ABCC2p.R393W variant contributes to Dubin-Johnson syndrome by targeting MRP2 to proteasome degradation

Rong-Yue Sun, Yi-Ming Chen, Mian-Mian Zhu, Ji-an Sun, Hong-Wei Wang, Chen-Yu Wu, Ting Zhu, Yu-Jing Gong, Chao-Sheng Lu, L. Ronzoni, Luca Valenti, M. Zheng, Dan Wang
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Abstract

Dubin-Johnson syndrome (DJS), a rare autosomal recessive liver condition, is caused by biallelic loss-of-function mutations of theABCC2gene. This study aimed to investigate genetic variations in the drug efflux transporterABCC2(MRP2) gene in patients with DJS and to characterise the expression and mechanism of theABCC2gene variant.Trio whole exome sequencing was performed in the family to identify the genetic causes. Bioinformatics analysis was performed to assess pathogenicity. Inin vitroexperiments, site-directed mutagenesis was used to introduceABCC2variants in constructs then expressed in HEK293T, HuH-7 and HepG2 cell lines. The expression of total and cell membrane MRP2 was quantified in cells expressing the wild-type or variant forms. Chloroquine and MG132 were used to evaluate the effects of p.R393W on lysosomal and/or proteasomal degradation.The twin probands carry DJS-associated variants c.1177C>T (rs777902199) in theABCC2gene inherited from the father and the c.3632T>C mutation in the other allele inherited from the mother. TheABCC2variant, c.1177C>T, results in a p.R393W substitution in MRP2 that is highly conserved among vertebrates, drastically decreasing the expression of mutant protein by promoting proteasomal degradation. Another variant c.3632T>C results in a p.L1211P substitution in MRP2, decreasing the expression of membrane MRP2 but not changing the expression of total protein.These results strongly suggest that the p.R393W variant affects the stability of the MRP2 protein and decreases its expression by ubiquitin-mediated proteasomal degradation, and the p.L1211P decreases the expression of membrane MRP2, indicating that these two variants, respectively, cause a loss-of-function of the MRP2 protein and membrane MRP2 ultimately leading to DJS development.
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ABCC2p.R393W变体通过靶向蛋白酶体降解MRP2而导致杜宾-约翰逊综合征
杜宾-约翰逊综合征(Dubin-Johnson Syndrome,DJS)是一种罕见的常染色体隐性肝病,由ABCC2基因的双倍功能缺失突变引起。本研究旨在调查DJS患者药物外排转运体ABCC2(MRP2)基因的遗传变异,并描述ABCC2基因变异的表达和机制。对该家族进行了三重全外显子组测序,以确定遗传原因,并进行了生物信息学分析以评估致病性。在体外实验中,使用定点突变技术在构建体中引入ABCC2变体,然后在HEK293T、HuH-7和HepG2细胞系中表达。在表达野生型或变异型的细胞中,对总 MRP2 和细胞膜 MRP2 的表达进行了量化。这对双胞胎携带了从父亲那里遗传的ABCC2基因中的c.1177C>T(rs777902199)变异和从母亲那里遗传的另一个等位基因中的c.3632T>C变异。ABCC2变异体c.1177C>T导致MRP2中的p.R393W置换,该置换在脊椎动物中高度保守,通过促进蛋白酶体降解而大幅降低突变蛋白的表达。另一个变体 c.3632这些结果有力地表明,p.R393W 变体会影响 MRP2 蛋白的稳定性,并通过泛素介导的蛋白酶体降解降低其表达量,而 p.L1211P 则会降低膜 MRP2 的表达量。L1211P降低了膜MRP2的表达,这表明这两个变异分别导致了MRP2蛋白和膜MRP2的功能缺失,最终导致了DJS的发生。
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