ABCMdb

PMID: 20233947

He L, Aleksandrov LA, Cui L, Jensen TJ, Nesbitt KL, Riordan JR
Restoration of domain folding and interdomain assembly by second-site suppressors of the DeltaF508 mutation in CFTR.
FASEB J. 2010 Aug;24(8):3103-12. Epub 2010 Mar 16., [PubMed]

Sentences

No. Mutations Sentence Comment

27 ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys These suppressor mutations (I539T, G550E, R553M/Q, and R555K) promote ⌬F508-CFTR maturation and trafficking to the cell surface, and also restore channel activity (16). Login to comment 28 ABCC7 p.Arg555Lys The R555K mutation alone increases the channel activity of both WTand ⌬F508-CFTR by extending the open-channel burst duration (15). Login to comment 29 ABCC7 p.Gly550Glu In addition to a similar effect, G550E also decreases the interburst interval of the ⌬F508-CFTR channel, such that its open probability is higher than that of WT-CFTR (17). Login to comment 72 ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys RESULTS Suppressor mutations restore folding mutations in NBD1 but not elsewhere Four suppressor mutations (I539T, G550E, R553M, and R555K) were originally found to rescue ⌬F508-CFTR maturation in a yeast mating screen using STE6/CFTR chimeras (14-16). Login to comment 79 ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys ABCC7 p.Arg555Lys B, C) HEK293 cells were transiently transfected with CFTR variants with maturation-compromising mutations introduced in different domains, in the presence or absence of single or combined suppressor mutations (4S: I539T/G550E/R4553M/R555K; 3S: G550E/R553M/R555K). Login to comment 84 ABCC7 p.Arg553Met ABCC7 p.Arg555Lys Among these four suppressor mutations, R555K had the largest effect in promoting ⌬F508-CFTR maturation, while R553M had the least effect. Login to comment 85 ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys The addition of G550E and R553M to R555K (3S) further increased its maturation, but no additional effect was detected by the addition of the fourth mutation I539T (4S) (Fig. 1A). Login to comment 89 ABCC7 p.Arg560Thr To test this hypothesis, we introduced suppressor mutations into another NBD1-processing mutant, R560T (17). Login to comment 91 ABCC7 p.Arg560Thr ABCC7 p.Gly550Glu As shown in Fig. 1B, R560T-CFTR, although not rescued by G550E alone (17), was very effectively rescued by the 4S combination. Login to comment 92 ABCC7 p.Arg560Thr Interestingly, while lowering the temperature alone did not promote R560T-CFTR maturation, it augmented the extent of rescue by 4S (Fig. 1C). Login to comment 95 ABCC7 p.Arg1070Trp Indeed, some substitutions on the CL4 side of this interface such as R1070W have been reported to improve ⌬F508-CFTR maturation (23). Login to comment 97 ABCC7 p.Leu1065Pro ABCC7 p.Arg1066Cys Therefore, we tested whether the suppressor mutations could also reverse the processing defects caused by disease-associated mutations (R1066C and L1065P; ref. 24) on the CL4 side of the NBD1/CL4 interface. Login to comment 98 ABCC7 p.Leu1065Pro ABCC7 p.Arg1066Cys Introduction of the 4S mutations into R1066C or the 3S mutations into L1065P did not promote CFTR maturation (Fig. 1B). Login to comment 101 ABCC7 p.Asn1303Lys In yet another variant harboring N1303K, which is the only known mutation in NBD2 that impairs CFTR maturation, the introduction of suppressor mutations did not increase the proportion of the mature form either. Login to comment 102 ABCC7 p.Asn1303Lys However, the suppressors did result in slightly increased amounts of both mature and immature N1303K-CFTR, and this effect was enhanced at reduced temperature (Fig. 1B, C). Login to comment 112 ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys Stable BHK cells overexpressing WT-CFTR and ⌬F508-CFTR with and without 4 suppressor mutations (I539T/G550E/R553M/R555K, ⌬F/ 4S) were pulse labeled with 100 ␮Ci/ml [35 S] methionine for 20 min, followed by 0, 1, 2, and 4 h chase. Login to comment 122 ABCC7 p.Gln1280Cys ABCC7 p.Lys1284Cys As shown in Fig. 3A, when ⌬F508 was introduced into Cys-less CFTR containing pairs 276C/Q1280C or 276C/K1284C at the NBD2/CL2 interface, no mature CFTR was detected, and neither M3M nor M8M (200 ␮M) caused cross-linking of the immature band. Login to comment 123 ABCC7 p.Val510Cys ABCC7 p.Gly1069Cys ABCC7 p.Gly1069Cys ABCC7 p.Lys564Cys Cys- pair cross-linking also was not observed with Cys pairs at V510C/G1069C and K564C/G1069C in the NBD1/ CL4 interface of ⌬F508-CFTR (Fig. 3B). Login to comment 124 ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys To test whether these NBD/CL interfaces not formed in ⌬F508-CFTR could be restored by the suppressor mutations, the 4 combined suppressor mutations, I539T/G550E/R553M/R555K (4S) were introduced into the ⌬F508-CFTR constructs with the Cys pairs at the potential interfaces. Login to comment 125 ABCC7 p.Gln1280Cys ABCC7 p.Lys1284Cys As shown in Fig. 3A, ⌬F508-CFTR maturation was restored as was the cross-linking of the Cys pairs 276C/Q1280C and 276C/ K1284C at NBD2/CL2, as indicated by the slower migration rate of the mature bands in SDS-PAGE in samples treated with M3M and M8M (18). Login to comment 126 ABCC7 p.Val510Cys ABCC7 p.Gly1069Cys ABCC7 p.Gly1069Cys ABCC7 p.Lys564Cys In addition, Cys pairs V510C/G1069C and K564C/G1069C at the NBD1/CL4 interface also were able to be cross-linked by M3M and M8M (Fig. 3B). Login to comment 128 ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu The rescue of ⌬F508 by suppressor mutations is diminished in the absence of NBD2 According to a structural model of CFTR (18), some of the suppressor mutations (I539T and G550E) are located at the NBD1/NBD2 interface (Fig. 1A), and ⌬F508 is known to destabilize NBD2 (6, 7). Login to comment 139 ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys HEK293 cells were transiently transfected with Cys-less ⌬F508-CFTR in the presence or absence of suppressor mutations I539T/G550E/R553M/R555K, with Cys pairs introduced at CL2/NBD2 (A) or CL4/NBD1 (B) interfaces. Login to comment 146 ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys However, when suppressor mutations (3S: G550E/R553M/R555K) were introduced into the N-half ⌬F508-CFTR, they did not promote complex glycosylation of the C half (Fig. 5A, lane 4), as they did in full-length CFTR (Fig. 1). Login to comment 154 ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys ABCC7 p.Arg555Lys HEK293 cells were transiently transfected with 1172X-CFTR or ⌬F508-1172X-CFTR in the presence or absence of single or combined suppressor mutations (4S: I539T/G550E/R4553M/R555K; 3S: G550E/R553M/R555K). Login to comment 162 ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys N halves were either WT or carried the ⌬F508 mutation, in the absence or presence of suppressor mutations (3S: G550E/R553M/R555K). Login to comment 181 ABCC7 p.Val510Cys Using this assay, we compared the accessibility of 491C and V510C in NBD1 of full-length CFTR in the absence or presence of ⌬F508. Login to comment 202 ABCC7 p.Val510Cys In contrast to the buried 491C, V510C was clearly labeled in both immature and mature forms of WT-CFTR (lane 5). Login to comment 205 ABCC7 p.Val510Cys ABCC7 p.Gly1069Cys This may reflect the involvement of this loop in the NBD1/CL4 interface, as shown with the cross-linking of V510C/ G1069C in the mature but not in the immature form (18). Login to comment 206 ABCC7 p.Val510Cys In contrast to the more buried 491C, introduction of ⌬F508 did not make V510C more accessible to labeling compared to the immature WT-CFTR (lane 6). Login to comment 207 ABCC7 p.Val510Cys Introduction of the suppressor mutations did not appear to reduce exposure of V510C in the immature form, as it had the more buried 491C. Login to comment 208 ABCC7 p.Val510Cys However, the accessibility of V510C in the mature form, which was restored by the suppressors, was similar to that in the WT-CFTR (lane 7). Login to comment