ABCMdb

PMID: 19625452

Grove DE, Rosser MF, Ren HY, Naren AP, Cyr DM
Mechanisms for rescue of correctable folding defects in CFTRDelta F508.
Mol Biol Cell. 2009 Sep;20(18):4059-69. Epub 2009 Jul 22., [PubMed]

Sentences

No. Mutations Sentence Comment

10 ABCC7 p.Val232Asp In contrast, misfolding caused by the rare CF-causing mutation V232D in MSD1 was highly correctable by Corr-4a. Login to comment 183 ABCC7 p.Asn1303Lys ABCC7 p.Gly85Glu ABCC7 p.Gly91Arg ABCC7 p.Val232Asp ABCC7 p.Met1137Arg Thus, we asked to what extent can disease related folding defects caused by mutations in MSD1 (G85E, G91R, and V232D), MSD2 (M1137R), and NBD2 (N1303K) be corrected relative to those caused by deletion of F508 in NBD1 (Figure 4A). Login to comment 184 ABCC7 p.Asn1303Lys ABCC7 p.Gly85Glu ABCC7 p.Gly91Arg ABCC7 p.Met1137Arg The G85E, G91R, M1137R, and N1303K mutations all hinder folding of the nascent B-form of CFTR via a mechanism that involves insertion of a charged amino acid into an inappropriate region (Gregory et al., 1991; Xiong et al., 1997; Vankeerberghen et al., 1998). Login to comment 185 ABCC7 p.Val232Asp The V232D mutation has been proposed to introduce an abnormal hydrogen bond within the transmembrane (TM) region of MSD1, which impedes normal TM assembly (Therien et al., 2001), but the affect of this mutation on CFTR biogenesis is unknown. Login to comment 186 ABCC7 p.Asn1303Lys The effect of Corr-4a on the NBD2 mutant N1303K was similar to that observed with CFTR⌬F508 in that a small degree of correction was observed (Figure 4A). Login to comment 187 ABCC7 p.Asn1303Lys The levels of the immature form of CFTR N1303K were slightly elevated while accumulation of the mature form was enhanced upon addition of Corr-4a. Login to comment 189 ABCC7 p.Gly85Glu ABCC7 p.Gly91Arg ABCC7 p.Val232Asp The CFTR G85E and G91R point mutations are contained within TM1, whereas the V232D mutation lies within TM4 of CFTR`s MSD1 domain. Login to comment 190 ABCC7 p.Met1137Arg The CFTR M1137R mutation exists within TM12 of CFTR`s MSD2 domain. Login to comment 191 ABCC7 p.Gly85Glu The addition of Corr-4a slightly increased the steady-state levels of CFTR G85E B-form, but Corr-4a-dependent folding of this mutant was not detected. Login to comment 192 ABCC7 p.Gly91Arg ABCC7 p.Gly91Arg ABCC7 p.Met1137Arg ABCC7 p.Met1137Arg Furthermore, chemical treatment of CFTR G91R or CFTR M1137R did not significantly affect the accumulation of the immature B-form; however, the mature C-forms of both CFTR G91R and CFTR M1137R were apparent. Login to comment 194 ABCC7 p.Val232Asp Intriguingly, introduction of the V232D mutation generates an unstable CFTR biogenic mutant with decreased levels of the immature B-form and no apparent maturation product (Figure 4A). Login to comment 195 ABCC7 p.Val232Asp Yet, treatment with Corr-4a resulted in a dramatic increase in accumulation of both the immature and mature forms of CFTR V232D. Login to comment 196 ABCC7 p.Val232Asp However, folding defects in CFTR V232D were not significantly rescued by low temperature incubations (data not shown). Login to comment 197 ABCC7 p.Val232Asp Pulse-chase analysis indicated that Corr-4a strongly increases the folding efficiency of CFTR V232D, and does not simply increase the synthesis of this point mutant (Figure 4B). Login to comment 198 ABCC7 p.Val232Asp The ability of Corr-4a to dramatically enhance CFTR V232D folding suggests that this mutant may have a single folding defect in MSD assembly that is correctable by Corr-4a. Login to comment 199 ABCC7 p.Val232Asp ABCC7 p.Val232Asp In agreement with this idea, Corr-4a is able to enhance the accumulation of the V232D-containing MSD1 fragment, CFTR 370X V232D (data not shown). Login to comment 209 ABCC7 p.Val232Asp (B) Corr-4a increases the biosynthetic maturation of CFTR V232D. Login to comment 210 ABCC7 p.Val232Asp HEK293 cells transfected with CFTR V232D (1 ␮g) were preincubated with Corr-4a or DMSO for 2 h, labeled with [35 S]methionine, and chased for the indicated amounts of time in the continuous presence of chemical treatment. Login to comment 227 ABCC7 p.Val232Asp Next, we probed the ability of Corr-4a to enhance the proper association of CFTR 837X V232D with CFTR 837-1480. Login to comment 228 ABCC7 p.Val232Asp We observed that, when compared with the wild-type 837X fragment, the steady-state levels of CFTR 837X V232D were decreased (Figure 5B). Login to comment 229 ABCC7 p.Val232Asp Yet, the levels of CFTR 837X V232D were increased upon Corr-4a treatment. Login to comment 230 ABCC7 p.Val232Asp Furthermore, pulse-chase analysis demonstrated that Corr-4a stabilizes CFTR 837X V232D (Figure 5C). Login to comment 231 ABCC7 p.Val232Asp In addition, the levels of the C-terminal fragment, 837-1480, were increased when coexpressed with the CFTR 837X V232D mutant fragment (Figure 5D). Login to comment 233 ABCC7 p.Val232Asp Yet, when Corr-4a was present, a large subpopulation of the 837X V232D and 837- Figure 5. Login to comment 238 ABCC7 p.Val232Asp (B) HEK293 cells transfected individually with CFTR 837X (1 ␮g), CFTR 837X⌬F508 (1 ␮g), CFTR 837X V232D (1 ␮g), or CFTR 837-1480 (1 ␮g) were cultured for 18 h before addition of Corr-4a or DMSO. Login to comment 242 ABCC7 p.Val232Asp (C) Corr-4a increases the stability of CFTR 837X V232D and CFTR 837-1480. Login to comment 243 ABCC7 p.Val232Asp HEK293 cells transfected with either CFTR 837X V232D or CFTR 837-1480 were preincubated with Corr-4a or DMSO for 2 h, labeled with [35 S]methionine, and chased for the indicated amounts of time in the continuous presence of chemical treatment. Login to comment 247 ABCC7 p.Val232Asp (D) HEK293 cells were transfected with either CFTR 837-1480, CFTR 837X and CFTR 837-1480, CFTR 837X⌬F508 and CFTR 837-1480, or CFTR 837X V232D and CFTR 837-1480. Login to comment 254 ABCC7 p.Val232Asp Thus, Corr-4a is able to correct defects in MSD1 folding to the degree that it facilitates the proper assembly of CFTR 837X V232D and CFTR 837-1480. Login to comment 257 ABCC7 p.Val232Asp Yet, it appears that multiple folding defects limit the rescue of CFTR⌬F508, whereas a less complex mutant, such as CFTR V232D, is readily rescued. Login to comment 261 ABCC7 p.Val510Asp ABCC7 p.Val510Asp ABCC7 p.Val510Asp Indeed, introduction of the V510D suppressor point mutation into CFTR⌬F508 partially restored folding of some CFTR⌬F508 V510D (Wang et al., 2007), but the majority of CFTR⌬F508 V510D accumulated in its B-form (Figure 6A), and the level of repair remained below the observed levels of folded wild-type CFTR (compare to CFTR panel in Figure 3B). Login to comment 262 ABCC7 p.Val510Asp Corr-4a was able to enhance the accumulation of the folded C-form of CFTR⌬F508 V510D (Figure 6A). Login to comment 263 ABCC7 p.Val510Asp Pulse-chase analysis indicated that Corr-4a stabilizes the B-form and enhances the folding efficiency of CFTR⌬F508 V510D (Figure 6B). Login to comment 265 ABCC7 p.Val510Asp Overall, the V510D suppressor mutation and Corr-4a appear to act in an additive manner to restore the folding of CFTR⌬F508 toward wild-type levels. Login to comment 268 ABCC7 p.Val510Asp To test this model, we asked if the V510D suppressor mutation permits Corr-4a to correct the misfolding of CFTR 1172X⌬F508. Login to comment 269 ABCC7 p.Val510Asp ABCC7 p.Val510Asp Suppression of CFTR 1172X⌬F508 misfolding upon introduction of the V510D mutation was similar to the folding correction observed with CFTR⌬F508 V510D (Figure 6A). Login to comment 270 ABCC7 p.Val510Asp Corr-4a was now able to promote the proper folding of a small pool of CFTR 1172X⌬F508 V510D and enhance the accumulation of its maturely glycosylated C-form. Login to comment 271 ABCC7 p.Val510Asp However, again the level of correction was low and an increase in the folding efficiency of CFTR 1172X⌬F508 V510D was not detected in pulse-chase experiments (Figure 6B). Login to comment 276 ABCC7 p.Val510Asp ABCC7 p.Val510Asp (A) HEK293 cells were transfected with 1 ␮g of either CFTR⌬F508, CFTR⌬F508 V510D, CFTR 1172X⌬F508, or CFTR 1172X⌬F508 V510D. Login to comment 309 ABCC7 p.Val232Asp Corr-4a is also able to act on CFTR 837X V232D, and enhance its accumulation. Login to comment 312 ABCC7 p.Val510Asp Yet, Corr-4a is significantly more effective at promoting the folding of CFTR⌬F508 V510D than CFTR⌬F508. Login to comment 313 ABCC7 p.Val510Asp The V510D mutant is designed to facilitate interactions between NBD1 and MSD2 that are defective when F508 is deleted (Mornon et al., 2008). Login to comment 316 ABCC7 p.Gly91Arg ABCC7 p.Met1137Arg Corr-4a-dependent rescue of MSD biogenic mutants G91R and M1137R was no greater than that observed with corrector-treated CFTR⌬F508. Login to comment 317 ABCC7 p.Gly85Glu Furthermore, the folding defect of the MSD1 mutant G85E appeared to be largely noncorrectable by Corr-4a. Login to comment 318 ABCC7 p.Val232Asp Surprisingly, there was a dramatic enhancement of CFTR V232D folding by Corr-4a. Login to comment 319 ABCC7 p.Val232Asp ABCC7 p.Val232Asp In addition, Corr-4a was able to enhance the accumulation of CFTR 837X V232D and to increase levels of the mature, highly glycosylated form of CFTR 837-1480 with CFTR 837X V232D. Login to comment 320 ABCC7 p.Val232Asp The V232D mutant presents a mild form of CF (Alonso et al., 2007); however, the molecular basis for CF associated with this mutation has not been well-defined. Login to comment 321 ABCC7 p.Val232Asp The V232D mutation is proposed to form an aberrant hydrogen bond in MSD1 (Therien et al., 2001; Choi et al., 2004) that may hinder formation of interdomain contacts between MSD1 and MSD2 that are proposed to occur in the folded CFTR channel (Dawson and Locher, 2006; Serohijos et al., 2008). Login to comment 325 ABCC7 p.Val232Asp Yet, rare mutations, such as V232D, which cause more specific folding defects (Therien et al., 2001; Choi et al., 2004) are easier to correct. Login to comment