Rescue of NBD2 Mutants N1303K and S1235R of CFTR by Small-Molecule Correctors and Transcomplementation

Abstract

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.

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

“…An increasing number of correctors have been evaluated for their ability to rescue CFTR bearing ΔF508, as well as other NBD mutations [18, 23, 24]. The impact of these small-molecules to restoring CFTR processing and trafficking in cells bearing the mutants G85E, E92K, L1077P, and M1101K has notyet been established.…”

“…To answer this question, we incubated the cells bearing these mutants for 16 h with several correctors (see supplementary material, Fig. S2-S5): C3 and C18, discovered by Vertex Pharmaceuticals [25]; C4, developed by the Verkman lab [21]; and the CFFT compounds 002 and 003 [19, 24]. …”

Inhibition of histone deacetylase 6 activity reduces cyst growth in polycystic kidney disease

“…Given that CFTR-mediated chloride secretion is positively regulated by cAMP, 31, 32 we added the adenylate cyclase activator forskolin (10 μM) and potentiated the CFTR-mediated chloride secretion with the tyrosine kinase inhibitor genistein (30 μM). Thiazolidonone, a specific CFTR inhibitor (CFTR inh 172), 33, 34 was added at 10 μM to inhibit the I SC , indicating that the measured current was indeed generated by the CFTR. Pretreatment with tubacin significantly reduced the CFTR-dependent I SC in MDCK.2 cells when compared to DMSO-treated cells (Fig.…”

CFTR Controls the Activity of NF-κB by Enhancing the Degradation of TRADD

“…To test the hypothesis that TRADD regulation might be mediated by a proteolytic mechanism, we treated cells with MG132, or E64 to inhibit proteosomal or lysosomal degradation, respectively (see [40] for a description of both inhibitors). Figure 10 C & D shows that in cells transfected with Wt CFTR, there is a significant increase in TRADD protein in response to either MG132 or E64; the greater increase was noted with MG132.…”