benzyloxycarbonylleucyl-leucyl-leucine-aldehyde and Cholestasis--Intrahepatic

Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a hepatic disorder occurring predominantly in childhood and is difficult to diagnose. PFIC3, being a rare autosomal recessive disease, is caused by genetic mutations in both alleles of

Topics: Adolescent; ATP Binding Cassette Transporter, Subfamily B; Cholestasis, Intrahepatic; Down-Regulation; Exome Sequencing; Female; Hep G2 Cells; Homozygote; Humans; Leupeptins; Mutation, Missense; Pedigree

Multidrug resistance 3 (MDR3), encoded by the ATP-binding cassette, subfamily B, member 4 gene (ABCB4), localizes to the canalicular membrane of hepatocytes and translocates phosphatidylcholine from the inner leaflet to the outer leaflet of the canalicular membrane. Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a rare hepatic disease caused by genetic mutations of ABCB4. In this study, we characterized 8 ABCB4 mutations found in PFIC3 patients, using in vitro molecular assays. First, we examined the transport activity of each mutant by measuring its ATPase activity using paclitaxel or phosphatidylcholine. Then, the pathogenic mechanisms by which these mutations affect MDR3 were examined through immunoblotting, cell surface biotinylation, and immunofluorescence. As a result, three ABCB4 mutants showed significantly reduced transport activity. Among these mutants, one mutation A364V, located in intracellular domains, markedly decreased MDR3 expression on the plasma membrane, while the others did not affect the expression. The expression of MDR3 on the plasma membrane and transport activity of A364V was rescued by a pharmacological chaperone, cyclosporin A. Our study provides the molecular mechanisms of ABCB4 mutations and may contribute to the understanding of PFIC3 pathogenesis and the development of a mutation-specific targeted treatment for PFIC3.

Topics: ATP Binding Cassette Transporter, Subfamily B; Cholestasis, Intrahepatic; Cyclosporine; Gene Expression; Genetic Association Studies; Genetic Predisposition to Disease; HEK293 Cells; Humans; Kinetics; Leupeptins; Macrolides; Mutation, Missense; Paclitaxel; Protein Transport

Multidrug resistance-associated protein 2 (MRP2) is a member of a family of efflux transporters that are involved in biliary excretion of organic anions from hepatocytes. Disrupted canalicular localization and decreased protein expression of MRP2 have been observed in patients with chronic cholestatic disorder and hepatic failure without a change in its mRNA expression. We have previously demonstrated that post-transcriptional regulation of the rapid retrieval of rat MRP2 from the canalicular membrane to the intracelluar compartment occurs under conditions of acute (~30min) oxidative stress. However, it is unclear whether MRP2 expression is decreased during its sustained internalization during chronic oxidative stress. The present study employed buthionine sulfoximine (BSO) to induce chronic oxidative stress in the livers of Sprague-Dawley rats and then examined the protein expression and localization of MRP2. Canalicular MRP2 localization was altered by BSO treatment for 2h without changing the hepatic protein expression of MRP2. While the 8h after exposure to BSO, hepatic MRP2 protein expression was decreased, and the canalicular localization of MRP2 was disrupted without changing the mRNA expression of MRP2. The BSO-induced reduction in MRP2 protein expression was suppressed by pretreatment with N-benzyloxycarbonyl (Cbz)-Leu-Leu-leucinal ( MG-132), a proteasomal inhibitor. Furthermore, the modification of MRP2 by small ubiquitin-relatedmodifier 1 (SUMO-1) was impaired in BSO-treated rat liver,while that by ubiquitin (Ub) and MRP2 was enhanced. Taken together, the results of this study suggest the sustained periods of low GSH content coupled with altered modification of MRP2 by Ub/SUMO-1 were accompanied by proteasomal degradation of MRP2.

Topics: Animals; Buthionine Sulfoximine; Cholestasis, Intrahepatic; Glutathione; Hepatocytes; Leupeptins; Liver; Liver Failure; Male; Membrane Transport Proteins; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Oxidative Stress; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Rats; Rats, Sprague-Dawley; RNA, Messenger; SUMO-1 Protein; Ubiquitin

Human BSEP (ABCB11) mutations are the molecular basis for at least three clinical forms of liver disease, progressive familial intrahepatic cholestasis type 2 (PFIC2), benign recurrent intrahepatic cholestasis type 2 (BRIC2), and intrahepatic cholestasis of pregnancy (ICP). To better understand the pathobiology of these disease phenotypes, we hypothesized that different mutations may cause significant differences in protein defects. Therefore we compared the effect of two PFIC2 mutations (D482G, E297G) with two BRIC2 mutations (A570T and R1050C) and one ICP mutation (N591S) with regard to the subcellular localization, maturation, and function of the rat Bsep protein. Bile salt transport was retained in all but the E297G mutant. Mutant proteins were expressed at reduced levels on the plasma membrane of transfected HEK293 cells compared with wild-type (WT) Bsep in the following order: WT > N591S > R1050C approximately A570T approximately E297G >> D482G. Total cell protein and surface protein expression were reduced to the same extent, suggesting that trafficking of these mutants to the plasma membrane is not impaired. All Bsep mutants accumulate in perinuclear aggresome-like structures in the presence of the proteasome inhibitor MG-132, suggesting that mutations are associated with protein instability and ubiquitin-dependent degradation. Reduced temperature, sodium butyrate, and sodium 4-phenylbutyrate enhanced the expression of the mature and cell surface D482G protein in HEK293 cells. These results suggest that the clinical phenotypes of PFIC2, BRIC2, and ICP may directly correlate with the amount of mature protein that is expressed at the cell surface and that strategies to stabilize cell surface mutant protein may be therapeutic.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B, Member 11; ATP-Binding Cassette Transporters; Bile Acids and Salts; Butyrates; Cell Line; Cell Membrane; Cholestasis, Intrahepatic; Cysteine Proteinase Inhibitors; Dogs; Genotype; Glycosylation; Humans; Kinetics; Leupeptins; Mutation, Missense; Phenotype; Phenylbutyrates; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Protein Transport; Rats; Severity of Illness Index; Temperature; Transfection; Ubiquitin