ABCMdb

PMID: 23865422

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., [PubMed]

Sentences

No. Mutations Sentence Comment

17 ABCC7 p.Gly126Asp ABCC7 p.Val232Asp ABCC7 p.Phe337Arg ABCC7 p.Ser1141Arg 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). Login to comment 18 ABCC7 p.Gly126Asp ABCC7 p.Val232Asp ABCC7 p.Phe337Arg ABCC7 p.Ser1141Arg 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. Login to comment 20 ABCC7 p.Gly126Asp ABCC7 p.Val232Asp ABCC7 p.Ser1141Arg Figure 2B shows that full-length and ƊNBD2 forms of G126D, V232D, and S1141R could be efficiently rescued with all the correctors such that the mature protein was the major product (Figure 2C,D). Login to comment 21 ABCC7 p.Phe337Arg By contrast, only VX-809 promoted maturation of mutant F337R (Figure 2B). Login to comment 22 ABCC7 p.Phe337Arg ABCC7 p.Phe337Arg The bithiazoles did not promote maturation of the F337R ƊNBD2 or full-length F337R CFTR (Figure 2C,D). Login to comment 24 ABCC7 p.Arg553Gln ABCC7 p.Gly550Glu ABCC7 p.Arg555Lys ABCC7 p.Phe494Asn It should be noted, however, that the ƊNBD2 CFTR used in this study was different from that used by Okiyoneda et al.18 The ƊNBD2 CFTR constructs in our study did not contain the G550E, R553Q, R555K, and F494N mutations or the three hemagglutinin tags in the fourth extracellular loop that could influence CFTR corrector interactions. Login to comment 25 ABCC7 p.Gly126Asp ABCC7 p.Val232Asp ABCC7 p.Phe337Arg ABCC7 p.Ser1141Arg 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. Login to comment 26 ABCC7 p.Phe337Arg 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. Login to comment