ABCB4 p.Ile541Phe
| Predicted by SNAP2: | A: D (59%), C: N (66%), D: D (80%), E: D (80%), F: D (59%), G: D (71%), H: D (71%), K: D (80%), L: N (72%), M: N (57%), N: D (75%), P: D (80%), Q: D (71%), R: D (75%), S: D (63%), T: D (63%), V: N (93%), W: D (75%), Y: D (71%), |
| 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] Ubiquitin-mediated proteasomal degradation of ABC ... J Pharm Sci. 2011 Sep;100(9):3602-19. doi: 10.1002/jps.22615. Epub 2011 May 12. Nakagawa H, Toyoda Y, Wakabayashi-Nakao K, Tamaki H, Osumi M, Ishikawa T
Ubiquitin-mediated proteasomal degradation of ABC transporters: a new aspect of genetic polymorphisms and clinical impacts.
J Pharm Sci. 2011 Sep;100(9):3602-19. doi: 10.1002/jps.22615. Epub 2011 May 12., [PMID:21567408]
Abstract [show]
The interindividual variation in the rate of drug metabolism and disposition has been known for many years. Pharmacogenomics dealing with heredity and response to drugs is a part of science that attempts to explain variability of drug responses and to search for the genetic basis of such variations or differences. Genetic polymorphisms of drug metabolizing enzymes and drug transporters have been found to play a significant role in the patients' responses to medication. Accumulating evidence demonstrates that certain nonsynonymous polymorphisms have great impacts on the protein stability and degradation, as well as the function of drug metabolizing enzymes and transporters. The aim of this review article is to address a new aspect of protein quality control in the endoplasmic reticulum and to present examples regarding the impact of nonsynonymous single-nucleotide polymorphisms on the protein stability of thiopurine S-methyltransferase as well as ATP-binding cassette (ABC) transporters including ABCC4, cystic fibrosis transmembrane conductance regulator (CFTR, ABCC7), ABCC11, and ABCG2. Furthermore, we will discuss the molecular mechanisms underlying posttranslational modifications (intramolecular and intermolecular disulfide bond formation and N-linked glycosylation) and ubiquitin-mediated proteasomal degradation of ABCG2, one of the major drug transporter proteins in humans.
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No. Sentence Comment
155 Effect of Mutations and Nonsynonymous SNPs on Protein Trafficking, Maturation, or ERAD of ABC Transporters Protein AA Mutation/SNP Effect on Protein Reference ABCA1 W590S Mutation Functional defect 115 R587W Mutation Impaired glycol processing 115 Q597R Mutation Impaired glycol processing, ERAD 115,116 Y1532C Mutation Altered protein trafficking 117 R1925Q Mutation Altered protein trafficking 118 ABCA3 R43L Mutation Altered protein trafficking 119 L101P Mutation Altered protein trafficking 119 R280C Mutation Altered protein trafficking 119 ABCA4 L541P Mutation Mislocalization 120 R602W Mutation Mislocalization 120 A1038V Mutation Mislocalization 120 C1490Y Mutation Mislocalization 120 ABCB1a G268V Mutation ERAD 121 G341C Mutation ERAD 121 I1196S Mutation Reduced glycosylation 122 ABCB4 I541F Mutation Accumulation in ER 123 ABCB11a E135K Mutation Reduced level of mature protein 124 L198P Mutation Reduced level of mature protein 124 E297G Mutation Reduced level of mature protein 124 L413W Mutation Reduced level of mature protein 124 R432T Mutation Reduced level of mature protein 124 D482G Mutation Immature protein in ER 124,125 N490D Mutation Reduced level of mature protein 124 A570T Mutation Reduced level of mature protein 124 T655I Mutation Reduced level of mature protein 124 Y818F SNP Moderate reduction of protein 124 G982R Mutation Retention in ER 125 R1153C Mutation ERAD 125 R1286Q Mutation Retention in ER 125 ABCC2a R768W Mutation Impaired protein trafficking 126 I1173F Mutation Impaired protein maturation 127 R1392 Mutation Impaired protein maturation 128 M1393 Mutation Impaired protein maturation 129 ABCC4a E757K SNP Altered protein trafficking 23 ABCC7 F508 Mutation Misfolding, ERAD 36-39,130 G85E Mutation Impaired protein maturation 130-132 G91R Mutation Impaired protein maturation 130-132 N1303K Mutation Impaired protein maturation 130-132 ABCC8 WT Wild type Ubiquitin-proteasome degradation 133 A116P Mutation Ubiquitin-proteasome degradation 133 V187D Mutation Ubiquitin-proteasome degradation 133 F1388 Mutation Impaired protein trafficking 134 L1544P Mutation Impaired protein trafficking 135,136 ABCC11a G180R SNP Ubiquitin-proteasome degradation 50 27 Mutation Ubiquitin-proteasome degradation 50 ABCG2a V12M SNP Altered protein localization 96 Q141K SNP Ubiquitin-proteasome degradation 102 F208S SNP Ubiquitin-proteasome degradation 78,99 S441N SNP Ubiquitin-proteasome degradation 78,99 Mutations of ABCA1, ABCA3, ABCA4, ABCB4, ABCB11, ABCC2, ABCC7 (CFTR), and ABCC8 are associated with Tangier disease, fatal surfactant deficiency, Stargardt disease, progressive familial intrahepatic cholestasis type 3 (PFIC-3), progressive familial intrahepatic cholestasis type 2 (PFIC-2), Dubin-Johnson syndrome, cystic fibrosis, and familial hyperinsulinism, respectively.
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ABCB4 p.Ile541Phe 21567408:155:797
status: NEW[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
133 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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ABCB4 p.Ile541Phe 19185004:133:201
status: NEW4 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.
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ABCB4 p.Ile541Phe 19185004:4:152
status: NEW5 In MDCK cells, ABCB1-I541F also accumulated intracellularly in compartments, which were identified as the endoplasmic reticu- lumandcis-Golgi,andremainedpartiallyendoH-sensitive.Aftershiftingcellsto27°C,ABCB1-I541F wasexpressedattheapicalcellsurfaceinamatureandactiveform.Similarly,ABCB4wassignificantly trafficked to the membrane of bile canaliculi in HepG2 cells.
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ABCB4 p.Ile541Phe 19185004:5:21
status: NEWX
ABCB4 p.Ile541Phe 19185004:5:214
status: NEW6 Conclusion: Mutation I541F causes mislocalizationofbothABCB4andABCB1.IntracellularretentionofABCB4-I541Fcanexplainthe disease in PFIC3 patients bearing this mutation.
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ABCB4 p.Ile541Phe 19185004:6:21
status: NEW31 We have studied the effect of mutation I541F, located in the first nucleotide-binding domain (NBD), which has been described in a homozygous patient with PFIC3.4 This female patient experienced chronic cholestasis since 1 year of age and developed cirrhosis.
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ABCB4 p.Ile541Phe 19185004:31:39
status: NEW100 Results Expression of ABCB4 and ABCB4-I541F in HepG2 and MDCK Cells.
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ABCB4 p.Ile541Phe 19185004:100:38
status: NEW102 To examine the cellular localization of ABCB4 and ABCB4-I541F, HepG2 cells were transiently transfected with the pcDNA3 plasmid encoding wild-type ABCB4 or ABCB4-I541F or with the empty plasmid.
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ABCB4 p.Ile541Phe 19185004:102:56
status: NEWX
ABCB4 p.Ile541Phe 19185004:102:162
status: NEW105 Cells transfected with the plasmid encoding ABCB4 expressed the protein exclusively on the membranes of bile canaliculi, which correspond to the apical surface (Fig. 1A), consistent with the expected canalicular localization of ABCB4 in hepatocytes.15 By contrast, cells transfected with the plasmid encoding ABCB4-I541F did not express the protein at the membrane of bile canaliculi.
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ABCB4 p.Ile541Phe 19185004:105:315
status: NEW109 MDCK cells were transfected with the plasmids encoding ABCB4 and ABCB4-I541F or the empty plasmid.
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ABCB4 p.Ile541Phe 19185004:109:71
status: NEW114 By contrast, ABCB4-I541F was detected around the nuclei (Fig. 1D).
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ABCB4 p.Ile541Phe 19185004:114:19
status: NEW116 These expression studies show that ABCB4 is expressed at the apical membrane, whereas ABCB4-I541F leads to a major trafficking defect and accumulates within the cell cytoplasm, in both HepG2 and MDCK cells.
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ABCB4 p.Ile541Phe 19185004:116:92
status: NEW117 ABCB1-I541F-GFP Is Also Intracellular.
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ABCB4 p.Ile541Phe 19185004:117:6
status: NEW118 The I541F mutation is localized next to the ABC signature (LSSGGQ) in the first nucleotide-binding domain.
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ABCB4 p.Ile541Phe 19185004:118:4
status: NEW123 Expression of ABCB4 and ABCB4-I541F in HepG2 and MDCK cells.
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ABCB4 p.Ile541Phe 19185004:123:30
status: NEW124 HepG2 cells transiently expressing ABCB4 (A) or ABCB4-I541F (B), and filter-grown MDCK cells stably expressing ABCB4 (C, E) or ABCB4-I541F (D, F) were fixed with methanol/acetone and processed for immunofluorescence using the P3II-26 monoclonal antibody and Alexa 488-conjugated anti-mouse immunoglobulin G.
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ABCB4 p.Ile541Phe 19185004:124:54
status: NEWX
ABCB4 p.Ile541Phe 19185004:124:133
status: NEW127 Bars, 10 m. activity.20 In MDCK cells, ABCB1-GFP was exclusively expressed at the apical surface (Fig. 3A), as already reported.24 In contrast, ABCB1-I541F-GFP was intracellular, as observed for ABCB4-I541F.
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ABCB4 p.Ile541Phe 19185004:127:159
status: NEWX
ABCB4 p.Ile541Phe 19185004:127:210
status: NEW132 ABCB1-I541F-GFP accumulated around the nuclei, in membrane compartments that appeared sometimes granular (Fig. 3B).
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ABCB4 p.Ile541Phe 19185004:132:6
status: NEW134 ABCB1-I541F-GFP Is Retained in an Endoplasmic Reticulum/Golgi Compartment.
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ABCB4 p.Ile541Phe 19185004:134:6
status: NEW135 To precisely determine the sites of intracellular accumulation, different cellular compartments were identified with specific antibodies in MDCK/ABCB1-I541F-GFP cells grown to subconfluence.
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ABCB4 p.Ile541Phe 19185004:135:151
status: NEW137 In all of these colocalization experiments, ABCB1-I541F-GFP was observed exclusively in the cytoplasm, especially around the nuclei, whereas little ABCB1-GFP was intracellular (Fig. 4A).
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ABCB4 p.Ile541Phe 19185004:137:50
status: NEW138 ABCB1-I541F-GFP colocalized strongly with calnexin and to a lesser extent with protein disulfide isomerase, two endoplasmic reticulum (ER) markers (Fig. 4B).
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ABCB4 p.Ile541Phe 19185004:138:6
status: NEW140 In nontransfected cells, the distribution of calnexin was finely granular, whereas in ABCB1-I541F cells, bundles strongly labeled for calnexin colocalized with the fluorescent mutant (Fig. 4B).
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ABCB4 p.Ile541Phe 19185004:140:92
status: NEW197 To check whether the temperature rescue of ABCB1-I541F in MDCK cells might be relevant for PFIC3 therapy, we studied the effect of low temperature on ABCB4 expression in HepG2 cells.
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ABCB4 p.Ile541Phe 19185004:197:49
status: NEW198 Cells were transiently transfected with either ABCB4 or ABCB4-I541F cDNA.
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ABCB4 p.Ile541Phe 19185004:198:62
status: NEW200 Figure 7A shows that ABCB4-I541F was detected in significant amount at the membrane of bile canaliculi in cells grown at 27°C.
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ABCB4 p.Ile541Phe 19185004:200:27
status: NEW204 Low temperature rescues functional ABCB1-I541F at the apical membrane.
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ABCB4 p.Ile541Phe 19185004:204:41
status: NEW205 (A) MDCK/ABCB1-I541F-GFP cells were grown on filters until confluence at 37°C, then shifted or not to 27°C for 24 hours.
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ABCB4 p.Ile541Phe 19185004:205:15
status: NEW211 MDCK/ABCB1-I541F-GFP cells were grown at 37°C then shifted or not to 27°C 24 hours before the experiment.
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ABCB4 p.Ile541Phe 19185004:211:11
status: NEW216 ABCB4-I541F migrated as a single 140-kDa polypeptide in cells grown at 37°C.
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ABCB4 p.Ile541Phe 19185004:216:6
status: NEW218 These results show that, for ABCB1-I541F in MDCK cells, low temperature is effective on processing and bile canalicular delivery of the ABCB4 mutant in hepatic cells.
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ABCB4 p.Ile541Phe 19185004:218:35
status: NEW221 Homozygous I541F ABCB4 mutation was described in a patient with PFIC3 who presented with persistent cholestasis from the age of 1 year and developed severe portal hypertension.4 In the patient`s liver, immunohistochemistry did not show any canalicular staining for ABCB4.4 This suggested that in the liver in vivo, the mutant protein could not reach the canalicular membrane because of a targeting defect or protein degradation.
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ABCB4 p.Ile541Phe 19185004:221:11
status: NEW222 In both HepG2 and MDCK cells, we found that ABCB4-I541F accumulated intracellularly and was not detected at the canalicular membrane, as in the previously reported mutated patient.
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ABCB4 p.Ile541Phe 19185004:222:50
status: NEW227 The location of the I541F mutation, in the first NBD domain, next to the LSSGQ signature, predicted that ATP-binding or hydrolysis could be affected.
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ABCB4 p.Ile541Phe 19185004:227:20
status: NEW235 Many inherited diseases are known to arise because of point mutations within a gene that result in the production of proteins unable to assume a stable conformation within the cell.28,29 Mutations lead to non-native protein folding intermediates that are recognized by specialized chaperones and eventually targeted for destruction by the quality control machinery of the cell.30 The fact that trafficking of I541F-mutant can be rescued by lowering the temperature suggests that this mutation causes a folding defect.
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ABCB4 p.Ile541Phe 19185004:235:409
status: NEW237 In the case of I541F, both isoleucine and phenylalanine are hydrophobic amino acids, but isoleucine has a branched chain, whereas phenylalanine has a Fig. 7.
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ABCB4 p.Ile541Phe 19185004:237:15
status: NEW238 ABCB4-I541F reaches the bile canalicular membrane at low temperature.
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ABCB4 p.Ile541Phe 19185004:238:6
status: NEW239 HepG2 cells were transfected with either ABCB4 or ABCB4-I541F cDNA.
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ABCB4 p.Ile541Phe 19185004:239:56
status: NEW247 Our observation that folding of ABCB1-I541F at 27°C allows translocation of calcein indicates that, despite the mutation, the NBD domain is able to adopt a native transport-competent conformation.
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ABCB4 p.Ile541Phe 19185004:247:38
status: NEW249 Response to UDCA therapy is variable among PFIC3 patients.4 The patient harboring the I541F mutation was not improved by UDCA therapy and required liver transplantation.
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ABCB4 p.Ile541Phe 19185004:249:86
status: NEW252 The observation that the I541F-mutant is transport-competent if it successfully transits to the plasma membrane raises the possibility that strategies to influence protein folding inside cells might prove to have therapeutic value.
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ABCB4 p.Ile541Phe 19185004:252:25
status: NEW[hide] Cholestatic liver disease in children. Curr Gastroenterol Rep. 2010 Feb;12(1):30-9. Santos JL, Choquette M, Bezerra JA
Cholestatic liver disease in children.
Curr Gastroenterol Rep. 2010 Feb;12(1):30-9., [PMID:20425482]
Abstract [show]
Inherited syndromes of intrahepatic cholestasis and biliary atresia are the most common causes of chronic liver disease and the prime indication for liver transplantation in children. Our understanding of the pathogenesis of these diseases has increased substantially by the discovery of genetic mutations in children with intrahepatic cholestasis and the findings that inflammatory circuits are operative at the time of diagnosis of biliary atresia. Building on this solid foundation, recent studies provide new insight into genotype-phenotype relationships and how mutations produce altered bile composition and cholestasis. New evidence exists that although liver transplantation is curative for patients with end-stage liver disease owing to cholestasis, some patients may develop recurrence of cholestasis because of the emergence of autoantibodies that disrupt canalicular function in the new graft. Progress is also evident in biliary atresia, with recent studies identifying candidate modifier genes and directly implicating lymphocytes and inflammatory signals in the pathogenesis of bile duct injury and obstruction.
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No. Sentence Comment
82 This mechanism was illustrated in one study by tracking the signal produced by the I541F MDR3 mutant in liver and kidney cell lines, which was detected in the cytoplasm because of trapping within the ER and cis-Golgi [21].
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ABCB4 p.Ile541Phe 20425482:82:83
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
5 Variations in the ABCB4 gene sequence cause progressivefamilialintrahepaticcholestasistype3.Wehaveshown previously that the I541F mutation, when reproduced either in ABCB1orinABCB4,ledtoretentionintheendoplasmicreticulum (ER)/Golgi.
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ABCB4 p.Ile541Phe 22184139:5:124
status: NEW12 Glycerol improved maturation and exit of the mutant from theER.CyclosporinA,acompetitivesubstrateforABCB1,restored maturation, plasma membrane expression, and activity of ABCB1-I541F.
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ABCB4 p.Ile541Phe 22184139:12:177
status: NEW13 Cyclosporin A also improved maturation of ABCB4-I541F in Madin-Darby canine kidney cells.
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ABCB4 p.Ile541Phe 22184139:13:48
status: NEW14 In HepG2 cells transfected with ABCB4-I541F cDNA, cyclosporin A allowed a significant amount of the mutant protein to reach the membrane of bile canaliculi.
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ABCB4 p.Ile541Phe 22184139:14:38
status: NEW44 We have previously investigated effects of the I541F ABCB4 mutation that has been identified in a progressive familial intrahepatic cholestasis type 3 patient.
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ABCB4 p.Ile541Phe 22184139:44:47
status: NEW47 Nevertheless, intracellular traffic was restored when cells were grown at 27 °C, and the rescued ABCB1-I541F mutant was functional.
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ABCB4 p.Ile541Phe 22184139:47:108
status: NEW48 The aim of this work was to test other means to provide functional rescue for the I541F mutation.
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ABCB4 p.Ile541Phe 22184139:48:82
status: NEW49 We have taken the ABCB1-I541F mutant as a model to explore different strategies.
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ABCB4 p.Ile541Phe 22184139:49:24
status: NEW65 The generation of MDCK cells stably expressing GFP-ABCB1, GFP-ABCB1-I541F, ABCB4 and ABCBA-I541F has been previously described (10).
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ABCB4 p.Ile541Phe 22184139:65:68
status: NEWX
ABCB4 p.Ile541Phe 22184139:65:91
status: NEW102 RESULTS The I541F Mutant Is More Susceptible to Protease Degradation-We previously showed that the I541F mutant was retained in the ER and that retention could be rescued by low temperature, suggesting that the mutant had a folding defect (10).
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ABCB4 p.Ile541Phe 22184139:102:12
status: NEWX
ABCB4 p.Ile541Phe 22184139:102:99
status: NEW104 We therefore studied whether the ABCB1-I541F was more susceptible to in situ proteolysis, using isolated microsomes from MDCK cells stably transfected with either GFP-ABCB1-WT or GFP-ABCB1-I541F.
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ABCB4 p.Ile541Phe 22184139:104:39
status: NEWX
ABCB4 p.Ile541Phe 22184139:104:189
status: NEW122 Immunoprecipitation experiments showed that calnexin coimmunoprecipitated with both ABCB1-WT and ABCB1-I541F (Fig. 2B).
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ABCB4 p.Ile541Phe 22184139:122:103
status: NEW156 In these conditions, the expression of ABCB1-I541F was notably increased but remained immature, indicating that it did not exit the ER (Fig. 5B).
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ABCB4 p.Ile541Phe 22184139:156:45
status: NEW157 Effect of Chemical Chaperones on ABCB1-I541F Expression-Different compounds referred to as chemical chaperones have been shown to rescue folding of mutant proteins.
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ABCB4 p.Ile541Phe 22184139:157:39
status: NEW159 ABCB1-I541F expressing cells were treated with thapsigargin or 4-PB at concentrations up to 100 M for 24 h.
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ABCB4 p.Ile541Phe 22184139:159:6
status: NEW160 The Western blot analysis pattern of ABCB1-I541F did not change in treated cells (Fig. 6A).
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ABCB4 p.Ile541Phe 22184139:160:43
status: NEW163 On the other hand, treatment with 7.5% glycerol increased the mature form of ABCB1-I541F on Western blot analyses (Fig. 6C).
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ABCB4 p.Ile541Phe 22184139:163:83
status: NEW197 A substantial amount of matured ABCB4-I541F was detected in cells treated with cyclosporin A at a concentration of 2 M (Fig. 8, A and B).
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ABCB4 p.Ile541Phe 22184139:197:38
status: NEW199 To confirm that a similar rescue would also occur in hepatic cells, HepG2 cells were transfected with the ABCB4-I541F plasmid.
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ABCB4 p.Ile541Phe 22184139:199:112
status: NEW203 In control cells, ABCB4-I541F was detected exclusively in the cytoplasm and was not detected at the membrane of bile canaliculi.
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ABCB4 p.Ile541Phe 22184139:203:24
status: NEW207 We found that the ABCB1-I541F mutant was more susceptible to the in situ action of proteolytic enzymes, especially in the first moiety of the molecule, where the mutation is located.
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ABCB4 p.Ile541Phe 22184139:207:24
status: NEW209 In keeping with this conclusion, it was shown previously that ABCB4 was undetectable by immunohistochemistry in the liver tissue from a patient bearing the ABCB4-I541F mutation (16).
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ABCB4 p.Ile541Phe 22184139:209:162
status: NEW213 Effect of chemical chaperones on the expression pattern of ABCB1-I541F.
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ABCB4 p.Ile541Phe 22184139:213:65
status: NEW214 A, MDCK cells stably transfected with GFP-tagged ABCB1-WT or ABCB1-I541F were treated with vehicle (-), 4-PB, or thapsigargin (Thap) at the indicated concentrations for 24 h.
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ABCB4 p.Ile541Phe 22184139:214:67
status: NEW216 B, filter-grown MDCK cells stably transfected with GFP-tagged ABCB1-I541F were treated with vehicle (control), 100 M thapsigargin, or 1 mM 4-phenylbutyrate for 24 h.
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ABCB4 p.Ile541Phe 22184139:216:68
status: NEW219 D, MDCK cells stably transfected with GFP-tagged ABCB1-I541F were treated with 7.5% glycerol for 24 h.
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ABCB4 p.Ile541Phe 22184139:219:55
status: NEW223 Scale bar ϭ 10 m. E, the colocalization of GFP-tagged ABCB1-I541F with GM130 was quantified using the ImageJ 1.41 measure colocalization function on multiple confocal sections of at least 30 cells in three independent experiments.
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ABCB4 p.Ile541Phe 22184139:223:74
status: NEW256 In our system, treatment with thapsigargin and 4-PB had no effect on the I541F mutant.
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ABCB4 p.Ile541Phe 22184139:256:73
status: NEW257 Only glycerol did improve the maturation of the ABCB1-I541F mutant, which was able to exit the ER.
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ABCB4 p.Ile541Phe 22184139:257:54
status: NEW262 Finally, rescue of the I541F mutant was obtained with a specific substrate.
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ABCB4 p.Ile541Phe 22184139:262:23
status: NEW[hide] Function and pathophysiological importance of ABCB... Pflugers Arch. 2007 Feb;453(5):601-10. Epub 2006 Apr 19. Oude Elferink RP, Paulusma CC
Function and pathophysiological importance of ABCB4 (MDR3 P-glycoprotein).
Pflugers Arch. 2007 Feb;453(5):601-10. Epub 2006 Apr 19., [PMID:16622704]
Abstract [show]
Like several other ATP-binding cassette (ABC) transporters, ABCB4 is a lipid translocator. It translocates phosphatidylcholine (PC) from the inner to the outer leaflet of the canalicular membrane of the hepatocyte. Its function is quite crucial as evidenced by a severe liver disease, progressive familial intrahepatic cholestasis type 3, which develops in persons with ABCB4 deficiency. Translocation of PC makes the phospholipid available for extraction into the canalicular lumen by bile salts. The primary function of biliary phospholipid excretion is to protect the membranes of cells facing the biliary tree against these bile salts: the uptake of PC in bile salt micelles reduces the detergent activity of these micelles. In this review, we will discuss the functional aspects of ABCB4 and the regulation of its expression. Furthermore, we will describe the clinical and biochemical consequences of complete and partial deficiency of ABCB4 function.
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No. Sentence Comment
141 Canalicular lipid transport defects can cause gallstone formation Cholesterol supersaturation of bile, which occurs in a large proportion of humans, leads to the formation of cholesterol Walker B; L556R 571del Truncation PFIC3 LPAC ICP 27 splice Truncation 132 del Truncation TM 2; W138R TM 12; 981 del Truncation Linker; Q636X Truncation TM 11; R957X Truncation TM 6; S346I E395G Walker B; I541F TM 12; G983S Walker A; V425M Walker A; T424A Walker B; D564G TM 7; F711S 180 del truncation 336 delT truncation Exon 22-23 del truncation F165I T175A TM 5; M301T TM 5; S320F 336 insT truncation Walker A; 432 insA truncation E528D L591Q W658stop 757 insT R788E A934T P1161S TM 5; S320F TM 8; G762ER144X Walker B; A546D Walker B; G535AALL 96 del Truncation Walker B; L556R 571del Truncation PFIC3 LPAC ICP 27 splice Truncation 132 del Truncation TM 2; W138R TM 12; 981 del Truncation Linker; Q636X Truncation TM 11; R957X Truncation TM 6; S346I E395G Walker B; I541F TM 12; G983S Walker A; V425M Walker A; T424A Walker B; D564G TM 7; F711S 180 del truncation 336 delT truncation Exon 22-23 del truncation F165I T175A TM 5; M301T TM 5; S320F 336 insT truncation Walker A; 432 insA truncation E528D L591Q W658stop 757 insT R788E A934T P1161S TM 5; S320F TM 8; G762ER144X Walker B; A546D Walker B; G535AALL 96 del Truncation Fig. 3 Summary of the known mutations and their localization in the protein, as identified in patients with PFIC type 3, LPAC syndrome (intrahepatic gallstone formation), and intrahepatic cholestasis of pregnancy (ICP).
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ABCB4 p.Ile541Phe 16622704:141:391
status: NEWX
ABCB4 p.Ile541Phe 16622704:141:956
status: NEW[hide] The wide spectrum of multidrug resistance 3 defici... Gastroenterology. 2001 May;120(6):1448-58. Jacquemin E, De Vree JM, Cresteil D, Sokal EM, Sturm E, Dumont M, Scheffer GL, Paul M, Burdelski M, Bosma PJ, Bernard O, Hadchouel M, Elferink RP
The wide spectrum of multidrug resistance 3 deficiency: from neonatal cholestasis to cirrhosis of adulthood.
Gastroenterology. 2001 May;120(6):1448-58., [PMID:11313315]
Abstract [show]
BACKGROUND & AIMS: We have specified the features of progressive familial intrahepatic cholestasis type 3 and investigated in 31 patients whether a defect of the multidrug resistance 3 gene (MDR3) underlies this phenotype. METHODS: MDR3 sequencing, liver MDR3 immunohistochemistry, and biliary phospholipid dosage were performed. RESULTS: Liver histology showed a pattern of biliary cirrhosis with patency of the biliary tree. Age at presentation ranged from the neonatal period to early adulthood. Sequence analysis revealed 16 different mutations in 17 patients. Mutations were identified on both alleles in 12 patients and only on 1 allele in 5. Four mutations lead to a frame shift, 2 are nonsense, and 10 are missense. An additional missense mutation probably representing a polymorphism was found in 5 patients. MDR3 mutations were associated with abnormal MDR3 canalicular staining and a low proportion of biliary phospholipids. Gallstones or episodes of cholestasis of pregnancy were found in patients or parents. Children with missense mutations had a less severe disease and more often a beneficial effect of ursodeoxycholic acid therapy. CONCLUSIONS: At least one third of the patients with a progressive familial intrahepatic cholestasis type 3 phenotype have a proven defect of MDR3. This gene defect should also be considered in adult liver diseases.
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No. Sentence Comment
126 MDR3 Mutations, Immunohistochemistry, and Biliary Phospholipids in Patients With High GGT PFIC (PFIC3) Patients Exon Nucleotide mutation Amino acid change Protein consequence Family MDR3 liver Phospholipids (%) bile Homozygous Deletion/Insertion M Y M I (sister of M Y) 2 111 A Ͼ G, splice donor site, exon/intron boundary 2/3 27 Frame shift in NH2 terminus, then truncation ND Absent ND 1.6 B Ka 6 426-432 del 132 Frame shift in TMD 2, 29 novel amino acids then truncation Mother ϩ/- Father ϩ/- 1 sibling ϩ/ϩ Absent ND B Sa 14 1744 del T 571 Frame shift, 15 novel amino acids then truncation Mother ϩ/- (ICP) Father ϩ/- 3 siblings ϩ/ϩ Absent ND P G 24 2975-2984 del Compound heterozygous 981 Frame shift in TMD 12, 3 novel amino acids then truncation Mother ϩ/ϩ Father ϩ/- ND 14.9 Bo S Bo N (sister of Bo S) 16 Nonsense 1938 C Ͼ T Q636X Linker region Truncation Mother ϩ/- Father ϩ/- 2 siblings ϩ/- Absent 2 ND B Aa 23 2901 C Ͼ T R957X TMD 11 Truncation Mother ϩ/- (ICP) Father ϩ/- 4 siblings ϩ/- or ϩ/ϩ Absent ND S F 10 Missense 1069 G Ͼ T S346I Serine to isoleucine in TMD6 Mother ϩ/- Father ϩ/- Faint 1 B H 11 1216 A Ͼ G E395G Glutamate to glycine between TMD 6 and first Walker A motif ND ND ND N I 14 1653 A Ͼ T I541F Isoleucine to phenylalanine in first Walker B motif Mother ϩ/- Father ϩ/- Absent ND M M 14 1699 T Ͼ G L556R Leucine to arginine in first Walker B motif ND ND ND P G 24 2979 G Ͼ A Compound heterozygous G983S Glycine to serine in TMD 12 Mother ϩ/- Father ϩ/ϩ ND 14.9 P A 6 Heterozygous Missense 444 T Ͼ C W138R Trytophan to arginine in TMD 2 Mother ϩ/- Father ϩ/ϩ ND 6.7 M Aa 12 1302 A Ͼ G T424A Threonine to alanine in first Walker A motif ND gallbladder lithiasis in father Faint 6.3 P K 12 1307 G Ͼ A V425M Valine to methionine in first Walker A motif ND Normal ND G A 14 1723 A Ͼ G D564G Aspartate to glycine in first Walker B motif ND ND ND G M 17 2132 T Ͼ C F711S Phenylalanine to serine in TMD 7 ND ND ND L M 16 Polymorphism 1986 A Ͼ G Homozygous R652G Arginine to glycine in linker region Mother ϩ/- (suspicion of ICP) Father ϩ/- ND ND Ga M " Heterozygous " " Mother ϩ/- Father ϩ/ϩ 1 sibling ϩ/- ND 9.7 L H L F (sister of L H) " Heterozygous " " ND ND ND VH C " Heterozygous " " Mother ϩ/ϩ Father ϩ/- ND ND NOTE.
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ABCB4 p.Ile541Phe 11313315:126:1380
status: NEW[hide] Functional characterization of genetic variations ... Biochem Biophys Res Commun. 2013 Jan 25;430(4):1312-8. doi: 10.1016/j.bbrc.2012.12.041. Epub 2012 Dec 19. Jang GH, Kim TH, Choe Y, Ham A, Choi JH
Functional characterization of genetic variations in the MDR3 promoter.
Biochem Biophys Res Commun. 2013 Jan 25;430(4):1312-8. doi: 10.1016/j.bbrc.2012.12.041. Epub 2012 Dec 19., [PMID:23261441]
Abstract [show]
Multidrug resistance 3 (MDR3) is present on the canalicular membrane of the hepatocyte and plays an important role in protecting the liver from bile acids. In this study, we characterized the transcriptional effects of four common haplotypes and four polymorphic variants in the promoter region of MDR3 that were identified in 126 DNA samples from Koreans. We measured the luciferase activities of the four MDR3 promoter haplotypes using in vitro reporter assays. Among them, two haplotypes showed a significant decrease in reporter activity compared to the reference. One of the mechanisms by which these haplotypes might decrease MDR3 transcriptional activity was determined: one of the polymorphisms that are present in haplotype 3, was associated with a significant reduction in the promoter activity of MDR3, and the transcription factor NF-Y was predicted to bind to the promoter in the region of g.-1584C>T. Electrophoretic mobility shift assays showed that the g.-1584C allele exhibited greater binding to NF-Y than did the g.-1584T allele. Through the measurement of promoter activity after the overexpression of NF-Y, we found that NF-Y can act as a transcriptional activator of MDR3. These data suggest that the reduced transcriptional activity of g.-1584C>T results from a reduction in the binding affinity of the activator NF-Y to the MDR3 promoter region. Our study suggests that two common haplotypes of MDR3 can regulate the transcriptional rate of MDR3 and that NF-Y may be one of the transcriptional factors involved in this regulation.
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No. Sentence Comment
19 For example, a nonsynonymous mutation, p.I541F, found in a PFIC3 patient showed decreased transport activity owing to reduced membrane expression [8].
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ABCB4 p.Ile541Phe 23261441:19:41
status: NEW[hide] ABCB4: Insights from pathobiology into therapy. Clin Res Hepatol Gastroenterol. 2014 Oct;38(5):557-63. doi: 10.1016/j.clinre.2014.03.001. Epub 2014 Jun 19. Falguieres T, Ait-Slimane T, Housset C, Maurice M
ABCB4: Insights from pathobiology into therapy.
Clin Res Hepatol Gastroenterol. 2014 Oct;38(5):557-63. doi: 10.1016/j.clinre.2014.03.001. Epub 2014 Jun 19., [PMID:24953525]
Abstract [show]
Adenosine triphosphate (ATP)-binding cassette, sub-family B, member 4 (ABCB4), also called multidrug resistance 3 (MDR3), is a member of the ATP-binding cassette transporter superfamily, which is localized at the canalicular membrane of hepatocytes, and mediates the translocation of phosphatidylcholine into bile. Phosphatidylcholine secretion is crucial to ensure solubilization of cholesterol into mixed micelles and to prevent bile acid toxicity towards hepatobiliary epithelia. Genetic defects of ABCB4 may cause progressive familial intrahepatic cholestasis type 3 (PFIC3), a rare autosomic recessive disease occurring early in childhood that may be lethal in the absence of liver transplantation, and other cholestatic or cholelithiasic diseases in heterozygous adults. Development of therapies for these conditions requires understanding of the biology of this transporter and how gene variations may cause disease. This review focuses on our current knowledge on the regulation of ABCB4 expression, trafficking and function, and presents recent advances in fundamental research with promising therapeutic perspectives.
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No. Sentence Comment
83 Wild-type ABCB4 (A) or I541F missense mutant (B, C) were expressed in HepG2 cells.
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ABCB4 p.Ile541Phe 24953525:83:23
status: NEW105 The I541F mutation led to improper folding, retention in the endoplasmic reticulum and premature degradation [59,60] (Fig. 2).
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ABCB4 p.Ile541Phe 24953525:105:4
status: NEW127 We recently showed that the ER-retained I541F missense mutant of ABCB4, identified in patients with PFIC3, was rescued by cyclosporin A treatment, while 4-phenylbutyrate had no effect [60] (Fig. 2).
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ABCB4 p.Ile541Phe 24953525:127:40
status: NEW[hide] Functional Characterization of ABCB4 Mutations Fou... Korean J Physiol Pharmacol. 2013 Dec;17(6):525-30. doi: 10.4196/kjpp.2013.17.6.525. Epub 2013 Dec 16. Kim TH, Park HJ, Choi JH
Functional Characterization of ABCB4 Mutations Found in Low Phospholipid-Associated Cholelithiasis (LPAC).
Korean J Physiol Pharmacol. 2013 Dec;17(6):525-30. doi: 10.4196/kjpp.2013.17.6.525. Epub 2013 Dec 16., [PMID:24381502]
Abstract [show]
Multidrug resistance 3 (MDR3) is expressed on the canalicular membrane of the hepatocytes and plays an important role in protecting the liver from bile acids. Altered ABCB4 gene expression can lead to a rare hepatic disease, low phospholipid-associated cholelithiasis (LPAC). In this study, we characterized 3 ABCB4 mutations in LPAC patients using various in vitro assay systems. We first measured the ability of each mutant to transport paclitaxel and then the mechanisms by which these mutations might change MDR3 transport activity were determined using immunoblotting, cell surface protein biotinylation, and immunofluorescence. Through a membrane vesicular transport assay, we observed that the uptake of paclitaxel was significantly reduced in membrane vesicles expressing 2 ABCB4 mutations, F165I and S320F. Both mutants showed significantly decreased total and cell surface MDR3 expression. These data suggest two missense mutations of ABCB4 may alter function of MDR3 and ultimately can be determined as LPAC-causing mutations.
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No. Sentence Comment
20 For example, a ABCB4 mutation found in PFIC3 patients, I541F, was shown to decrease transport activity through reduction of membrane MDR3 expression [10,11].
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ABCB4 p.Ile541Phe 24381502:20:55
status: NEW123 One of the ABCB4 mutations, I541F, was found in PFIC3 patients and was shown to be a trafficking-defective mutation [10,11].
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ABCB4 p.Ile541Phe 24381502:123:28
status: NEW125 2 mutants, F165I and S320F, examined in this study also showed decreased transport activity and protein expression, although the extent of reduction was less than that of I541F.
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ABCB4 p.Ile541Phe 24381502:125:171
status: NEW