title: Molecular mechanistic explanation for the spectrum of cholestatic disease caused by the S320F variant of ABCB4.
abstract: ABCB4 flops phosphatidylcholine into the bile canaliculus to protect the biliary tree from the detergent activity of bile salts. Homozygous-null ABCB4 mutations cause the childhood liver disease, progressive familial intrahepatic cholestasis, but cause and effect is less clear, with many missense mutations linked to less severe cholestatic diseases. ABCB4(S320F), in particular, is described in 13 patients, including in heterozygosity with ABCB4(A286V), ABCB4(A953D), and null mutants, whose symptoms cover the spectrum of cholestatic disease. We sought to define the impact of these mutations on the floppase, explain the link with multiple conditions at the molecular level, and investigate the potential for reversal. ABCB4(S320F), ABCB4(A286V), and ABCB4(A953D) expression was engineered in naïve cultured cells. Floppase expression, localization, and activity were measured by western blot, confocal microscopy, and lipid transport assays, respectively. ABCB4(S320F) was fully active for floppase activity but expression at the plasma membrane was reduced to 50%. ABCB4(A286V) expressed and trafficked efficiently but could not flop lipid, and ABCB4(A953D) expressed poorly and was impaired in floppase activity. Proteasome inhibition stabilized nascent ABCB4(S320F) and ABCB4(A953D) but did not improve plasma membrane localization. Cyclosporin-A improved plasma membrane localization of both ABCB4(S320F) and ABCB4(A953D), but inhibited floppase activity.
  
  
  CONCLUSIONS
  The level of ABCB4 functionality correlates with, and is the primary determinant of, cholestatic disease severity in these patients. ABCB4(S320F) homozygosity, with half the normal level of ABCB4, is the tipping point between more benign and potentially fatal cholestasis and makes these patients more acutely sensitive to environmental effects. Cyclosporin-A increased expression of ABCB4(S320F) and ABCB4(A953D), suggesting that chemical chaperones could be exploited for therapeutic benefit to usher in a new era of personalized medicine for patients with ABCB4-dependent cholestatic disease.
authors.affiliation: Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary, University of London, London, UK.
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Andress
authors.forename: Michael
authors.initials: M
authors.lastname: Nicolaou
authors.forename: Marta R
authors.initials: MR
authors.lastname: Romero
authors.forename: Sandhia
authors.initials: S
authors.lastname: Naik
authors.forename: Peter H
authors.initials: PH
authors.lastname: Dixon
authors.forename: Catherine
authors.initials: C
authors.lastname: Williamson
authors.forename: Kenneth J
authors.initials: KJ
authors.lastname: Linton
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000737:chemistry / Q000235:genetics*; D002779:Cholestasis / Q000235:genetics*; D016572:Cyclosporine / Q000494:pharmacology; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D020641:Polymorphism, Single Nucleotide; D061988:Proteasome Inhibitors / Q000494:pharmacology; D017510:Protein Folding; D021381:Protein Transport
chemical_list: D018435:ATP Binding Cassette Transporter, Subfamily B; D061988:Proteasome Inhibitors; D016572:Cyclosporine; C095122:multidrug resistance protein 3