研究调控机制(Regulatory Mechanism)发现，在解除CSN的抑制功能后，β细胞内CRL4的拟素化增加，CRL4COP1 E3酶活化，而转录因子ETV5的降解增强。ETV5通过调控Ins1基因和囊泡释放相关因子Sytl3和Exoc6的转录来抑制胰岛素分泌( Insulin Secretion)。因此，CSN2-K70E杂合突变体小鼠会持续过度分泌胰岛素，诱发高胰岛素血症和肥胖。CRL拟素化抑制剂MLN4924可能成为靶向抑制CRL4COP1-ETV5信号轴而减轻高胰岛素血症诱发的肥胖/糖尿病的新兴药物。同时，我们筛选出有效的IP5K抑制剂Suramin，尝试使用这种IP6生成调控工具来调节胰岛素分泌，进一步拓展糖尿病治疗方法领域。