ABCC7 p.Phe337Arg
| Predicted by SNAP2: | A: D (71%), C: D (66%), D: D (91%), E: D (91%), G: D (75%), H: D (75%), I: D (71%), K: D (91%), L: D (53%), M: D (66%), N: D (85%), P: D (91%), Q: D (85%), R: D (85%), S: D (85%), T: D (85%), V: D (75%), W: D (85%), Y: D (71%), |
| 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] Corrector VX-809 stabilizes the first transmembran... Biochem Pharmacol. 2013 Sep 1;86(5):612-9. doi: 10.1016/j.bcp.2013.06.028. Epub 2013 Jul 5. Loo TW, Bartlett MC, Clarke DM
Corrector VX-809 stabilizes the first transmembrane domain of CFTR.
Biochem Pharmacol. 2013 Sep 1;86(5):612-9. doi: 10.1016/j.bcp.2013.06.028. Epub 2013 Jul 5., [PMID:23835419]
Abstract [show]
Processing mutations that inhibit folding and trafficking of CFTR are the main cause of cystic fibrosis (CF). A potential CF therapy would be to repair CFTR processing mutants. It has been demonstrated that processing mutants of P-glycoprotein (P-gp), CFTR's sister protein, can be efficiently repaired by a drug-rescue mechanism. Many arginine suppressors that mimic drug-rescue have been identified in the P-gp transmembrane (TM) domains (TMDs) that rescue by forming hydrogen bonds with residues in adjacent helices to promote packing of the TM segments. To test if CFTR mutants could be repaired by a drug-rescue mechanism, we used truncation mutants to test if corrector VX-809 interacted with the TMDs. VX-809 was selected for study because it is specific for CFTR, it is the most effective corrector identified to date, but it has limited clinical benefit. Identification of the VX-809 target domain will help to develop correctors with improved clinical benefits. It was found that VX-809 rescued truncation mutants lacking the NBD2 and R domains. When the remaining domains (TMD1, NBD1, TMD2) were expressed as separate polypeptides, VX-809 only increased the stability of TMD1. We then performed arginine mutagenesis on TM6 in TMD1. Although the results showed that TM6 had distinct lipid and aqueous faces, CFTR was different from P-gp as no arginine promoted maturation of CFTR processing mutants. The results suggest that TMD1 contains a VX-809 binding site, but its mechanism differed from P-gp drug-rescue. We also report that V510D acts as a universal suppressor to rescue CFTR processing mutants.
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No. Sentence Comment
168 Examples of the different effects of the arginines such as no effect (I344R), small reduction in maturation (V345R), large reduction in maturation (F337R), and no maturation (L346R) are shown in Fig. 4B. The TM6 arginine mutagenesis results were projected on a helical wheel (Fig. 4C).
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ABCC7 p.Phe337Arg 23835419:168:148
status: NEW204 (B) Examples of immunoblots of wild-type CFTR and mutants I344R, V345R, F337R, and L346R showing the various effects of arginines introduced into TM6.
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ABCC7 p.Phe337Arg 23835419:204:72
status: NEW[hide] Bithiazole correctors rescue CFTR mutants by two d... Biochemistry. 2013 Aug 6;52(31):5161-3. doi: 10.1021/bi4008758. Epub 2013 Jul 22. Loo TW, Bartlett MC, Clarke DM
Bithiazole correctors rescue CFTR mutants by two different mechanisms.
Biochemistry. 2013 Aug 6;52(31):5161-3. doi: 10.1021/bi4008758. Epub 2013 Jul 22., [PMID:23865422]
Abstract [show]
Better correctors are needed to repair cystic fibrosis transmembrane conductance regulator (CFTR) processing mutants that cause cystic fibrosis. Determining where the correctors bind to CFTR would aid in the development of new correctors. A recent study reported that the second nucleotide-binding domain (NBD2) was involved in binding of bithiazole correctors. Here, we show that bithiazole correctors could also rescue CFTR mutants that lacked NBD2. These results suggest that bithiazoles rescue CFTR mutants by two different mechanisms.
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No. Sentence Comment
17 VX-809 appears to rescue CFTR processing mutants through direct interactions with TMD1.12,18 Bithiazoles, however, appear to rescue CFTR processing mutants by a different mechanism because bithiazoles have an additive effect on maturation when used in combination with VX-809.15 Identification of the bithiazole rescue site in CFTR is controversial as there is evidence that these compounds bind to NBD218 or to the TMDs.9 Evidence that bithiazoles interact with the TMDs was that 4a inhibited cross-linking between cysteines introduced into TMD1 and TMD219 and that 4a appears to stabilize TMD2.20 In addition, it was found that bithiazoles enhanced core glycosylation of a truncation mutant that contained only the TMDs.19 By contrast, it was recently reported that bithiazoles must interact with NBD2 because ƊF508 CFTR missing NBD2 was not rescued with bithiazoles and in silico docking predicted the presence of a bithiazole-binding site in NBD2.18 To test whether rescue of other CFTR processing mutants with bithiazoles (see Figure 1 for structures) was mediated by the TMDs or NBD2, we tested if bithiazoles could rescue full-length or ƊNBD2 CFTR mutants that had processing mutations in the TMDs such as G126D in TM2,21 V232D in Received: July 3, 2013 Revised: July 17, 2013 Published: July 18, 2013 Rapid Report pubs.acs.org/biochemistry (c) 2013 American Chemical Society dx.doi.org/10.1021/bi4008758 | Biochemistry 2013, 52, 5161-5163 Terms of Use TM4,20 F337R in TM6,12 and S1141R in TM12 (see Figure 2A).
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ABCC7 p.Phe337Arg 23865422:17:1479
status: NEW18 Mutants G126D and V232D are naturally occurring CF mutants, whereas F337R and S1141R mutants were constructed to map the orientation of the TM segments.12 The mutants were expressed for 18 h with or without VX-809 or the 4a, 4d, or 15Jf bithiazole correctors.
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ABCC7 p.Phe337Arg 23865422:18:68
status: NEW21 By contrast, only VX-809 promoted maturation of mutant F337R (Figure 2B).
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ABCC7 p.Phe337Arg 23865422:21:55
status: NEW22 The bithiazoles did not promote maturation of the F337R ƊNBD2 or full-length F337R CFTR (Figure 2C,D).
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ABCC7 p.Phe337Arg 23865422:22:50
status: NEWX
ABCC7 p.Phe337Arg 23865422:22:82
status: NEW25 The second bithiazole rescue mechanism appears to involve the TMDs because removal of NBD2 had little effect on bithiazole rescue of mutants G126D, V232D, and S1141R and mutation F337R in TM6 inhibited rescue of full-length CFTR with bithiazoles.
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ABCC7 p.Phe337Arg 23865422:25:179
status: NEW26 Other pieces of evidence suggesting the bithiazoles affect folding of the TMDs were the observations that 4a blocked cross-linking between cysteines introduced into TM segments 6 and 1219 and 15Jf promoted core glycosylation of a CFTR truncation mutant consisting of TMD1 and TMD2.9 In addition, it was reported that 4a appeared to stabilize TMD2.20 VX-809 and bithiazoles appear to interact at different sites in the TMDs as the F337R mutant only prevented rescue with bithiazoles.
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ABCC7 p.Phe337Arg 23865422:26:430
status: NEW