Dubin-Johnson综合征（DJS）以直接胆红素升高为特征，与ABCC2基因突变导致其编码的MRP2蛋白功能障碍有关。课题组前期研究发现，中国DJS病人中存在高频率的ABCC2基因NBD区突变，并引起MRP2蛋白亚细胞定位改变及表达降低，但具体机制尚不清楚。本项目拟针对ABCC2基因NBD区热点突变，首先，构建ABCC2稳定敲除细胞模型及定点突变质粒，初步探讨上述突变对MRP2蛋白的表达水平、亚细胞定位以及有机阴离子转运功能的影响；其次，构建突变体腺相关病毒并感染ABCC2-/-小鼠，研究NBD突变对小鼠的DJS表型的影响及机制；再次，通过研究突变影响MRP2与支架蛋白PDZK1之间相互作用以及MRP2泛素化修饰的蛋白酶体降解，明确NBD区突变影响MRP2的亚细胞定位及降解的机制；最后，从临床层面分析基因型与表型的关系。本项目将为我国DJS患者的发病机制及临床诊断提供有效的实验依据。

Dubin-Johnson syndrome (DJS) is a rare, autosomal recessive disorder characterized by predominantly conjugated hyperbilirubinemia, caused by mutations in ABCC2 gene coding MRP2 protein. Our previous study indicated that the ABCC2 gene NBD mutation resulted in subcellular mislocalization and degeneration of MRP2. But the specific molecular mechanism is not clear. Therefore, this project attempts to focus on the ABCC2 NBD mutation. Firstly, we will construct plasmids with ABCC2 wild type and ABCC2 NBD mutant type, and then transfect the cell lines with different plasmids to detect the expression of MRP2, the changes of subcellular localization of MRP2 and the changes of transport function of MRP2 protein. Secondly, we explore the phenotype, serum total and direct bilirubin, MRP2 immunohistochemical staining of liver tissue in ABCC2 knock out animal models. Thirdly, Co-IP will be performed to detect the protein-protein interactions between wild-type/mutant MRP2 and PDZK1. Besides, Co-IP and proteasome inhibitor MG132 will be used to detect the differential performance of ubiquitination modification between wild-type and mutant MRP2. This project will reveal the molecular mechanisms of subcellular mislocalization and degeneration of MRP2 that caused by ABCC2 gene NBD mutation, mainly through protein-protein interaction between mutant MRP2 and PDZK1, and ubiquitination pathway. It is a preliminary study on the pathogenicity and mechanism of ABCC2 gene NBD mutation in China, and try to provide valuable experimental basis for the study of DJS and differential diagnosis of hyperbilirubinemia.