Functional Rescue of Trafficking-Impaired ABCB4 Mutants by Chemical Chaperones

RESEARCH ARTICLE Functional Rescue of Trafficking-Impaired ABCB4 Mutants by Chemical Chaperones Raquel Gordo-Gilart1, Sara Andueza1, Loreto Hierro1,2, Paloma Jara1,2, Luis Alvarez1* 1 La Paz University Hospital Health Research Institute-IdiPAZ, Madrid, Spain, 2 Pediatric Liver Service, La Paz Children’s University Hospital, Madrid, Spain * Abstract OPEN ACCESS Citation: Gordo-Gilart R, Andueza S, Hierro L, Jara P, Alvarez L (2016) Functional Rescue of TraffickingImpaired ABCB4 Mutants by Chemical Chaperones. PLoS ONE 11(2): e0150098. doi:10.1371/journal. pone.0150098 Editor: Christopher Beh, Simon Fraser University, CANADA Received: October 19, 2015 Accepted: February 9, 2016 Published: February 22, 2016 Copyright: © 2016 Gordo-Gilart et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Funding: This work was supported by grants from the Instituto de Salud Carlos III (ISCIII)-Fondo Europeo de Desarrollo Regional (FEDER) (FIS PI12/ 01196) and from Fundación ACS. Competing Interests: The authors have declared that no competing interests exist. Multidrug resistance protein 3 (MDR3, ABCB4) is a hepatocellular membrane protein that mediates biliary secretion of phosphatidylcholine. Null mutations in ABCB4 gene give rise to severe early-onset cholestatic liver disease. We have previously shown that the diseaseassociated mutations p.G68R, p.G228R, p.D459H, and p.A934T resulted in retention of ABCB4 in the endoplasmic reticulum, thus failing to target the plasma membrane. In the present study, we tested the ability of two compounds with chaperone-like activity, 4-phenylbutyrate and curcumin, to rescue these ABCB4 mutants by assessing their effects on subcellular localization, protein maturation, and phospholipid efflux capability. Incubation of transfected cells at a reduced temperature (30°C) or exposure to pharmacological doses of either 4-PBA or curcumin restored cell surface expression of mutants G228R and A934T. The delivery of these mutants to the plasma membrane was accompanied by a switch in the ratio of mature to inmature protein forms, leading to a predominant expression of the mature protein. This effect was due to an improvement in the maturation rate and not to the stabilization of the mature forms. Both mutants were also functionally rescued, displaying bile salt-dependent phospholipid efflux activity after addition of 4-PBA or curcumin. Druginduced rescue was mutant specific, given neither 4-PBA nor curcumin had an effect on the ABCB4 mutants G68R and A934T. Collectively, these data indicate that the functionality of selected trafficking-defective ABCB4 mutants can be recovered by chemical chaperones through restoration of membrane localization, suggesting a potential treatment for patients carrying such mutations. Introduction Multidrug resistance protein 3 (MDR3, ABCB4) deficiency is among those liver disorders associated with impairment of the canalicular transport of bile constituents [1]. ABCB4 is an ATPbinding cassette (ABC) transmembrane protein that translocates phosphatidylcholine (PC) from the inner to the outer leaflet of the canalicular membrane of hepatocytes [2–4]. By doing so, it makes possible the biliary secretion of PC. Genetic alterations of ABCB4 gene result in ABCB4 deficiency and hence in the formation of PC-deprived bile, which produces PLOS ONE | DOI:10.1371/journal.pone.0150098 February 22, 2016 1 / 14 Pharmacological Rescue of ABCB4 Mutants hepatobiliary injury [5]. The severity of the liver disease in ABCB4 deficiency ranges from biochemical abnormalities with annoying symptoms to life-threatening disease with the potential for progression to death due to hepatic failure [6]. The varying clinical phenotypes are in part related to the extent to which ABCB4 function is impaired by particular mutations. Patients carrying ABCB4 defects that result in a complete loss of ABCB4 function display progressive liver damage that typically requires liver transplantation during childhood. Patients with milder missense mutations or with heterozygous defects exhibit less severe phenotypes and might respond to ursodeoxycholic acid (UDCA) therapy [5,6]. To date, 35 ABCB4 diseasecausing missense variants have been characterized in vitro [7–17]. They preclude proper functioning of ABCB4 in various ways, affecting trafficking of the protein to the plasma membrane, protein expression, or PC-translocating activity. Nine out of these 35 variants cause intracellular retention of ABCB4 in the endoplasmic reticulum (ER), leading to mislocalization to the plasma membrane [7,8,11,13,16,17]. Such as mutations clearly result in a loss-of-function protein, and biallelic carriers are expected to develop progressive, end-stage liver disease [8,13]. In recent years, strategies to ameliorate the consequences of mutations in genes encoding hepatocanalicular transporters have shown some promise [18,19]. Drugs with chaperone-like activity are being tested to deal with ER-trapped mutant proteins, because these compounds might facilitate the folding and transport to the plasma membrane of misfolded, intracellularly retained proteins [20]. Molecules such as 4-phenylbutyrate (4-PBA) or curcumin, known as “chemical chaperones”, have been shown to rescue in vitro mislocalized mutant ABC transporters and canalicular membrane proteins, including ABCA1, ABCA3, ABCB11 (BSEP), ABCC6, ABCC7 (CFTR), ABCD2, ATP7B and ATP8B1 [21–30]. Beneficial effects of 4-PBA treatment have also been reported for patients bearing ER-retained ATP8B1 and BSEP protein mutants [31–35]. We recently reported that the ABCB4 mutations p.G68R, p.G228R, p.D459H, and p.A934T, which we found in children with progressive familial intrahepatic cholestasis type 3 (PFIC3), resulted in ABCB4 protein being trapped in the ER [13,16]. In this study, we investigated whether all these mutant proteins can be targeted by chaperone drugs and restore plasma membrane localization and activity. Our data indicated that some of these mutants could be functionally rescued by such compounds, providing a pharmacological option for the treatment of specific patients. Materials and Methods Plasmids and Reagents The generation of the ABCB4 mutants G68R, G228R, D459H and A934T has been previously described [13,16]. Madin-Darby canine kidney MDCK-II cells and human embryonic kidney AD-293 cells were obtained from ATCC (LGC Standards Barcelona, Spain) and Agilent Technologies (Santa Clara, CA, USA), respectively. The mouse monoclonal anti-ABCB4 antibody (clone P3II-26) was purchased from Millipore (Billerica, Masachussets, USA). The rabbit anti-calnexin antibody was obtained from StressMarq (Victoria, Canada), and the anti-Na/ K-ATPase antibody was obtained from Santa Cruz B (...truncated)