Background: Mutations in nucleotide binding domain 1 of cystic fibrosis (CF) transmembrane conductance regulator cause severe CF. Results: Correctors rescue trafficking of ⌬F508 by altering interaction with Hsp27 and Ϫ40. Conclusion: Rescue of trafficking mutants can be accomplished by combining correctors from different classes. Significance: ⌬F508 is the most common mutation. Information on the mechanism of corrector action is critical for treating the majority of patients.

Although, the most common Cystic Fibrosis mutation, ΔF508, in the cystic fibrosis transmembrane regulator. (CFTR), is located in nucleotide binding domain (NBD1), disease-causing mutations also occur in NBD2. To provide information on potential therapeutic strategies for mutations in NBD2, we studied, using a combination of biochemical approaches and newly created cell lines, two disease-causing NBD2 mutants, N1303K and S1235R. Surprisingly, neither was rescued by low temperature. Inhibition of proteasomes with MG132 or aggresomes with tubacin rescued the immature B and mature C bands of N1303K and S1235R, indicating that degradation occurs via proteasomes and aggresomes. We found no effect of the lysosome inhibitor E64. Thus, our results show that these NBD2 mutants are processing mutants with unique characteristics. Several known correctors developed to rescue ΔF508-CFTR, when applied either alone or in combination, significantly increased the maturation of bands B and C of both NBD 2 mutants. The best correction occurred with the combinations of C4 plus C18 or C3 plus C4. Co-transfection of truncated CFTR (∆27-264) into stably transfected cells was also able to rescue them. This demonstrates for the first time that transcomplementation with a truncated version of CFTR can rescue NBD2 mutants. Our results show that the N1303K mutation has a more profound effect on NBD2 processing than S1235R and that small-molecule correctors increase the maturation of bands B and C in NBD2 mutants. In addition, ∆27-264 was able to transcomplement both NDB2 mutants. We conclude that differences and similarities occur in the impact of mutations on NBD2 when compared to ΔF508-CFTR suggesting that individualized strategies may be needed to restore their function. Finally our results are important because they suggest that gene or corrector molecule therapies either alone or in combination individualized for NBD2 mutants may be beneficial for patients bearing N1303K or S1235R mutations.

Background/Aims: Premature degradation of mutated cystic fibrosis transmembrane conductance regulator (CFTR) protein causes cystic fibrosis (CF), the commonest Mendelian disease in Caucasians. Despite recent advances in precision medicines for CF patients, many CFTR mutants have not been characterized and the effects of these new therapeutic approaches are still unclear for those mutants. Methods: Cells transfected or stably expressing four CFTR transmembrane-domain mutants (G85E, E92K, L1077P, and M1101K) were used to: 1) characterize the mutants according to their protein expression, thermal sensitivity, and degradation pathways; 2) evaluate the effects of correctors in rescuing them; and 3) explore the effects of correctors on CFTR interactions with proteostasis components. Results: All four mutants exhibited lower protein expression than did wild type-CFTR, and they were degraded by proteasomes and aggresomes. At low temperature, only cells expressing the mutants L1077P and M1101K exhibited increased CFTR maturation. Co-administration of C4 and C18 showed the greatest effect, restoring functional expression and partial stability of CFTR bearing E92K, L1077P, or M1101K at the cell surface. However, this treatment was inefficient in rectifying the defect of CFTR bearing G85E. Correctors rescued CFTR mutants by reducing their interactions with proteostasis components associated with protein retention in the endoplasmic reticulum and ubiquitination. Conclusion: Co-administration of C4 and C18 rescued CFTR transmembrane-domain mutants by remodeling the CFTR interactome.

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We evaluated whether small molecule correctors could rescue four nucleotide-binding domain 1 (NBD1) mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (A455E, S492F, ΔI507, and R560T). We first transfected Cos-7 cells (green monkey kidney cells) with A455E, S492F, ΔI507, or R560T and created HEK-293 (human embryonic kidney cells) cell lines stably expressing these CFTR mutations. The mutants showed lowered protein expression, instability at physiological temperature, and rapid degradation. After treatment with correctors CFFT-002, CFFT-003, C3, C4, and/or C18, the combination of C18+C4 showed the most correction and resulted in increased CFTR residing in the plasma membrane. We found a profound decrease in binding of CFTR to histone deacetylases (HDAC) 6 and 7 and heat shock proteins (Hsps) 27 and 40. Silencing Hsp27 or 40 rescued the mutants, but no additional amount of CFTR was rescued when both proteins were knocked down simultaneously. Thus, CFTR mutations in NBD1 can be rescued by a combination of correctors, and the treatment alters the interaction between mutated CFTR and the endoplasmic reticulum machinery.

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The missing phenylalanine at position 508, located in nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane regulator (CFTR), is the most common cystic fibrosis mutation. Severe disease-causing mutations also occur in NBD2. To provide information on potential therapeutic strategies for mutations in NBD2, we used a combination of biochemical, cell biological and electrophysiological approaches and newly created cell lines to study two disease-causing NBD2 mutants, N1303K and S1235R. We observed that neither was sensitive to E64, a cysteine protease inhibitor. However, further investigation showed that when treated with a combination of correctors, C4 + C18, both mutants also responded to E64. Further exploration to assess aggresome throughput using the autophagy regulator LC3 as a marker showed that, in the absence of correctors, N1303K showed a stalled throughput of LC3-II to the aggresome. The throughput became active again after treatment with the corrector combination C4 + C18. Confocal microscopic studies showed that the N1303K and S1235R mutant proteins both co-localized with LC3, but this co-localization was abolished by the corrector combination and, to a lesser extent, by VX-809. Both the corrector combination and VX-809 increased the CFTR chloride channel function of both mutants. We conclude that correctors have a dual effect, particularly on N1303K: they improve trafficking and function at the plasma membrane and reduce the association with autophagosomes. After treatment with correctors persistent degradation by the autophagosome may limit restoration of function. Thus, mutations in NBD2 of CFTR, in contrast to ΔF508-CFTR, may require additional personalized strategies to rescue them.

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