Hepatology
Liver Biology/Pathobiology
Folding Defects in P-Type Atp 8B1 Associated With Hereditary Cholestasis Are Ameliorated by 4-Phenylbutyrate
van der Velden, Lieke M.1,*; Stapelbroek, Janneke M.1,2,*; Krieger, Elmar3; van den Berghe, Peter V. E.1; Berger, Ruud1; Verhulst, Patricia M.4; Holthuis, Joost C. M.4; Houwen, Roderick H. J.2; Klomp, Leo W. J.1,†,‡; van de Graaf, Stan F. J.1,†
1Department of Metabolic and Endocrine Diseases, University Medical Center (UMC) Utrecht, and Netherlands Metabolomics Centre, Utrecht, The Netherlands
2Department of Pediatric Gastroenterology, UMC Utrecht, Utrecht, The Netherlands
3Centre for Molecular and Biomolecular Informatics, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands
4Department of Membrane Enzymology, Bijvoet Center and Institute of Biomembranes, Utrecht University, The Netherlands
‡Address reprint requests to: University Medical Center Utrecht, Department of Metabolic and Endocrine Diseases, Room KC-02.069.1, P.O. Box 85090, 3508 AB Utrecht, The Netherlands. Email:[email protected]; fax: +31 30 2504295.
Received 30 December 2008; Accepted 26 August 2009
Published online 4 September 2009 in Wiley InterScience (www.interscience.wiley.com).
Financial support: Wilhelmina Children's Foundation (L.W.J.K.), Utrecht University (to J.C.M.H. and L.W.J.K.), NWO (VENI to S.F.J.v.d.G.) and Stichting BRIC, de leverziekte (to R.H.J.H.).
Potential conflict of interest: Nothing to report.
Additional Supporting Information may be found in the online version of this article.
*These authors contributed equally to this work.
†These authors have equal senior authorship
Abstract
Deficiency in P-type ATP8B1 is a severe and clinically highly variable hereditary disorder that is primarily characterized by intrahepatic cholestasis. It presents either as a progressive (progressive familial intrahepatic cholestasis type 1 [PFIC1]) or intermittent (benign recurrent intrahepatic cholestasis type 1 [BRIC1]) disease. ATP8B1 deficiency is caused by autosomal recessive mutations in the gene encoding ATP8B1, a putative aminophospholipid-translocating P-type adenosine triphosphatase. The exact pathogenesis of the disease is elusive, and no effective pharmacological therapy is currently available. Here, the molecular consequences of six distinctATP8B1missense mutations (p.L127P, p.G308V, p.D454G, p.D554N, p.I661T, and p.G1040R) and one nonsense mutation (p.R1164X) associated with PFIC1 and/or BRIC1 were systematically characterized. Except for the p.L127P mutation, all mutations resulted in markedly reduced ATP8B1 protein expression, whereas messenger RNA expression was unaffected. Five of seven mutations resulted in (partial) retention of ATP8B1 in the endoplasmic reticulum. Reduced protein expression was partially restored by culturing the cells at 30°C and by treatment with proteasomal inhibitors, indicating protein misfolding and subsequent proteosomal degradation. Protein misfolding was corroborated by predicting the consequences of most mutations onto a homology model of ATP8B1. Treatment with 4-phenylbutyrate, a clinically approved pharmacological chaperone, partially restored defects in expression and localization of ATP8B1 substitutions G308V, D454G, D554N, and in particular I661T, which is the most frequently identified mutation in BRIC1.Conclusion:A surprisingly large proportion ofATP8B1mutations resulted in aberrant folding and decreased expression at the plasma membrane. These effects were partially restored by treatment with 4-phenylbutyrate. We propose that treatment with pharmacological chaperones may represent an effective therapeutic strategy to ameliorate the recurrent attacks of cholestasis in patients with intermittent (BRIC1) disease. (Hepatology 2009.)