Combination of Correctors Rescues CFTR Transmembrane-Domain Mutants by Mitigating their Interactions with Proteostasis.

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.

Correctors Rescue CFTR Mutations in Nucleotide-Binding Domain 1 (NBD1) by Modulating Proteostasis.

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.

Rescuing Trafficking Mutants of the ATP-binding Cassette Protein, ABCA4, with Small Molecule Correctors as a Treatment for Stargardt Eye Disease.

Stargardt disease is the most common form of early onset macular degeneration. Mutations in ABCA4, a member of the ATP-binding cassette (ABC) family, are associated with Stargardt disease. Here, we have examined two disease-causing mutations in the NBD1 region of ABCA4, R1108C, and R1129C, which occur within regions of high similarity with CFTR, another ABC transporter gene, which is associated with cystic fibrosis. We show that R1108C and R1129C are both temperature-sensitive processing mutants that engage the cellular quality control mechanism and show a strong interaction with the chaperone Hsp 27. Both mutant proteins also interact with HDCAC6 and are degraded in the aggresome. We also demonstrate that novel corrector compounds that are being tested as treatment for cystic fibrosis, such as VX-809, can rescue the processing of the ABCA4 mutants, particularly their expression at the cell surface, and can reduce their binding to HDAC6. Thus, our data suggest that VX-809 can potentially be developed as a new therapy for Stargardt disease, for which there is currently no treatment.

Rescue of NBD2 mutants N1303K and S1235R of CFTR by small-molecule correctors and transcomplementation.

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.

Correcting the cystic fibrosis disease mutant, A455E CFTR.

Cystic fibrosis is caused by more than 1000 mutations, the most common being the ΔF508 mutation. These mutations have been divided into five classes [1], with ΔF508 CFTR in class II. Here we have studied the class V mutation A455E. We report that the mature and immature bands of A455E are rapidly degraded primarily by proteasomes; the short protein half-life of this mutant therefore resembles that of ΔF508 CFTR. A455E could be rescued by treatment of the cells with proteasome inhibitors. Furthermore, co-transfection of A455E with the truncation mutant Δ264 CFTR also rescued the mature C band, indicating that A455E can be rescued by transcomplementation. We found that Δ264 CFTR bound to A455E, forming a bimolecular complex. Treatment with the compound correctors C3 and C4 also rescued A455E. These results are significant because they show that although ΔF508 belongs to a different class than A455E, it can be rescued by the same strategies, offering therapeutic promise to patients with Class V mutations.

Growth failure in children with cystic fibrosis.

Poor linear growth and inadequate weight gain are very common problems in cystic fibrosis (CF) children. The most important factors involved in growth failure are undernutrition or malnutrition, chronic inflammation, lung disease, and corticosteroid treatment. Nutritional support and pharmacological therapy with recombinant human growth hormone are essential for a good management of children with CF, although these children are shorter and lighter than healthy children, and despite the catch-up growth observed after diagnosis, deficit in length/height and weight continues to be seen until adulthood. Early diagnosis is essential to ensure better nutritional status and growth, potentially associated with better respiratory function and prognosis. The aims of this review are try to explain etiology and pathogenetic mechanisms of growth failure in CF children and clarify their role in the disease morbidity and in clinical outcome, especially in relation to progressive decline of pulmonary function.