ABCMdb

ABCB4 p.Leu481Arg

Predicted by SNAP2:	A: D (66%), C: D (53%), D: D (91%), E: D (85%), F: D (66%), G: D (85%), H: D (80%), I: N (66%), K: D (85%), M: N (57%), N: D (85%), P: D (91%), Q: D (75%), R: D (85%), S: D (80%), T: D (75%), V: N (53%), W: D (80%), Y: D (75%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: N, K: D, M: N, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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[hide] Two ABCB4 point mutations of strategic NBD-motifs ... Eur J Hum Genet. 2014 May;22(5):633-9. doi: 10.1038/ejhg.2013.214. Epub 2013 Sep 18. Degiorgio D, Corsetto PA, Rizzo AM, Colombo C, Seia M, Costantino L, Montorfano G, Tomaiuolo R, Bordo D, Sansanelli S, Li M, Tavian D, Rastaldi MP, Coviello DA
Two ABCB4 point mutations of strategic NBD-motifs do not prevent protein targeting to the plasma membrane but promote MDR3 dysfunction.
Eur J Hum Genet. 2014 May;22(5):633-9. doi: 10.1038/ejhg.2013.214. Epub 2013 Sep 18., [PMID:24045840]

Abstract [show]
The ABCB4 gene encodes for MDR3, a protein that translocates phosphatidylcholine from the inner to the outer leaflet of the hepatocanalicular membrane; its deficiency favors the formation of 'toxic bile'. Several forms of hepatobiliary diseases have been associated with ABCB4 mutations, but the detrimental effects of most mutations on the encoded protein needs to be clarified. Among subjects with cholangiopathies who were screened for mutations in ABCB4 by direct sequencing, we identified the new mutation p.(L481R) in three brothers. According to our model of tertiary structure, this mutation affects the Q-loop, whereas the p.(Y403H) mutation, that we already described in two other families, involves the A-loop. This study was aimed at analyzing the functional relevance of these two ABCB4 mutations: MDR3 expression and lipid content in the culture supernatant were evaluated in cell lines stably transfected with the ABCB4 wild-type clone and corresponding mutants. No differences of expression were observed between wild-type and mutant gene products. Instead, both mutations caused a reduction of phosphatidylcholine secretion compared with the wild-type transfected cell lines. On the contrary, cholesterol (Chol) release, after 1 and 3 mM sodium taurocholate stimulation, was higher in the mutant-transfected cell lines than that in the wild-type and was particularly enhanced in cells transfected with the p.Y403H-construct.In summary, our data show that both mutations do not seem to affect protein expression, but are able to reduce the efflux of phosphatidylcholine associated with increase of Chol, thereby promoting the formation of toxic bile.

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2 Among subjects with cholangiopathies who were screened for mutations in ABCB4 by direct sequencing, we identified the new mutation p.(L481R) in three brothers. Login to comment
33 The plasmid pcDNA3.1/Hygro(&#fe; )/ABCB4 was also used to obtain clones harboring the p.(Y403H) or the p.(L481R) mutation. Login to comment
36 Bioinformatic analysis The three-dimensional models of the human MDR3 and of the p.Leu481Arg mutant were built by homology based on the structure of the murine multidrug resistance protein 1A (gene name, Abcb1a), determined with crystallographic methods (PDB: 3g5u).16 The models were built with an automated procedure with the SwissModel web server (http://www. Login to comment
54 RESULTS Molecular analysis and in silico evaluation The sequencing analysis of ABCB4 gene in the members of family A showed a new heterozygous mutation, c.1442T4G, which causes the p.(L481R) amino-acid change. Login to comment
56 The p.(L481R) replaces the hydrophobic Leu side chain with a bulkier and hydrophilic Arg residue in the first NBD. Login to comment
77 (b) Region surrounding the p.Leu481Arg mutation, with the neighboring residues represented in ball-and-stick. Login to comment
80 Wild-type cells secreted more PC and Chol than the mutant ones; PC: 3.417 nmol/mg proteins versus 1.410 for p.L481R and 2.346 for p.Y403H and Chol: 4.476 nmol/mg proteins versus 3.193 for p.L481R and 3.811 for p.Y403H (Figures 4a and b; Supplementary Table 1). Login to comment
82 Although wild-type cells and p.Y403H mutant showed similar fold increases in PC excretion, p.L481R mutant showed higher sensitivity to NaTC reaching 11-fold increase in PC secretion at 3 mM compared with the absence of NaTC (Supplementary Table 1). Login to comment
92 Lanes 5 and 6 show the stable expression of p.L481R-ABCB4 and p.Y403H, respectively. Login to comment
98 In this study, besides reporting the identification of a novel ABCB4 mutation (p.(L481R)) within the Q-loop in three siblings suffering from juvenile cholelithiasis, we demonstrate that this mutation, stably transfected in HUH28 cells, as well as the mutation p.(Y403H), previously described by our group,14 do not prevent protein targeting to the plasma membrane but induce an abnormal efflux of PC and Chol. Login to comment
102 The p.(L481R) mutation was identified in three out of seven affected siblings belonging to family A, whereas the already characterized p.(Y403H) mutation6,14 was identified in four affected members belonging to other two families. Login to comment
111 Analysis of PC (a) and Chol (b) secretion, and PC-Chol correlation (c) in MDR3 wild-type, MDR3 p.Y403H and MDR3 p.L481R cells. Login to comment

[hide] Functional analysis of ABCB4 mutations relates cli... Gut. 2015 Jan;64(1):147-55. doi: 10.1136/gutjnl-2014-306896. Epub 2014 Mar 4. Gordo-Gilart R, Andueza S, Hierro L, Martinez-Fernandez P, D'Agostino D, Jara P, Alvarez L
Functional analysis of ABCB4 mutations relates clinical outcomes of progressive familial intrahepatic cholestasis type 3 to the degree of MDR3 floppase activity.
Gut. 2015 Jan;64(1):147-55. doi: 10.1136/gutjnl-2014-306896. Epub 2014 Mar 4., [PMID:24594635]

Abstract [show]
OBJECTIVE: Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a potentially lethal autosomal recessive liver disease associated with mutations in ABCB4, the gene encoding the canalicular translocator of phosphatidylcholine MDR3. While some affected children benefit from ursodeoxycholic acid (UDCA) therapy, others evolve to end-stage liver disease. We aimed to evaluate whether these different outcomes are related to the impact of ABCB4 mutations. DESIGN: Six children with PFIC3 were investigated by sequencing of ABCB4 exons and flanking intron-exon boundaries and by immunohistochemistry. ABCB4 missense mutations were phenotyped in vitro by assessing their effects on MDR3 expression, subcellular localisation, and phosphatidylcholine-translocating activity. The resulting data were contrasted with the clinical outcomes. RESULTS: Eight distinct ABCB4 mutations were identified: one nonsense, one splicing and six missense mutations, four of which (G68R, T201M, P479L, D459H) affected MDR3 expression level. G68R and D459H also led to retention of the protein in endoplasmic reticulum. Phosphatidylcholine efflux assays indicated that T201M, P479L, S978P and E1118K mutations impaired MDR3 activity to variable degrees. Three children with mutations that caused a total loss of MDR3 expression/function manifested progressive liver disease refractory to UDCA treatment. This was also the case in a patient carrying two different mutations that, in combination, resulted in a 90% reduction in total MDR3 activity. A favourable response to UDCA was achieved in two patients with estimated MDR3 activities of 50% and 33%, respectively. CONCLUSIONS: These data provide experimental evidence of the correlation between the degree of MDR3 floppase activity and the clinical outcomes of PFIC3.

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175 Along these lines, it has recently been reported that the mutation of another amino acid at the Q-loop of the NBD1 of MDR3, L481R, lowers MDR3 activity.26 The S978P mutation completely abrogated the PC translocation capacity of MDR3. Login to comment
174 Along these lines, it has recently been reported that the mutation of another amino acid at the Q-loop of the NBD1 of MDR3, L481R, lowers MDR3 activity.26 The S978P mutation completely abrogated the PC translocation capacity of MDR3. Login to comment

[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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106 By contrast, the Y403H and L481R mutations did not alter membrane targeting but decreased ABCB4 activity [61]. Login to comment