ABCB1 p.Phe804Ala
| Predicted by SNAP2: | A: D (85%), C: D (80%), D: D (95%), E: D (91%), G: D (91%), H: D (71%), I: D (85%), K: D (95%), L: D (85%), M: D (71%), N: D (91%), P: D (95%), Q: D (91%), R: D (91%), S: D (91%), T: D (91%), V: D (85%), W: D (75%), Y: N (82%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: N, |
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[hide] Thapsigargin or curcumin does not promote maturati... Biochem Biophys Res Commun. 2004 Dec 10;325(2):580-5. Loo TW, Bartlett MC, Clarke DM
Thapsigargin or curcumin does not promote maturation of processing mutants of the ABC transporters, CFTR, and P-glycoprotein.
Biochem Biophys Res Commun. 2004 Dec 10;325(2):580-5., [PMID:15530432]
Abstract [show]
Misprocessed plasma membrane proteins of CFTR and P-glycoprotein (P-gp) are retained in the endoplasmic reticulum (ER) by molecular chaperones. Depletion of the calcium stores in the ER by the SERCA calcium pump inhibitors thapsigargin or curcumin inhibits these interactions and allows the protein to be trafficked to the plasma membrane [Nat. Med. 8 (2002) 485; Science 304 (2004) 600]. We tested this hypothesis by treating various cell lines expressing misprocessed mutants of CFTR or P-gp with thapsigargin or curcumin. Conversion of the immature core-glycosylated protein to mature product was detected by immunoblot analysis of whole cell extracts. Mature product was not detected in any of the misprocessed mutants. By contrast, all misprocessed P-gp mutants were rescued by the chemical chaperone/drug substrate cyclosporin A in a dose-dependent manner. These results show that thapsigargin or curcumin is not effective in rescuing misprocessed mutants of P-gp and CFTR.
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No. Sentence Comment
58 Mutations G251V and F804A are in the intracellular loops of the NH2- and COOH-terminal halves of P-gp, respectively.
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ABCB1 p.Phe804Ala 15530432:58:20
status: NEW70 Although the processing mutations (G251V, G300V, DY490, P709A, G722A, F804A, and P1194A) are located in different segments of P-gp, all the mutants could be rescued when expressed with drug substrates such as cyclosporin A.
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ABCB1 p.Phe804Ala 15530432:70:70
status: NEW90 We then compared the abilities of cyclosporin A, thapsigargin, and curcumin to induce maturation of P-gp processing mutants G251V, G300V, DY490, P709A, G722A, F804A, and P1194A.
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ABCB1 p.Phe804Ala 15530432:90:159
status: NEW[hide] Locking intracellular helices 2 and 3 together ina... J Biol Chem. 2014 Jan 3;289(1):229-36. doi: 10.1074/jbc.M113.527804. Epub 2013 Nov 25. Loo TW, Clarke DM
Locking intracellular helices 2 and 3 together inactivates human P-glycoprotein.
J Biol Chem. 2014 Jan 3;289(1):229-36. doi: 10.1074/jbc.M113.527804. Epub 2013 Nov 25., [PMID:24275649]
Abstract [show]
The P-glycoprotein (P-gp) drug pump (ABCB1) has two transmembrane domains and two nucleotide-binding domains (NBDs). Coupling of the drug-binding sites in the transmembrane domains to the NBDs occurs through interaction of the intracellular helices (IHs) with residues in the NBDs (IH1/IH4/NBD1 and IH2/IH3/NBD2). We showed previously that cross-linking of cysteines in IH3 and IH1 with a short cross-linker mimicked drug binding as it activated P-gp ATPase activity. Here we show that residue A259C(IH2) could be directly cross-linked to W803C(IH3). Cross-linking was inhibited by the presence of ATP and adenosine 5'-(beta,gamma-imino)triphosphate but not by ADP. Cross-linking of mutant A259C/W803C inhibited its verapamil-stimulated ATPase activity mutant, but activity was restored after addition of dithiothreitol. Because these residues are close to the ball-and-socket joint A266C(IH2)/Phe(1086)(NBD2), we mutated the adjacent Tyr(1087)(NBD2) close to IH3. Mutants Y1087A and Y1087L, but not Y1087F, were misprocessed, and all inhibited ATPase activity. Mutation of hydrophobic residues (F793A, L797A, L814A, and L818A) flanking IH3 also inhibited maturation. The results suggest that these residues, together with Trp(803) and Phe(804), form a large hydrophobic pocket. The results show that there is an important hydrophobic network at the IH2/IH3/NBD2 transmission interface that is critical for folding and activity of P-gp.
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No. Sentence Comment
79 It was Clamping IH2 to IH3 Inhibits P-gp 230 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289ߦNUMBER 1ߦJANUARY 3, found that two mutations in IH3 (W803A and F804A) inhibited maturation of P-gp (Fig. 1, B and C) so that immature 150-kDa P-gp was the major product.
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ABCB1 p.Phe804Ala 24275649:79:165
status: NEW83 Because W803A and F804A mutations in IH3 yielded the immature 150-kDa immature P-gp as the major product, we tested whether the mutants could be rescued with cyclosporine A.
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ABCB1 p.Phe804Ala 24275649:83:18
status: NEW85 Accordingly, the A52-tagged mutants W803A and F804A were expressed in HEK 293 cells in the absence or presence of 5 òe;M cyclosporine A, and whole cell SDS extracts were subjected to immunoblot analysis. Fig. 2A shows that expression of either W803A or F804A in the presence of cyclosporine A yielded the mature 170-kDa P-gp as the major product.
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ABCB1 p.Phe804Ala 24275649:85:46
status: NEWX
ABCB1 p.Phe804Ala 24275649:85:257
status: NEW89 To determine whether the W803A and F804A mutants rescued with cyclosporine A were active, the histidine-tagged mutants W803A and F804A were expressed in HEK 293 cells in the presence of cyclosporine A, and P-gp was isolated by nickel-chelate chromatography.
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ABCB1 p.Phe804Ala 24275649:89:35
status: NEWX
ABCB1 p.Phe804Ala 24275649:89:129
status: NEW101 Drug rescue of processing mutants and activity of IH3 mutants W803A and F804A.
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ABCB1 p.Phe804Ala 24275649:101:72
status: NEW102 A52-tagged WT P-gp, mutants W803A and F804A (A), or IH3 flanking mutants (F793A, L797A, L814A, and L818A) (B) were expressed in HEK293cellsintheabsence(afa;)orpresence(af9;)of5òe;M cyclosporineA(Cyclo) for 18 h, and whole cell extracts were subjected to immunoblot analysis.
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ABCB1 p.Phe804Ala 24275649:102:38
status: NEW103 The positions of mature (170-kDa) and immature (150-kDa) forms of P-gp are shown. C, verapamil-stimulated ATPase activities of histidine-tagged wild-type P-gp and W803A and F804A mutants.
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ABCB1 p.Phe804Ala 24275649:103:173
status: NEW109 Mutating Phe804 to Leu, Ser, or Asp inhibited maturation, and only the F804A or F804S mutants could be rescued with cyclosporine A to yield mature 170-kDa P-gp as the major product (Fig. 3, C and D).
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ABCB1 p.Phe804Ala 24275649:109:71
status: NEW