title: Levels of plasma membrane expression in progressive and benign mutations of the bile salt export pump (Bsep/Abcb11) correlate with severity of cholestatic diseases.
abstract: 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.
authors.affiliation: Liver Center and Department of Medicine, Yale University School of Medicine, New Haven, Connecticut 06520-8019, USA.
authors.forename: Ping
authors.initials: P
authors.lastname: Lam
authors.forename: Claire L
authors.initials: CL
authors.lastname: Pearson
authors.forename: Carol J
authors.initials: CJ
authors.lastname: Soroka
authors.forename: Shuhua
authors.initials: S
authors.lastname: Xu
authors.forename: Albert
authors.initials: A
authors.lastname: Mennone
authors.forename: James L
authors.initials: JL
authors.lastname: Boyer
mesh_terms: D000074020:ATP Binding Cassette Transporter, Subfamily B, Member 11; D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001647:Bile Acids and Salts / Q000378:metabolism*; D002087:Butyrates / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002780:Cholestasis, Intrahepatic / Q000235:genetics / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004285:Dogs; D005838:Genotype; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D007976:Leupeptins / Q000494:pharmacology; D020125:Mutation, Missense; D010641:Phenotype; D010654:Phenylbutyrates / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000187:drug effects / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D021381:Protein Transport; D051381:Rats; D012720:Severity of Illness Index; D013696:Temperature; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism
chemical_list: D000074020:ATP Binding Cassette Transporter, Subfamily B, Member 11; D018528:ATP-Binding Cassette Transporters; C490548:Abcb11 protein, rat; D001647:Bile Acids and Salts; D002087:Butyrates; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D010654:Phenylbutyrates; D025801:Ubiquitin; C075773:4-phenylbutyric acid; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde