ABCB1 p.Ile541Phe
| Predicted by SNAP2: | A: D (59%), C: N (61%), D: D (85%), E: D (80%), F: D (59%), G: D (80%), H: D (80%), K: D (80%), L: N (61%), M: D (53%), N: D (80%), P: D (85%), Q: D (75%), R: D (80%), S: D (71%), T: D (71%), V: N (82%), W: D (80%), Y: D (75%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, H: D, K: D, L: N, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: N, W: D, Y: D, |
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[hide] A missense mutation in ABCB4 gene involved in prog... Hepatology. 2009 Apr;49(4):1218-27. Delaunay JL, Durand-Schneider AM, Delautier D, Rada A, Gautherot J, Jacquemin E, Ait-Slimane T, Maurice M
A missense mutation in ABCB4 gene involved in progressive familial intrahepatic cholestasis type 3 leads to a folding defect that can be rescued by low temperature.
Hepatology. 2009 Apr;49(4):1218-27., [PMID:19185004]
Abstract [show]
Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a rare liver disease characterized by early onset of cholestasis that leads to cirrhosis and liver failure before adulthood. PFIC3 may be improved by chronic administration of ursodeoxycholic acid, although in many cases liver transplantation is the only therapy. The disease is caused by mutations of the adenosine triphosphate (ATP)-binding cassette, sub-family B, member 4 (ABCB4) [multidrug resistance 3 (MDR3)] gene encoding a specific hepatocellular canalicular transporter involved in biliary phosphatidylcholine secretion. Several mutations have been reported; however, the effect of individual mutations has not been investigated. ABCB4 is highly homologous to ATP-binding cassette, sub-family B, member 1 (ABCB1) (MDR1), the multidrug transporter responsible for drug resistance of cancer cells. We have studied the effect of mutation I541F localized to the first nucleotide-binding domain, which is highly conserved between ABCB4 and ABCB1. Plasmids encoding the wild-type human ABCB4 or rat ABCB1-green fluorescing protein (GFP) construct, and corresponding I541F-mutants, were expressed in hepatocellular carcinoma, human (HepG2) and Madin-Darby canine kidney (MDCK) cells. Expression studies showed that ABCB4 was localized at the bile canalicular membrane in HepG2 cells and at the apical surface in MDCK cells, whereas the I541F mutant was intracellular. In MDCK cells, ABCB1-I541F also accumulated intracellularly in compartments, which were identified as the endoplasmic reticulum and cis-Golgi, and remained partially endoH-sensitive. After shifting cells to 27 degrees C, ABCB1-I541F was expressed at the apical cell surface in a mature and active form. Similarly, ABCB4 was significantly trafficked to the membrane of bile canaliculi in HepG2 cells. CONCLUSION: Mutation I541F causes mislocalization of both ABCB4 and ABCB1. Intracellular retention of ABCB4-I541F can explain the disease in PFIC3 patients bearing this mutation. The observation that plasma membrane expression and activity can be rescued by low temperature opens perspectives to develop novel therapies for the treatment of PFIC3.
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No. Sentence Comment
3 ABCB4 is highly homologous to ATP-binding cassette, sub-family B, member 1 (ABCB1) (MDR1), themultidrugtransporterresponsiblefordrugresistanceofcancercells.Wehavestudiedtheeffectof mutation I541F localized to the first nucleotide-binding domain, which is highly conserved between ABCB4andABCB1.Plasmidsencodingthewild-typehumanABCB4orratABCB1-greenfluoresc- ing protein (GFP) construct, and corresponding I541F-mutants, were expressed in hepatocellular carcinoma, human (HepG2) and Madin-Darby canine kidney (MDCK) cells.
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ABCB1 p.Ile541Phe 19185004:3:190
status: NEWX
ABCB1 p.Ile541Phe 19185004:3:405
status: NEW61 For construction of the ABCB4-I541F and ABCB1-I541F-GFP mutants, site-directed mutagenesis was performed using the QuikChange XL mutagenesis kit (Stratagene Europe, Amsterdam Zvidoost, The Netherlands).
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ABCB1 p.Ile541Phe 19185004:61:30
status: NEWX
ABCB1 p.Ile541Phe 19185004:61:46
status: NEW68 Enrichment of the cell population with ABCB1-I541F-GFP-expressing cells was performed by fluorescence-activated cell sorting.
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ABCB1 p.Ile541Phe 19185004:68:45
status: NEW133 These expression experiments show that mutation I541F causes a trafficking defect both in ABCB1 and ABCB4, and that ABCB1-GFP chimera provides an interesting model to further investigate the effect of I541F mutation.
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ABCB1 p.Ile541Phe 19185004:133:48
status: NEW145 The location of mutation I541F is indicated by an arrow.
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ABCB1 p.Ile541Phe 19185004:145:25
status: NEW148 Expression of ABCB1-GFP and ABCB1-I541F-GFP in MDCK cells.
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ABCB1 p.Ile541Phe 19185004:148:34
status: NEW149 (A) Confluent filter-grown MDCK cells stably expressing ABCB1-GFP (A, C) or ABCB1-I541F-GFP (B, D) were fixed with paraformaldehyde and examined for the fluorescence of GFP.
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ABCB1 p.Ile541Phe 19185004:149:82
status: NEW152 (B) Subconfluent MDCK cells stably expressing ABCB1-I541F-GFP (B, D) were fixed with paraformaldehyde and ethanol, and processed for immunofluorescence using the monoclonal C219 antibody and Cy3-conjugated anti-mouse immunoglobulin G.
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ABCB1 p.Ile541Phe 19185004:152:52
status: NEW158 ABCB1-I541F-GFP Is Not Processed Correctly.
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ABCB1 p.Ile541Phe 19185004:158:6
status: NEW161 To assess more precisely the processing defect caused by the mutation, the glycosylation status of ABCB1 and ABCB1-I541F was studied with endoglycosidases.
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ABCB1 p.Ile541Phe 19185004:161:115
status: NEW166 In contrast, ABCB1-I541F-GFP migrated as two bands of roughly equal intensity of 180 and 190 kDa (Fig. 5).
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ABCB1 p.Ile541Phe 19185004:166:19
status: NEW168 Thus, biochemical studies confirmed that the I541F-mutant does not mature as the wild-type protein during the biosynthetic process, and remains largely in a high-mannose endoH-sensitive form.
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ABCB1 p.Ile541Phe 19185004:168:45
status: NEW172 Colocalization of ABCB1 and ABCB1-I541F with markers of specific cellular compartments.
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ABCB1 p.Ile541Phe 19185004:172:34
status: NEW173 Subconfluent MDCK cells stably expressing ABCB1-GFP (A) or ABCB1-I541F-GFP (B) were fixed with 4% paraformaldehyde, permeabilized with 0.075% saponin, and processed for immunofluorescence using antibodies to calnexin, protein disulfide isomerase, giantin, Golgi matrix protein, early endosomal antigen, or lysosomal-associated membrane protein 2, and appropriate Cy3-conjugated secondary antibodies.
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ABCB1 p.Ile541Phe 19185004:173:65
status: NEW176 Note that the calnexin pattern (B, upper middle picture) is different in ABCB1-I541F-GFP-expressing cells (large asterisks) than in nontransfected cells (small asterisks), and colocalizes with the GFP signal.
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ABCB1 p.Ile541Phe 19185004:176:79
status: NEW178 Expression and glycosylation status of ABCB1-GFP and ABCB1-I541F-GFP in MDCK cells.
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ABCB1 p.Ile541Phe 19185004:178:59
status: NEW179 MDCK/ABCB1-GFP and MDCK/ ABCB1-I541F-GFP cells were lysed and immunoprecipitation was performed using a polyclonal anti-GFP antibody absorbed onto protein A-sepharose.
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ABCB1 p.Ile541Phe 19185004:179:31
status: NEW183 defects that can be overcome by lowering the temperature.25 MDCK-ABCB1-I541F-GFP cells were filter grown to confluence at 37°C, then the cells were switched or not to 27°C for 24 hours.
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ABCB1 p.Ile541Phe 19185004:183:71
status: NEW184 Figure 6A shows that in control cells grown at 37°C, ABCB1-I541F-GFP was intracellular, whereas in cells grown at 27°C, ABCB1-I541F-GFP was expressed at the apical surface.
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ABCB1 p.Ile541Phe 19185004:184:64
status: NEWX
ABCB1 p.Ile541Phe 19185004:184:136
status: NEW187 These results show that I541F is a temperature-sensitive mutation and that processing and trafficking of the mutant reverts toward that of wild-type on lowering temperature.
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ABCB1 p.Ile541Phe 19185004:187:24
status: NEW188 ABCB1-I541F-GFP Is Functional When Expressed at the Apical Membrane.
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ABCB1 p.Ile541Phe 19185004:188:6
status: NEW194 Only very weak activity was measured in MDCK/ ABCB1-I541F-GFP cells grown at 37°C; however, when cells were grown at 27°C for 24 hours before the assay, they accumulated significantly less fluorescent calcein (Fig. 6C).
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ABCB1 p.Ile541Phe 19185004:194:52
status: NEW195 These results show that mutation I541F does not impair activity, provided that the transporter can reach the plasma membrane.
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ABCB1 p.Ile541Phe 19185004:195:33
status: NEW231 Here, we also used ABCB1 and showed that the I541F mutation caused similar intracellular retention in both ABCB1 and ABCB4.
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ABCB1 p.Ile541Phe 19185004:231:45
status: NEW[hide] Effects of cellular, chemical, and pharmacological... J Biol Chem. 2012 Feb 10;287(7):5070-8. Epub 2011 Dec 19. Gautherot J, Durand-Schneider AM, Delautier D, Delaunay JL, Rada A, Gabillet J, Housset C, Maurice M, Ait-Slimane T
Effects of cellular, chemical, and pharmacological chaperones on the rescue of a trafficking-defective mutant of the ATP-binding cassette transporter proteins ABCB1/ABCB4.
J Biol Chem. 2012 Feb 10;287(7):5070-8. Epub 2011 Dec 19., [PMID:22184139]
Abstract [show]
The ATP-binding cassette transporter ABCB4 is a phosphatidylcholine translocator specifically expressed at the bile canalicular membrane in hepatocytes, highly homologous to the multidrug transporter ABCB1. Variations in the ABCB4 gene sequence cause progressive familial intrahepatic cholestasis type 3. We have shown previously that the I541F mutation, when reproduced either in ABCB1 or in ABCB4, led to retention in the endoplasmic reticulum (ER)/Golgi. Here, Madin-Darby canine kidney cells expressing ABCB1-GFP were used as a model to investigate this mutant. We show that ABCB1-I541F is not properly folded and is more susceptible to in situ protease degradation. It colocalizes and coprecipitates with the ER chaperone calnexin and coprecipitates with the cytosolic chaperone Hsc/Hsp70. Silencing of calnexin or overexpression of Hsp70 have no effect on maturation of the mutant. We also tested potential rescue by chemical and pharmacological chaperones. Thapsigargin and sodium 4-phenyl butyrate were inefficient. Glycerol improved maturation and exit of the mutant from the ER. Cyclosporin A, a competitive substrate for ABCB1, restored maturation, plasma membrane expression, and activity of ABCB1-I541F. Cyclosporin A also improved maturation of ABCB4-I541F in Madin-Darby canine kidney cells. In HepG(2) cells transfected with ABCB4-I541F cDNA, cyclosporin A allowed a significant amount of the mutant protein to reach the membrane of bile canaliculi. These results show that the best strategy to rescue conformation-defective ABCB4 mutants is provided by pharmacological chaperones that specifically target the protein. They identify cyclosporin A as a potential novel therapeutic tool for progressive familial intrahepatic cholestasis type 3 patients.
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No. Sentence Comment
1 Results: The I541F mutation induces misfolding and intracellular retention that is rescued by the ABCB1-competitive substrate cyclosporin A but not by modulating the chaperones calnexin or Hsp/Hsc70.
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ABCB1 p.Ile541Phe 22184139:1:13
status: NEW50 ABCB1-I541F has the advantage that it can be expressed at detectable level as a GFP fusion protein and ABCB1 activity is easy to measure.
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ABCB1 p.Ile541Phe 22184139:50:6
status: NEW52 We found that cyclosporin A, an ABCB1 substrate, induced the best rescue of the ABCB1-I541F mutant and also provides rescue of the ABCB4-I541F mutant.
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ABCB1 p.Ile541Phe 22184139:52:86
status: NEWX
ABCB1 p.Ile541Phe 22184139:52:137
status: NEW110 An ϳ40-kDa fragment was resistant to kallikrein both in ABCB1 and ABCB1-I541F, again indicating that the C terminus of the molecule was less affected by the mutation.
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ABCB1 p.Ile541Phe 22184139:110:78
status: NEW111 These proteolysis experiments provided evidence for a conformational defect of the I541F mutant.
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ABCB1 p.Ile541Phe 22184139:111:83
status: NEW112 The I541F Mutant Colocalizes and Coprecipitates with Calnexin-Calnexin is a chaperone of the ER that is involved in the folding of ABCB1 (19).
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ABCB1 p.Ile541Phe 22184139:112:4
status: NEW113 To study whether the mutant interacts with calnexin, we performed colocalization and coimmunoprecipitation experiments using MDCK cells stably transfected with ABCB1-WT or ABCB1-I541F.
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ABCB1 p.Ile541Phe 22184139:113:178
status: NEW115 By contrast, the ABCB1-I541F mutant colocalized strongly with calnexin, which appeared to be relocalized near the nucleus, together with the mutant (Fig. 2A).
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ABCB1 p.Ile541Phe 22184139:115:23
status: NEW117 In situ protease susceptibility of ABCB1-WT and ABCB1-I541F.
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ABCB1 p.Ile541Phe 22184139:117:54
status: NEW118 Microsomes obtained from MDCK cells expressing GFP-tagged ABCB1-WT or ABCB1-I541F were submitted to limited proteolysis at the indicated concentrations of thermolysin or kallikrein for 30 min at 4 °C.
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ABCB1 p.Ile541Phe 22184139:118:76
status: NEW119 Samples (ABCB1, 30 g and ABCB1-I541F, 50 g protein per lane) were immunoblotted with the anti-GFP monoclonal antibody.
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ABCB1 p.Ile541Phe 22184139:119:39
status: NEW123 ABCB1 migrated as two bands of ϳ190 kDa and 175 kDa (bands A and B), whereas ABCB1-I541F presented only the 175-kDa band.
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ABCB1 p.Ile541Phe 22184139:123:89
status: NEW125 Immunoprecipitation of calnexin also pulled down ABCB1-WT and ABCB1-I541F.
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ABCB1 p.Ile541Phe 22184139:125:68
status: NEW127 Effect of Calnexin Down-expression-Interaction between ABCB1-I541F and calnexin suggested that the chaperone might retain the I541F mutant in the ER.
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ABCB1 p.Ile541Phe 22184139:127:61
status: NEWX
ABCB1 p.Ile541Phe 22184139:127:126
status: NEW131 However, it had little or no effect on the expression of ABCB1-I541F (Fig. 3) and did not induce the appearance of the mature form, which would have indicated a rescue effect.
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ABCB1 p.Ile541Phe 22184139:131:63
status: NEW132 The I541F Mutant Coprecipitation with Hsc70 and Effect of Hsp70 Overexpression-The cognate Hsp70 family member Hsc70 is another ubiquitous chaperone that is involved in ABCB1 folding (20).
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ABCB1 p.Ile541Phe 22184139:132:4
status: NEW135 However, Hsc70 coprecipitated significantly with the mutant ABCB1-I541F, and in higher amounts than with FIGURE 2.
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ABCB1 p.Ile541Phe 22184139:135:66
status: NEW136 ABCB1-I541F colocalizes and coprecipitates with calnexin.
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ABCB1 p.Ile541Phe 22184139:136:6
status: NEW137 A, MDCK cells stably transfected with GFP-tagged ABCB1-WT or ABCB1-I541F were fixed, and immunolocalization of calnexin was performed using a Cy3-conjugated secondary antibody.
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ABCB1 p.Ile541Phe 22184139:137:67
status: NEW139 Note that the staining pattern of calnexin in ABCB1-I541F cells is different from that in ABCB1-WT cells because of ER accumulation of the mutant.
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ABCB1 p.Ile541Phe 22184139:139:52
status: NEW140 Scale bars ϭ 10 m. B, MDCK cells stably transfected with GFP-tagged ABCB1-WT or ABCB1-I541F and control MDCK cells were lysed, and immunoprecipitation (IP) was performed with the anti-GFP antibody. Samples were analyzed by immunoblotting using anti-GFP or anti-calnexin monoclonal antibodies.
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ABCB1 p.Ile541Phe 22184139:140:100
status: NEW143 D, MDCK cells stably transfected with GFP-tagged ABCB1-WT or ABCB1-I541F and control MDCK cells were lysed, and immunoprecipitation was performed with the anti-calnexin antibody. Samples were analyzed by immunoblotting using the anti-GFP antibody.
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ABCB1 p.Ile541Phe 22184139:143:67
status: NEW145 Effect of calnexin silencing on ABCB1-I541F expression.
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ABCB1 p.Ile541Phe 22184139:145:38
status: NEW146 MDCK cells stably transfected with GFP-tagged ABCB1-WT or ABCB1-I541F were transfected with control or calnexin (CLNX) siRNA.
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ABCB1 p.Ile541Phe 22184139:146:64
status: NEW169 ABCB1-I541F Is Functionally Rescued by Cyclosporin A-Cyclosporin A is an immunosuppressant known to be a substrate for and a competitive inhibitor of ABCB1 (28).
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ABCB1 p.Ile541Phe 22184139:169:6
status: NEW171 We therefore tested the effect of cyclosporin A on the I541F mutant at concentrations ranging from 0.
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ABCB1 p.Ile541Phe 22184139:171:55
status: NEW172 1 to 10 M. Cyclosporin A given for 24 h caused a dose-dependent increase in the mature form of ABCB1-I541F (Fig. 7A and B).
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ABCB1 p.Ile541Phe 22184139:172:109
status: NEW174 Study of the localization of the rescued mutant by confocal microscopy showed that after cyclosporin A treatment, the I541F mutant was expressed at the apical membrane like the wild-type protein (Fig. 7C).
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ABCB1 p.Ile541Phe 22184139:174:118
status: NEW176 ABCB1-I541F coprecipitates with Hsc70.
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ABCB1 p.Ile541Phe 22184139:176:6
status: NEW177 A, MDCK cells stably transfected with GFP-tagged ABCB1-I541F were fixed, and Hsc70 was detected by immunofluorescence using a Cy3-conjugated secondary antibody.
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ABCB1 p.Ile541Phe 22184139:177:55
status: NEW179 Scale bar ϭ 10 m. B, MDCK cells stably transfected with GFP-tagged ABCB1-WT or ABCB1-I541F and control MDCK cells were lysed, and immunoprecipitation (IP) was performed using the monoclonal anti-GFP antibody. Samples were analyzed by immunoblotting using anti-GFP or anti-Hsc70 monoclonal antibodies. Shown is one representative of three experiments.
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ABCB1 p.Ile541Phe 22184139:179:99
status: NEW181 Effect of Hsp70 overexpression on ABCB1-I541F expression.
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ABCB1 p.Ile541Phe 22184139:181:40
status: NEW182 A, MDCK cells stably transfected with GFP-tagged ABCB1-WT or ABCB1-I541F were maintained at 37 °C or exposed at 42 °C for 2 h and then returned to 37 °C for 24 h.
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ABCB1 p.Ile541Phe 22184139:182:67
status: NEW184 B, MDCK cells stably transfected with GFP-tagged ABCB1-WT or ABCB1-I541F were transfected with the plasmid encoding Hsp70.
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ABCB1 p.Ile541Phe 22184139:184:67
status: NEW190 Fig. 7D shows that ABCB1-I541F MDCK cells treated with cyclosporin A extruded more fluorescent calcein than control cells in a dose-dependent manner.
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ABCB1 p.Ile541Phe 22184139:190:25
status: NEW192 ABCB4-I541F Is Also Rescued by Cyclosporin A-Because cyclosporin A was very efficient at rescuing ABCB1-I541F, we also tested its potential effect on ABCB4-I541F.
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ABCB1 p.Ile541Phe 22184139:192:6
status: NEWX
ABCB1 p.Ile541Phe 22184139:192:104
status: NEWX
ABCB1 p.Ile541Phe 22184139:192:156
status: NEW193 ABCB4-I541F was expressed at a low level in stably transfected MDCK cells and was not reproducibly detected by Western blotting.
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ABCB1 p.Ile541Phe 22184139:193:6
status: NEW194 We therefore studied the effect of cyclosporin A in MDCK cells transiently transfected with the ABCB4-I541F plasmid.
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ABCB1 p.Ile541Phe 22184139:194:102
status: NEW231 Similarly, we did not observe that calnexin silencing induced significant change in the expression of the ABCB1-I541F mutant and that it did not rescue ER retention.
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ABCB1 p.Ile541Phe 22184139:231:112
status: NEW234 We found an increase in the ABCB1-I541F mutant when Hsp70 was overexpressed by transfection of the Hsp70 cDNA but not after heat shock treatment.
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ABCB1 p.Ile541Phe 22184139:234:34
status: NEW266 Here, we found that the ABCB1 inhibitor cyclosporin A was remarkably effective at restoring the traffic of ABCB1 bearing the I541F mutation.
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ABCB1 p.Ile541Phe 22184139:266:125
status: NEW273 However, the fact that the ABCB1 mutant was able to extrude calcein after cyclosporin A treatment suggests that rescued ABCB4-I541F may be functional as well.
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ABCB1 p.Ile541Phe 22184139:273:126
status: NEW