title: 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.
authors: Department of Medicine, Johns Hopkins University, School of Medicine, Baltimore, Maryland, United States of America; Department of Physiology, Johns Hopkins University, School of Medicine, Baltimore, Maryland, United States of America.
authors: Daniele
authors: D
authors: Rapino
authors: Department of Medicine, Johns Hopkins University, School of Medicine, Baltimore, Maryland, United States of America; Department of Physiology, Johns Hopkins University, School of Medicine, Baltimore, Maryland, United States of America.
authors: Inna
authors: I
authors: Sabirzhanova
authors: Department of Medicine, Johns Hopkins University, School of Medicine, Baltimore, Maryland, United States of America; Department of Physiology, Johns Hopkins University, School of Medicine, Baltimore, Maryland, United States of America.
authors: Miquéias
authors: M
authors: Lopes-Pacheco
authors: Department of Medicine, Johns Hopkins University, School of Medicine, Baltimore, Maryland, United States of America; Department of Physiology, Johns Hopkins University, School of Medicine, Baltimore, Maryland, United States of America.
authors: Rahul
authors: R
authors: Grover
authors: Department of Physiology, Johns Hopkins University, School of Medicine, Baltimore, Maryland, United States of America.
authors: William B
authors: WB
authors: Guggino
authors: Department of Medicine, Johns Hopkins University, School of Medicine, Baltimore, Maryland, United States of America.
authors: Liudmila
authors: L
authors: Cebotaru
mesh_terms: D000818:Animals; D001692:Biological Transport; D019556:COS Cells; D002522:Chlorocebus aethiops; D003550:Cystic Fibrosis / Q000188:drug therapy* / Q000235:genetics* / Q000473:pathology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics* / Q000378:metabolism; D005816:Genetic Complementation Test; D057809:HEK293 Cells; D006801:Humans; D009154:Mutation / Q000235:genetics*; D011485:Protein Binding; D054852:Small Molecule Libraries / Q000494:pharmacology*
chemical_list: C505032:CFTR protein, human; D054852:Small Molecule Libraries; C093349:cystic fibrosis transmembrane conductance regulator delta F508; D019005:Cystic Fibrosis Transmembrane Conductance Regulator