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.
Highlights
Cystic Fibrosis is a recessive autosomal disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an ATP-binding cassette protein (ABC, subfamily C, member 7) composed of two transmembrane domains, two nucleotide-binding domains, and a unique regulatory domain
The results differed for the NBD2 mutants: Temperature rescue had no effect on the maturation of band C of either N1303K or S1235R (Fig. 1)
We have studied two mutations in NBD2, S1235R and N1303K, and showed that both can be rescued by small-molecule correctors, used in combination, and transcomplementation using a truncated version of cystic fibrosis transmembrane regulator. (CFTR)
Summary
Cystic Fibrosis is a recessive autosomal disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an ATP-binding cassette protein (ABC, subfamily C, member 7) composed of two transmembrane domains, two nucleotide-binding domains, and a unique regulatory domain. CFTR functions as a chloride channel in epithelial cells where, depending on the tissue, transports chloride ions either into or out of the cells [1]. Numerous disease-causing mutations (over 1000) have been described; the most common is a deletion of phenylalanine at position 508 (ΔF508) in the NBD1 domain, [3] which results in severe CF. This amino acid deletion diminishes both the thermal stability of NBD1 and its ability to interact properly with the transmembrane domains [4]. Either a new class of correctors will have to be identified, or VX-809 will have to be combined with other small molecules to reach therapeutic level
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
