Neddylation介导的Nedd4-2激活在肾脏水电解质代谢中的作用机制

Neddylation is a process of the covalent attachment of ubiquitin-like protein Nedd8 to target proteins. The relevant research has been focused on neddylation modification of Cullin-RING family ubiquitin ligases E3 and its substrates regulating degradation of proteins. However, regulation of non-Cullin HECT family ligases E3 by neddylation has been rarely reported to date. It is still unknown whether Nedd4-2, a member of HECT family ligases, is modified by neddylation, and what is role of its neddylation in water/electrolyte metabolism and in pathogenesis of hypertension. Therefore, the project, in one hand, is going to identify Nedd4-2 activation by neddylation and its active site, and to clarify the mechanism by which neddylation-mediated Nedd4-2 regulates ion channels and ion transporters through ubiquitin-proteasome pathway in kidney. On the other hand, according to our preliminary finding that the different expression of Nedd4-2 and Nedd8 appears in liver and kidney of the CYP4F2 transgenic mice, the project is going to verify the neddylation and deneddylation of Nedd4-2 regulated by 20-HETE play a role in pathogenesis of hypertension. The study will provide new therapeutic targets for the treatment of hypertension.

Neddylation是类泛素蛋白Nedd8共价结合靶蛋白的过程。目前研究多集中在Neddylation修饰Cullin-RING家族泛素E3调节泛素-蛋白质降解，而与非Cullin 的HECT家族泛素E3及其底物的研究鲜有报道。HECT家族泛素E3成员之一Nedd4-2是否被Neddylation修饰及其在调节水电解质代谢的生理作用和在高血压的病理生理机制仍未知。因此，本课题一方面将鉴定Neddylation修饰Nedd4-2及其活性位点，阐明其对肾脏离子通道和离子转运体的泛素化水平的调节机制。另一方面，根据前期研究结果，Nedd4-2和Nedd8在CYP4F2转基因小鼠肝和肾组织的差异性表达，将阐述20-HETE对Neddylation和Deneddylation修饰Nedd4-2在高血压发病机制中的调节作用，为治疗高血压提供新的治疗靶点。

Neddylation是类泛素蛋白Nedd8共价结合靶蛋白的过程。围绕HECT家族泛素E3成员之一Nedd4-2的Neddylation修饰及其在调节水电解质代谢的生理作用和在高血压的病理生理机制展开一系列研究。本课题主要研究内容包括1）证明20-HETE可调节Nedd4-2的Neddylation修饰，并且证明该修饰作用具有组织特异性；2）NBCe1为泛素E3连接酶Nedd4-2的全新作用底物，构建Nedd4-2肾脏特异性敲除小鼠Nedd4-2，小鼠肾脏中 NBCe1表达显著上调。从细胞及动物水平证明 Nedd4-2可以介导NBCe1发生泛素化修饰并调节表达水平。3）通过改变Nedd4-2的Neddylation状态调控Nedd4-2活性，进而调节水盐代谢相关的下游靶分子，为研发治疗盐敏感性高血压的药物研究提供新的策略。4）高盐喂养的小鼠，WNK4蛋白表达水平显著高于野生型的小鼠，导致这一改变的原因是一方面高盐刺激上调泛素E3连接酶Cul3, KLHL3，同时高盐促进WNK4在S1022位点发生磷酸化，导致WNK4与Ub结合下降，进而影响WNK4的泛素化修饰，泛素化修饰的减弱导致WNK4蛋白降解的减少。5)针对先天性心脏病致病机制的研究方向，先天性心脏病胎儿羊水中cMyBP-C蛋白表达水平下降且泛素E3连接酶KLHL3 蛋白表达水平上升，实验证明同型半胱氨酸经由KLHL3介导的泛素-蛋白酶体途径诱导cMyBP-C 蛋白表达的降低。