Mécanismes de tri et d’adressage des protéines vers les deux domaines membranaires des cellules épithéliales polarisées

La polarité est une caractéristique fondamentale de la plupart des cellules eucaryotes. Le caractère primordial de cette polarité est l’individualisation de la membrane plasmique en domaines séparés: le domaine apical et le domaine basolatéral. Ces deux surfaces exercent des fonctions différentes qui correspondent à une composition très spécifique de chaque domaine en lipides et en protéines. Pour établir et maintenir leur polarité, les cellules épithéliales doivent trier et envoyer les protéines et les lipides vers la surface cellulaire correcte. Les données récentes concernant le transport polarisé montrent que le trafic intracellulaire des lipides et des protéines sont deux événements étroitement liés et qui ne doivent donc pas être abordés séparément. Dans cette revue nous discuterons de quelques mécanismes impliqués dans le transport polarisé, nous aborderons plus particulièrement les données récentes concernant le rôle des microdomaines lipidiques (Rafts) dans le trafic des protéines vers la surface apicale des cellules épithéliales polarisées

Raft-mediated Trafficking of Apical Resident Proteins Occurs in Both Direct and Transcytotic Pathways in Polarized Hepatic Cells: Role of Distinct Lipid Microdomains

In polarized hepatic cells, pathways and molecular principles mediating the flow of resident apical bile canalicular proteins have not yet been resolved. Herein, we have investigated apical trafficking of a glycosylphosphatidylinositol-linked and two single transmembrane domain proteins on the one hand, and two polytopic proteins on the other in polarized HepG2 cells. We demonstrate that the former arrive at the bile canalicular membrane via the indirect transcytotic pathway, whereas the polytopic proteins reach the apical membrane directly, after Golgi exit. Most importantly, cholesterol-based lipid microdomains (“rafts”) are operating in either pathway, and protein sorting into such domains occurs in the biosynthetic pathway, largely in the Golgi. Interestingly, rafts involved in the direct pathway are Lubrol WX insoluble but Triton X-100 soluble, whereas rafts in the indirect pathway are both Lubrol WX and Triton X-100 insoluble. Moreover, whereas cholesterol depletion alters raft-detergent insolubility in the indirect pathway without affecting apical sorting, protein missorting occurs in the direct pathway without affecting raft insolubility. The data implicate cholesterol as a traffic direction-determining parameter in the direct apical pathway. Furthermore, raft-cargo likely distinguishing single vs. multispanning membrane anchors, rather than rafts per se (co)determine the sorting pathway

A PDZ-Like Motif in the Biliary Transporter ABCB4 Interacts with the Scaffold Protein EBP50 and Regulates ABCB4 Cell Surface Expression

International audienceABCB4/MDR3, a member of the ABC superfamily, is an ATP-dependent phosphatidylcholine translocator expressed at the canalicular membrane of hepatocytes. Defects in the ABCB4 gene are associated with rare biliary diseases. It is essential to understand the mechanisms of its canalicular membrane expression in particular for the development of new therapies. The stability of several ABC transporters is regulated through their binding to PDZ (PSD95/DglA/ZO-1) domain-containing proteins. ABCB4 protein ends by the sequence glutamine-asparagine-leucine (QNL), which shows some similarity to PDZ-binding motifs. The aim of our study was to assess the potential role of the QNL motif on the surface expression of ABCB4 and to determine if PDZ domain-containing proteins are involved. We found that truncation of the QNL motif decreased the stability of ABCB4 in HepG2-transfected cells. The deleted mutant ABCB4-ΔQNL also displayed accelerated endocytosis. EBP50, a PDZ protein highly expressed in the liver, strongly colocalized and coimmunoprecipitated with ABCB4, and this interaction required the QNL motif. Down-regulation of EBP50 by siRNA or by expression of an EBP50 dominant-negative mutant caused a significant decrease in the level of ABCB4 protein expression, and in the amount of ABCB4 localized at the canalicular membrane. Interaction of ABCB4 with EBP50 through its PDZ-like motif plays a critical role in the regulation of ABCB4 expression and stability at the canalicular plasma membrane

Comparison of in silico prediction and experimental assessment of ABCB4 variants identified in patients with biliary diseases

International audienceGenetic variations of the phosphatidylcholine transporter, ABCB4 cause several biliary diseases. The large number of reported variations makes it difficult to foresee a comprehensive study of each variation. To appreciate the reliability of in silico prediction programs, 1) we confronted them with the assessment in cell models of two ABCB4 variations (E528D and P1161S) identified in patients with low phospholipid-associated cholelithiasis (LPAC); 2) we extended the confrontation to 19 variations that we had previously characterized in cellulo. Four programs (Provean, Polyphen-2, PhD-SNP and MutPred) were used to predict the degree of pathogenicity. The E528D and P1161S variants were studied in transfected HEK293 and HepG2 cells by immunofluorescence, immunoblotting and measurement of phosphatidylcholine secretion. All prediction tools qualified the P1161S variation as deleterious, but provided conflicting results for E528D. In cell models, both mutants were expressed and localized as the wild type but their activity was significantly reduced, by 48% (P1161S) and 33% (E528D). These functional defects best correlated with MutPred predictions. MutPred program also proved the most accurate to predict the pathogenicity of the 19 ABCB4 variants that we previously characterized in cell models, and the most sensitive to predict the pathogenicity of 65 additional mutations of the Human Gene Mutation Database. These results confirm the pathogenicity of E528D and P1161S variations and suggest that even a moderate decrease (by less than 50%) of phosphatidylcholine secretion can cause LPAC syndrome. They highlight the reliability of in silico prediction tools, most notably MutPred, as a first approach to predict the pathogenicity of ABCB4 variants

Comparison of in silico prediction and experimental assessment of ABCB4 variants identified in patients with biliary diseases

International audienceGenetic variations of the phosphatidylcholine transporter, ABCB4 cause several biliary diseases. The large number of reported variations makes it difficult to foresee a comprehensive study of each variation. To appreciate the reliability of in silico prediction programs, 1) we confronted them with the assessment in cell models of two ABCB4 variations (E528D and P1161S) identified in patients with low phospholipid-associated cholelithiasis (LPAC); 2) we extended the confrontation to 19 variations that we had previously characterized in cellulo. Four programs (Provean, Polyphen-2, PhD-SNP and MutPred) were used to predict the degree of pathogenicity. The E528D and P1161S variants were studied in transfected HEK293 and HepG2 cells by immunofluorescence, immunoblotting and measurement of phosphatidylcholine secretion. All prediction tools qualified the P1161S variation as deleterious, but provided conflicting results for E528D. In cell models, both mutants were expressed and localized as the wild type but their activity was significantly reduced, by 48% (P1161S) and 33% (E528D). These functional defects best correlated with MutPred predictions. MutPred program also proved the most accurate to predict the pathogenicity of the 19 ABCB4 variants that we previously characterized in cell models, and the most sensitive to predict the pathogenicity of 65 additional mutations of the Human Gene Mutation Database. These results confirm the pathogenicity of E528D and P1161S variations and suggest that even a moderate decrease (by less than 50%) of phosphatidylcholine secretion can cause LPAC syndrome. They highlight the reliability of in silico prediction tools, most notably MutPred, as a first approach to predict the pathogenicity of ABCB4 variants

Molecular Regulation of Canalicular ABC Transporters

International audienceThe ATP-binding cassette (ABC) transporters expressed at the canalicular membrane of hepatocytes mediate the secretion of several compounds into the bile canaliculi and therefore play a key role in bile secretion. Among these transporters, ABCB11 secretes bile acids, ABCB4 translocates phosphatidylcholine and ABCG5/G8 is responsible for cholesterol secretion, while ABCB1 and ABCC2 transport a variety of drugs and other compounds. The dysfunction of these transporters leads to severe, rare, evolutionary biliary diseases. The development of new therapies for patients with these diseases requires a deep understanding of the biology of these transporters. In this review, we report the current knowledge regarding the regulation of canalicular ABC transporters' folding, trafficking, membrane stability and function, and we highlight the role of molecular partners in these regulating mechanisms

Ivacaftor-Mediated Potentiation of ABCB4 Missense Mutations Affecting Critical Motifs of the NBDs: Repositioning Perspectives for Hepatobiliary Diseases

ABCB4 (ATP-binding cassette subfamily B member 4) is a hepatocanalicular floppase involved in biliary phosphatidylcholine (PC) secretion. Variations in the ABCB4 gene give rise to several biliary diseases, including progressive familial intrahepatic cholestasis type 3 (PFIC3), an autosomal recessive disease that can be lethal in the absence of liver transplantation. In this study, we investigated the effect and potential rescue of ten ABCB4 missense variations in NBD1:NBD2 homologous positions (Y403H/Y1043H, K435M/K1075M, E558K/E1200A, D564G/D1206G and H589Y/H1231Y) all localized at the conserved and functionally critical motifs of ABC transporters, six of which are mutated in patients. By combining structure analysis and in vitro studies, we found that all ten mutants were normally processed and localized at the canalicular membrane of HepG2 cells, but showed dramatically impaired PC transport activity that was significantly rescued by treatment with the clinically approved CFTR potentiator ivacaftor. Our results provide evidence that functional ABCB4 mutations are rescued by ivacaftor, paving the way for the repositioning of this potentiator for the treatment of selected patients with PFIC3 caused by mutations in the ATP-binding sites of ABCB4

Expression and localization of ABCB4-wt and ABCB4-ΔQNL.

<p>(A) ABCB4 was detected by immunoblotting from cell lysates of HepG2 cells stably expressing ABCB4-wt or ABCB4-ΔQNL. (B) HepG2 cells stably expressing ABCB4-wt or ABCB4-ΔQNL were fixed with methanol/acetone, processed for immunofluorescence using the monoclonal P3II-26 antibody and Alexa 488-conjugated anti-mouse IgG and visualized by confocal microscopy. Nuclei were stained with DRAQ 5 (blue). Asterisks indicate bile canaliculi. Cell contours are indicated by dotted lines. Bars, 10 μm. Representative of three experiments.</p

The C-terminus of ABCB4 ends by a PDZ-like motif.

<p>(A) Schematic representation of ABCB4. ABCB4 contains two transmembrane domains (TMD1 and TMD2) and two nucleotide binding domains (NBD1 and NBD2). The two glycosylation sites in the first extracellular loop are indicated. The amino acid sequence of the intracytoplasmic C-terminal domain of the human ABCB4 isoform A (NP_000434.1) and the human ABCB1 (NP_000918.2) are shown. Green letters indicate the PDZ-like motif of ABCB4. (B) A sequence alignment of known PDZ motifs found in ABC family members is shown. ABCC2, ABCC4 and ABCC7 display class I PDZ consensus motifs.</p

Stability of ABCB4-wt and ABCB4-ΔQNL.

<p>(A) Stability of ABCB4-wt or ABCB4-ΔQNL was analyzed in stably transfected HepG2, after inhibiting protein synthesis with cycloheximide (25 μg/mL). ABCB4 was detected at the indicated time points in cell lysates by immunoblotting, using equal amounts of total proteins per lane. (B) Amounts of ABCB4 were quantified from chase experiments. The amount of ABCB4 at time zero was considered as 100%. Remaining ABCB4 at later time points was expressed as percentage of time zero. Means (± SEM) of three independent experiments are shown. <i>* P</i><b><</b>0.05 at all points.</p