ABCMdb

PMID: 21152102

Hoelen H, Kleizen B, Schmidt A, Richardson J, Charitou P, Thomas PJ, Braakman I
The primary folding defect and rescue of DeltaF508 CFTR emerge during translation of the mutant domain.
PLoS One. 2010 Nov 30;5(11):e15458., [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCC7 p.Ile539Thr ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu Introduction of either the I539T or G550E suppressor mutation in NBD1 partially rescues DF508 CFTR to the cell surface, but only I539T repaired DF508 NBD1. Login to comment 5 ABCC7 p.Ile539Thr The co-translational rescue of DF508 NBD1 misfolding in CFTR by I539T advocates this domain as the most important drug target for cystic fibrosis. Login to comment 22 ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu Teem and coworkers [19] identified two mutations, G550E and I539T, that both significantly increased plasma membrane levels of DF508 CFTR and improved channel activity [19,20,21]. Login to comment 26 ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu Introduction of I539T, but not the G550E suppressor mutation, counteracted all folding defects within NBD1, whereas both mutations rescue CFTR trafficking to the cell surface. Login to comment 101 ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu Only I539T but not G550E suppresses the DF508 phenotype in NBD1 As the folding defect in DF508 CFTR arose in NBD1, we asked whether this defect could be rescued in NBD1 as well. Login to comment 102 ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu Teem and colleagues identified two suppressor mutations (G550E, I539T) in NBD1 that were located in the same subdomain as F508 (Figure 3B), and that each partially rescued DF508 CFTR from the ER [19]. Login to comment 103 ABCC7 p.Ile539Thr ABCC7 p.Ile539Thr We therefore examined whether the I539T mutation stabilized purified DF508 NBD1 (Figure 3A), and found that I539T completely rescued thermal stability of DF508 NBD1, and improved stability of wild-type NBD1 as well. Login to comment 105 ABCC7 p.Ile539Thr Indeed, thermal stability curves of mouse NBD1s were indistinguishable from those of the corresponding human I539T NBD1s (Figure 3A). Login to comment 106 ABCC7 p.Ile539Thr ABCC7 p.Ile539Thr To establish whether the in vitro suppression of DF508 by I539T leads to improved in vivo stability of NBD1, we expressed wild-type and DF508 NBD1 with or without suppressor mutation I539T in CHO cells, and measured each protein`s half-life (Figure 2E). Login to comment 107 ABCC7 p.Gly550Glu For comparison we included the G550E suppressor mutation. Login to comment 108 ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu The two mutations exerted very different effects in that G550E did not affect stability of wild-type or DF508 NBD1, while I539T measurably stabilized the domain (Figure 3D). Login to comment 109 ABCC7 p.Ile539Thr The amount of DF508 NBD1 remaining after 4 hours of chase improved from 10% to ,60% by insertion of I539T, whereas wild-type NBD1 improved from 30% to ,60%, consistent with the improved melting temperatures we measured. Login to comment 110 ABCC7 p.Ile539Thr We concluded that the I539T suppressor mutation rescues the thermodynamic and biological stability of DF508 NBD1. Login to comment 111 ABCC7 p.Ile539Thr ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu I539T but not G550E fully restores the conformational defect in DF508 NBD1 To establish whether the improved stability of the I539T mutant was due to rescued conformation, we in vitro translated wild-type and DF508 NBD1 with or without suppressor mutations and monitored changes in proteolytic digestion (Figure 4A). Login to comment 112 ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu Again, I539T, but not G550E, caused a dramatic effect on the proteinase K digest, particularly detectable at 5 mg/ml. Login to comment 113 ABCC7 p.Ile539Thr Notably, the I539T mutation restored the wild-type NBD1 pattern in DF508: both protease resistant bands of ,25 and 27 kDa that had been lost in DF508 NBD1 returned upon mutation of I539 to Threonine (Figure 4A, b). Login to comment 114 ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu Comparing longer exposures of the 100 mg/ml ProtK treatment of the NBD1 molecules revealed that only the I539T mutation restored the ,17 kDa protease resistant band that had been lost in DF508 NBD1, whereas G550E had no measurable impact (Figure 4B, N). Login to comment 115 ABCC7 p.Ile539Thr Performing similar limited proteolysis experiments on purified DF508 NBD1 with or without the I539T mutation confirmed that this suppressor mutation specifically restored protease resistance of the 25 and 17 kDa fragments (data not shown). Login to comment 118 ABCC7 p.Ile539Thr ABCC7 p.Ile539Thr The I539T mutation itself slightly decreased electrophoretic mobility of NBD1 and CFTR (not shown), but also of the 25 kDa fragment, suggesting that this fragment contained the I539T mutation. Login to comment 119 ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu We concluded that I539T but not G550E rescues the DF508 phenotype by completely restoring NBD1 conformation and stability. Login to comment 126 ABCC7 p.Ile539Thr ABCC7 p.Ile539Thr A similar experiment was done with I539T CFTR and DF508 I539T CFTR. Login to comment 127 ABCC7 p.Ile539Thr Lane quantitation of the proteolytic fragments after 30 minutes of synthesis showed a protease resistant 25 kDa fragment for both wild-type and DF508 CFTR containing the additional I539T mutation (Figure 5C). Login to comment 128 ABCC7 p.Ile539Thr These results show that the NBD1 conformation that leads to protection from limited proteolysis already formed co-translationally, and that DF508 CFTR was deficient in this process but was rescued by I539T during synthesis. Login to comment 129 ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu Both I539T and G550E partially restore ''band C`` levels of DF508 CFTR. Login to comment 132 ABCC7 p.Ile539Thr We found that only the I539T suppressor restored DF508 NBD1 domain stability suggesting that multiple mechanisms can contribute to rescue of DF508 CFTR. Login to comment 133 ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu To analyze whether the I539T suppressor improved CFTR maturation like G550E [20,21] we used a pulse-chase approach to monitor both rate and efficiency of DF508 CFTR rescue. Login to comment 136 ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu Introducing either G550E or I539T within DF508 CFTR partially countered misfolding and enhanced export from ER to Golgi (Figure 6). Login to comment 137 ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu ABCC7 p.Gly550Glu The I539T suppressor was much more effective than G550E in rescuing DF508 CFTR: the majority of CFTR molecules now reached the Golgi complex, whereas some loss of signal still occurred for G550E, implying some residual degradation. Login to comment 139 ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu We conclude that, while both mutations rescue full-length CFTR to the plasma membrane, the I539T mutation rescues the DF508 phenotype within the isolated NBD1 domain already during its synthesis whereas G550E practically bypasses the folding defect in NBD1 and rescues via an alternative mechanism. Login to comment 143 ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu The I539T suppressor mutation but not G550E in the same subdomain rescued the defect and restored NBD1 conformation to wild-type, Figure 3. Login to comment 144 ABCC7 p.Ile539Thr Stability of DF508 NBD1 is restored by introducing I539T. Login to comment 162 ABCC7 p.Ile539Thr Rescue of NBD1 conformation by the I539T suppressor mutation. Login to comment 163 ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu (A) Wild-type and DF508 NBD1 (top panel) mRNAs containing the G550E (middle panel) or I539T (bottom panel) mutation were in vitro translated in the presence of 35 S-labeled methionine and cysteine and analyzed by 15% SDS-PAGE after proteinase K treatment. Login to comment 165 ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu (B) Longer exposure of the 100 mg/ml proteinase K digest of in vitro translated NBD1, from same experiment as shown in B, showing the rescue of the 17 kDa band by the I539T but not by the G550E mutation. Login to comment 168 ABCC7 p.Ile539Thr The arrowhead indicates the 25 kDa fragment, which has slightly decreased mobility when the I539T mutation is present. Login to comment 190 ABCC7 p.Ile539Thr (C) Similar experimental conditions as in B but with the I539T mutation in wild-type and DF508 CFTR background. Login to comment 201 ABCC7 p.Ile539Thr Both 25 kDa and 17 kDa fragments did show increased protease susceptibility in the DF508 background, which was rescued completely by the I539T suppressor mutation, not only in terms of protease susceptibility but also its in vivo stability and function. Login to comment 203 ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu Reverting the DF508 phenotype While the I539T mutation rescued NBD1, G550E hardly affected the isolated DF508 NBD1 domain. Login to comment 210 ABCC7 p.Gly550Glu Where G550E does not measurably rescue NBD1 misfolding, it does rescue CFTR functioning and therefore is likely to rescue at least one of the downstream domain assembly steps. Login to comment 211 ABCC7 p.Gly550Glu The G550E mutation is located in the highly conserved ABC transporter signature motif of NBD1 (LSG550 GQ) and, according to the Sav1866 homology model [8], is in close proximity to NBD2. Login to comment 213 ABCC7 p.Gly550Glu While G550E only slightly increases steady state plasma membrane levels of DF508 CFTR, it does enhance CFTR mediated chloride currents [19] and restores CFTR channel activity by increasing the duration of channel opening and thereby open probability (Po) [20]. Login to comment 214 ABCC7 p.Ile539Thr The I539T mutation, by contrast, increases chloride channel activity at the plasma membrane (Eric J. Sorscher, Birmingham, personal communication) by increasing the number of CFTR molecules at the cell surface [19] (and Gergely Lukacs, Toronto, personal communication). Login to comment 216 ABCC7 p.Ile539Thr The crystal structure of NBD1 and the structural model of CFTR provides no obvious hint towards the molecular mechanism of rescue by the I539T mutation. Login to comment 239 ABCC7 p.Ile539Thr ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu ABCC7 p.Gly550Glu The deletion of F508 and the reverting mutations G550E and I539T were introduced both in full-length CFTR and NBD1 by side directed mutagenesis using primers (amino acid change underlined, I539T: 59-CCAAGTTTGCAGAGAAAGACAATACCG- TTCTTGGAGAAGGTGGAATC-39 G550E: 59-GGAGAAGG- TGGAATCACACTGAGTGAGGGTCAACGAGCAAGAATT- TCTTTAGC-39 DF508: 59-GGCACCATTAAAGAAAATATC- ATTGGTGTTTCCTATGATGAATATAG-39) and all constructs were sequence verified. Login to comment