Cullin3突变导致家族性高血钾型高血压的分子机制研究

Familial Hyperkalemic Hypertension (FHHt) is a Mendelian form of arterial hypertension that characterized by sodium chloride cotransporter(NCC) activation in renal distal convoluted tubule. Among all the 4 known FHHt-causing genes, mutations of Cullin3 (Cul3) has the earliest onset age and the most severe phenotype including hypertension, hyperkalemia and acidosis. Tremendous work has been done to elucidate the molecular mechanism of FHHt over a decade, however till now the mechanism of Cul3-FHHt remain unclear. Cul3 plays a critical role in the polyubiquitination and subsequent degradation of specific protein substrates as the core component and scaffold protein of an E3 ubiquitin ligase complex. It can ubiquitinate WNK kinases in distal nephron with the help of its adaptor protein, KLHL3. Our previous study published in The Journal of Clinical Investigation disclosed Cul3 disease mutant, Cul3△9 appeared to be much more neddylated, namely more activated than wild type Cul3, which subsequently caused the degradation of its adaptor protein, KLHL3. The loss of adaptor protein resulted in accumulated WNK kinases expression and hence NCC activation. However, the mechanism of increased neddylation of Cul3△9, and the reason for most severe phenotype of Cul3-FHHt among different disease-causing genes remain obscure. We continue to focus our efforts on the mechanism of Cul3-FHHt after the publication of JCI paper and our further investigation revealed impaired ability of Cul3△9 for binding COP9 signalosome (CSN), which serves as deneddylase and can remove nedd8 from Cul3. It strongly suggests the mechanism of over-neddylated state, namely excessive activation of Cul3△9. In addition, another adaptor of Cul3, KLHL2, which is also expressed in the distal tubule, can also be heavily degraded by Cul3△9. Presumably the degradation of both KLHL3 and KLHL2 by Cul3 △9 causes increased WNK kinase abundance in distal tubule, which likely accounts for the most severe phenotype of Cul3-FHHt. We will study the binding region of Cul3 to CSN, and the role of KLHL2 in Cul3-FHHt in mammalian cells and Cul3 △9 mouse we generated. We expect this study will be important in clarifying the mechanisms of FHHt and NCC regulation, and shed light on the pathogenesis of essential hypertension.

家族性高血钾型高血压（FHHt）是一种单基因遗传性高血压，表现为肾远曲小管钠氯共转运子（NCC）功能亢进，致病基因包括WNK激酶、KLHL3和Cul3，其中Cul3-FHHt表型最重。Cul3可在连接蛋白KLHL3的帮助下，泛素化降解WNK激酶。我们既往研究发现，Cul3致病突变△9比野生型Nedd化修饰明显增加，活性增强，转而显著降解KLHL3，造成WNK激酶降解受阻，最终激活NCC。然而为何Cul3△9 Nedd化修饰增加，以及Cul3-FHHt表型最重尚未搞清。我们根据预实验结果推测Cul3△9与去Nedd化修饰酶CSN结合减弱，是其Nedd化修饰增加的原因；远端肾小管还存在另一种连接蛋白KLHL2，也被Cul3△9显著降解，导致WNK激酶更多和/或更广泛的蓄积，是Cul3-FHHt表型最重的原因。本项目将进行体内体外研究，包括建立的Cul3 △9小鼠，阐明Cul3-FHHt机制。

高血压患病率高、危害性大，是目前人类健康的主要威胁之一。大部分高血压患者病因不清，为原发性高血压。单基因遗传高血压是探究高血压发病机制和肾离子重吸收调控机制的理想模型。家族性高血钾型高血压（FHHt）表现为高血压、高血钾、代谢性酸中毒，分子遗传学研究证实肾远曲小管上皮细胞表达的WNK激酶、KLHL3和Cul3发生突变，导致钠氯共转运子（NCC）功能亢进，重吸收钠离子增多而致病。Cul3可泛素化降解WNK激酶，KLHL3是其连接蛋白，然而Cul3突变导致FHHt的机制仍未搞清。本项目通过一系列体内体外实验研究Cul3致病突变的neddylation异常的机制，以及KLHL3的同源类似物——KLHL2被野生型和突变型Cul3降解的情况，深入探索Cul3-FHHt的分子发病机制。我们免疫荧光检测了去neddylation酶的核心组分CSN5在肾脏的表达，发现其在远曲小管和连接管处表达最为丰富，细胞定位在顶膜附近表达。我们构建了多个Cul3的片段， Co-IP证实并非Cul3致病突变缺失的片段，而是紧邻其后的α/β结构域是直接与CSN5结合的部位。在mRNA水平证实了KLHL2在肾小管各段的表达，KLHL2也具有降解各种WNK激酶的能力，也被Cul3所降解，且Cul3致病突变体降解明显增加，提示Cul3突变时，KLHL2也可作为其连接蛋白被清空，其结果造成WNK激酶的丰度增加，从而参与形成Cul3-FHHt的严重表型。