Phenotypic differences in PFIC2 and BRIC2 correlate with protein stability of mutant Bsep and impaired taurocholate secretion in MDCK II cells
Phenotypic differences in PFIC2 and BRIC2 correlate with protein stability of mutant Bsep and impaired taurocholate secretion in MDCK II cells
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DOI：
10.1152/ajpgi.00367.2007
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发表时间：
2008-01-01
影响因子：
4.5
通讯作者：
Mine, Tetsuya
中科院分区：
文献类型：
--
作者：
Kagawa, Tatehiro;Watanabe, Norihito;Mine, Tetsuya
进行性家族性胆汁淤积症(PFIC)2和良性复发性肝内胆汁淤积症(BRIC)2是由胆盐输出泵(BSEP，ABCB11)基因突变引起的，但它们的预后不同。PFIC2进展为肝硬变，需要肝移植，而BRIC2在临床上是良性的。为了确定导致表型差异的分子机制(S)，在大鼠BSEP中引入了8个PFIC2和2个BRIC2突变，并将其导入MDCK II细胞。在极化的MDCK II单层中研究了这些突变蛋白的牛磺胆酸盐转运活性、蛋白表达和亚细胞分布。牛磺胆酸转运活性在BRIC2突变体(A570T和R1050C)中约为野生型(WT)的一半，在两个PFIC2突变体(D482G和E297G)中显著低于野生型(WT)，在其他六个突变体(K461E、G982R、R1153C、R1268Q、3767-3768insC和R1057X)中几乎被取消。除R1057X外，BSEP蛋白表达水平与转运活性密切相关。D482G突变体的半衰期比WT突变体短(1.35h比3.49h)。BRIC2突变体和3个pFIC突变体(D482G、E297G和R1057X)主要分布在顶膜。其他的pFIC2突变体保持在细胞内。R1057X突变体蛋白稳定表达并运输到顶膜，表明COOH末端的尾巴是运输活性所必需的，但不是正确靶向所必需的。综上所述，牛磺胆酸转运功能受损与Bsep蛋白的快速降解成正比，其顺序依次为：A570T和R1050C>；D482G>；E297G>；K461E；G982R；R1153C；R1268Q；3767-3768insC和R1057X。这些结果可能解释了BRIC2和PFIC2的表型差异。
Progressive familial cholestasis (PFIC) 2 and benign recurrent intrahepatic cholestasis (BRIC) 2 are caused by mutations in the bile salt export pump (BSEP, ABCB11) gene; however, their prognosis differs. PFIC2 progresses to cirrhosis and requires liver transplantation, whereas BRIC2 is clinically benign. To identify the molecular mechanism(s) responsible for the phenotypic differences, eight PFIC2 and two BRIC2 mutations were introduced in rat Bsep, which was transfected in MDCK II cells. Taurocholate transport activity, protein expression, and subcellular distribution of these mutant proteins were studied in a polarized MDCK II monolayer. The taurocholate transport activity was approximately half of the wild-type (WT) in BRIC2 mutants (A570T and R1050C), was substantially less in two PFIC2 mutants (D482G and E297G), and was almost abolished in six other PFIC2 mutants (K461E, G982R, R1153C, R1268Q, 3767-3768insC, and R1057X). Bsep protein expression levels correlated closely with transport activity, except for R1057X. The half-life of the D482G mutant was shorter than that of the WT (1.35 h vs. 3.49 h in the mature form). BRIC2 mutants and three PFIC mutants (D482G, E297G, and R1057X) were predominantly distributed in the apical membrane. The other PFIC2 mutants remained intracellular. The R1057X mutant protein was stably expressed and trafficked to the apical membrane, suggesting that the COOH-terminal tail is required for transport activity but not for correct targeting. In conclusion, taurocholate transport function was impaired in proportion to rapid degradation of Bsep protein in the mutants, which were aligned in the following order: A570T and R1050C > D482G > E297G > K461E, G982R, R1153C, R1268Q, 3767 - 3768insC, and R1057X. These results may explain the phenotypic difference between BRIC2 and PFIC2.