ABCB1 p.Phe37Ala
| Predicted by SNAP2: | A: D (59%), C: N (61%), D: D (80%), E: D (75%), G: D (63%), H: N (53%), I: N (82%), K: D (66%), L: N (82%), M: N (66%), N: D (71%), P: D (75%), Q: D (63%), R: D (63%), S: D (59%), T: N (61%), V: N (61%), W: N (53%), Y: N (82%), |
| Predicted by PROVEAN: | A: N, C: D, D: D, E: D, G: D, H: D, I: N, K: D, L: N, M: N, N: D, P: D, Q: D, R: D, S: D, T: N, V: N, W: N, Y: N, |
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[hide] Gene expression profiling of ABC transporters in d... Lab Invest. 2008 Dec;88(12):1303-15. Epub 2008 Oct 20. Hendig D, Langmann T, Kocken S, Zarbock R, Szliska C, Schmitz G, Kleesiek K, Gotting C
Gene expression profiling of ABC transporters in dermal fibroblasts of pseudoxanthoma elasticum patients identifies new candidates involved in PXE pathogenesis.
Lab Invest. 2008 Dec;88(12):1303-15. Epub 2008 Oct 20., [PMID:18936737]
Abstract [show]
Mutations in the ABCC6 gene, encoding the multidrug resistance-associated protein 6 (MRP6), cause pseudoxanthoma elasticum (PXE). This heritable disorder leads to pathological alterations in connective tissues. The implication of MRP6 deficiency in PXE is still unknown. Moreover, nothing is known about a possible compensatory expression of other ATP binding-cassette (ABC) transporter proteins in MRP6-deficient cells. We investigated the gene expression profile of 47 ABC transporters in human dermal fibroblasts of healthy controls (n=2) and PXE patients (n=4) by TaqMan low-density array. The analysis revealed the expression of 37 ABC transporter genes in dermal fibroblasts. ABCC6 gene expression was not quantifiable in fibroblasts derived from PXE patients. Seven genes (ABCA6, ABCA9, ABCA10, ABCB5, ABCC2, ABCC9 and ABCD2) were induced, whereas the gene expression of one gene (ABCA3) was decreased, comparing controls and PXE patients (with at least twofold changes). We reanalyzed the gene expression of selected ABC transporters in a larger set of dermal fibroblasts from controls and PXE patients (n=6, each). Reanalysis showed high interindividual variability between samples, but confirmed the results obtained in the array analysis. The gene expression of ABC transporter genes, as well as lineage markers of PXE, was further examined after inhibition of ABCC6 gene expression by using specific small-interfering RNA. These experiments corroborated the observed gene expression alterations, most notably in the ABCA subclass (up to fourfold, P<0.05). We therefore conclude that MRP6-deficient dermal fibroblasts exhibit a distinct gene expression profile of ABCA transporters, potentially to compensate for MRP6 deficiency. Moreover, our results point to a function for ABCC6/MRP6 in sterol transport, as sterols are preferential regulators of ABCA transporter activity and expression. Further studies are now required to uncover the role of ABCA transporters in PXE.
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62 Table 1 Main characteristics of dermal fibroblasts derived from PXE patients and healthy controls used in the present study Sample ID Gender Agea Biopsy source ABCC6 genotypeb Statusc Age at disease onseta Number of involved organs PXE patients P60F Female 58 Axilla c.37-1G4A (SSM) c.37-1G4A (SSM) hm 56 3 P229F Female 50 NA c.1171A4G (p.R391G) c.1208C4A (p.A413N) c.2252T4A (p.M751K) cht NA NA P265F Female 62 Cervix c.1132C4T (p.Q378X) c.3421C4T (p.R1141X) cht 16 3 P3M Male 57 Cervix c.3421C4T (p.R1141X) c.3883-6G4A (SSM) cht 46 5 P128M Male 51 Cervix c.3769_3770insC (p.L1259fsX1277) c.3769_3770insC (p.L1259fsX1277) hm 48 3 P308M Male 42 NA c.3421C4T (p.R1141X) c.-90ins14 (c)ht NA NA P341M Male 41 NA c.1552C4T (p.R518X) ND ht NA NA Healthy controls F37A Female 37 Abdomen - - - wt - - F42A Female 42 Abdomen - - - wt - - F52C Female 52 Cheek - - - wt - - M2FS Male 2 Foreskin - - - wt - - M45D Male 45 Face - - - wt - - M56D Male 56 Face - - - wt - - hm, homozygote; cht, compound heterozygote; ht, heterozygote; wt, wild type; SSM, splice site mutation; NA, not applicable; ND, nondetected.
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ABCB1 p.Phe37Ala 18936737:62:758
status: NEW[hide] P-glycoprotein in blood-brain barrier endothelial ... J Neurochem. 2003 Nov;87(4):1010-23. Jodoin J, Demeule M, Fenart L, Cecchelli R, Farmer S, Linton KJ, Higgins CF, Beliveau R
P-glycoprotein in blood-brain barrier endothelial cells: interaction and oligomerization with caveolins.
J Neurochem. 2003 Nov;87(4):1010-23., [PMID:14622130]
Abstract [show]
P-glycoprotein (P-gp), an adenosine triphosphate (ATP)-binding cassette transporter which acts as a drug efflux pump, is highly expressed at the blood-brain barrier (BBB) where it plays an important role in brain protection. Recently, P-gp has been reported to be located in the caveolae of multidrug-resistant cells. In this study, we investigated the localization and the activity of P-gp in the caveolae of endothelial cells of the BBB. We used an in vitro model of the BBB which is formed by co-culture of bovine brain capillary endothelial cells (BBCEC) with astrocytes. Caveolar microdomains isolated from BBCEC are enriched in P-gp, cholesterol, caveolin-1, and caveolin-2. Moreover, P-gp interacts with caveolin-1 and caveolin-2; together, they form a high molecular mass complex. P-gp in isolated caveolae is able to bind its substrates, and the caveolae-disrupting agents filipin III and nystatin decrease P-gp transport activity. In addition, mutations in the caveolin-binding motif present in P-gp reduced the interaction of P-gp with caveolin-1 and increased the transport activity of P-gp. Thus, P-gp expressed at the BBB is mainly localized in caveolae and its activity may be modulated by interaction with caveolin-1.
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74 The mutated DNA was subcloned into the mammalian expression vector pcineo (Promega) to generate plasmid F37A-MDR1.
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ABCB1 p.Phe37Ala 14622130:74:104
status: NEW206 In order to further study the interaction between P-gp and caveolin-1, two P-gp proteins mutated in the caveolin-binding motif were produced: YWAA-MDR1 and F37A-MDR1.
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ABCB1 p.Phe37Ala 14622130:206:156
status: NEW208 In the other mutant, Phe37 was replaced with Ala (F37A-MDR1); nucleotide changes in the mutant genes were confirmed by sequencing.
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ABCB1 p.Phe37Ala 14622130:208:21
status: NEWX
ABCB1 p.Phe37Ala 14622130:208:50
status: NEW210 Immunodetection of P-gp in Cos-7 cells transiently transfected with the wild-type (WT)-MDR1, YWAA-MDR1, and F37A-MDR1 showed that WT-MDR1 and P-gp mutants were expressed to the same extent (Fig. 7a).
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ABCB1 p.Phe37Ala 14622130:210:108
status: NEW214 Figure 7(b) shows that relative fluorescence increased fourfold in WT-MDR1-, YWAA-MDR1-, and F37A-MDR1-expressing cells as compared to untransfected and f/s-MDR1-transfected cells.
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ABCB1 p.Phe37Ala 14622130:214:93
status: NEW217 Compared with WT-MDR1-transfected cells, the interaction between P-gp and caveolin-1 is reduced by 27% and 48% in cells expressing YWAA-MDR1 and F37A-MDR1, respectively (Fig. 8b).
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ABCB1 p.Phe37Ala 14622130:217:145
status: NEW221 In YWAA-MDR1and F37A-MDR1-transfected cells, Hoechst 33342 accumulation is lower than in WT-MDR1-transfected cells, indicating that mutation of caveolin-binding motif present in MDR1 P-gp affected its transport activity (Fig. 8b).
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ABCB1 p.Phe37Ala 14622130:221:16
status: NEW222 We also observed that P-gp is mainly located in caveolae in Cos-7 cells transfected with either WT-MDR1, YWAA-MDR1 or F37A-MDR1 (Fig. 8c).
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ABCB1 p.Phe37Ala 14622130:222:118
status: NEW248 (a) P-gp and caveolin-1 were immunodetected by western blot analysis in Cos-7 cells untransfected (cells were exposed to FuGENE 6 transfection reagent) or transiently transfected with WT-MDR1, YWAA-MDR1, F37A-MDR1, and f/s-MDR1 (n ¼ 4).
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ABCB1 p.Phe37Ala 14622130:248:204
status: NEW270 (a) Cos7 cells untransfected or transiently transfected with WT-MDR1, YWAA-MDR1, F37A-MDR1, and f/s-MDR1 were lysed in lysis buffer and cell lysate (500 lg of protein) was immunoprecipitated (IP) with 3 lg of mAb directed against P-gp (C219) followed by immunodetection (ID) of caveolin-1 (n ¼ 3).
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ABCB1 p.Phe37Ala 14622130:270:81
status: NEW309 Western blot and FACS analysis show that P-gp and caveolin-1 were expressed at constant levels in WT-MDR1-, YWAA-MDR1-, and F37A-MDR1-transfected cells, excluding the possibility of variation in protein expression.
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ABCB1 p.Phe37Ala 14622130:309:124
status: NEW[hide] Regulation of brain endothelial cells migration an... Biochem Biophys Res Commun. 2008 Aug 1;372(3):440-6. Epub 2008 May 15. Barakat S, Turcotte S, Demeule M, Lachambre MP, Regina A, Baggetto LG, Beliveau R
Regulation of brain endothelial cells migration and angiogenesis by P-glycoprotein/caveolin-1 interaction.
Biochem Biophys Res Commun. 2008 Aug 1;372(3):440-6. Epub 2008 May 15., [PMID:18485890]
Abstract [show]
We have investigated the involvement of P-glycoprotein (P-gp)/caveolin-1 interaction in the regulation of brain endothelial cells (EC) migration and tubulogenesis. P-gp overexpression in MDCK-MDR cells was correlated with enhanced cell migration whereas treatment with P-gp inhibitors CsA or PSC833 reduced it. Transfection of RBE4 rat brain endothelial cells with mutated versions of MDR1, in the caveolin-1 interaction motif, decreased the interaction between P-gp and caveolin-1, enhanced P-gp transport activity and cell migration. Moreover, down-regulation of caveolin-1 in RBE4 cells by siRNA against caveolin-1 stimulated cell migration. Interestingly, the inhibition of P-gp/caveolin-1 interaction increased also EC tubulogenesis. Furthermore, decrease of P-gp expression by siRNA inhibited EC tubulogenesis. These data indicate that the level of P-gp/caveolin-1 interaction can modulate brain endothelial angiogenesis and P-gp dependent cell migration.
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52 After 24 h, the cells were transiently transfected, in serum-free medium, with FuGENE 6 transfection reagent using 1 or 2 lg of DNA (either WT, YWAA or F37A).
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ABCB1 p.Phe37Ala 18485890:52:152
status: NEW132 In the other mutant, F37A, Phe37 (F) was replaced with Ala.
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ABCB1 p.Phe37Ala 18485890:132:21
status: NEW141 The caveolin-1/P-gp interaction was reduced by 25 and 35%, respectively, for the YWAA and F37A mutations (Fig. 3C).
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ABCB1 p.Phe37Ala 18485890:141:90
status: NEW142 Hoechst dye accumulation was decreased by similar amounts 22 and 28%, respectively, for the YWAA and F37A mutant transfected cells compared to the wild-type- transfected cells (Fig. 3D).
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ABCB1 p.Phe37Ala 18485890:142:101
status: NEW161 However, in the case of the mutants YWAA and F37A, the length of the capillary-like tubular structure was increased by about 75% compared to the wild-type MDR1 transfected cells.
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ABCB1 p.Phe37Ala 18485890:161:45
status: NEW186 However, the migration of cells transfected with either YWAA or F37A MDR1 was increased by 75% compared to the wild-type MDR1 transfected cells.
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ABCB1 p.Phe37Ala 18485890:186:64
status: NEW