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

Lopes‐Pacheco, Miquéias; Sabirzhanova, Inna; Rapino, Daniele; Morales, Marcelo M.; Guggino, William B.; Cebotaru, Liudmila

doi:10.1002/cbic.201500620

Cited by 31 publications

(34 citation statements)

References 41 publications

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“…Our data similarly show strongly reduced complex glycosylated CFTR (band C; Figure 1B), PM and total CFTR ( Figure 5B-D) at steady state conditions, underscoring rapid degradation of mutant protein by the ER and/or peripheral quality control. Cebotaru and colleagues identified the proteasome as the main source of protein degradation, indeed pointing towards a class II processing defect [37,38]. Both Kalydeco®(ivacaftor) and Symdeko®(tezacaftor, ivacaftor) are approved for patients with the A455E mutation (www.fda.gov, [9,10,14]).…”

Section: Discussionmentioning

confidence: 99%

Phenotyping of Rare CFTR Mutations Reveals Distinct Trafficking and Functional Defects

Ensinck

Keersmaecker

Heylen

et al. 2020

Cells

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Background. The most common CFTR mutation, F508del, presents with multiple cellular defects. However, the possible multiple defects caused by many rarer CFTR mutations are not well studied. We investigated four rare CFTR mutations E60K, G85E, E92K and A455E against well-characterized mutations, F508del and G551D, and their responses to corrector VX-809 and/or potentiator VX-770. Methods. Using complementary assays in HEK293T stable cell lines, we determined maturation by Western blotting, trafficking by flow cytometry using extracellular 3HA-tagged CFTR, and function by halide-sensitive YFP quenching. In the forskolin-induced swelling assay in intestinal organoids, we validated the effect of tagged versus endogenous CFTR. Results. Treatment with VX-809 significantly restored maturation, PM localization and function of both E60K and E92K. Mechanistically, VX-809 not only raised the total amount of CFTR, but significantly increased the traffic efficiency, which was not the case for A455E. G85E was refractory to VX-809 and VX-770 treatment. Conclusions. Since no single model or assay allows deciphering all defects at once, we propose a combination of phenotypic assays to collect rapid and early insights into the multiple defects of CFTR variants.

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Section: Discussionmentioning

confidence: 99%

Phenotyping of Rare CFTR Mutations Reveals Distinct Trafficking and Functional Defects

Ensinck

Keersmaecker

Heylen

et al. 2020

Cells

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“…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]. …”

Section: Resultsmentioning

confidence: 99%

“…Flp-In human embryonic kidney (HEK)-293 cells (catalog CRL-1573, Life Technologies) cultured in DMEM containing 10% FBS, penicillin (100 U/mL), streptomycin (100 µg/mL) and Zeocin (100 µg/mL) at 37°C were used to generate the stably transfected cell lines (see ref [19]). CFBE41o- cells maintained in Eagle's essential medium (MEM, Invitrogen) with 10% FBS, L-glutamate and penicillin/streptomycin were used to insert the Flp-In system and generate the stably transfected cell lines with each of the four CFTR mutants of interest.…”

Section: Methodsmentioning

confidence: 99%

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

Lopes‐Pacheco¹,

Boinot²,

Sabirzhanova³

et al. 2017

Cell Physiol Biochem

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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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“…2). Similarly, trivial Band C expression is observed in the CF mutant R560T‐ (30, 50, 71) and R560S‐CFTR (72). Taken together, our own and previous data highlight the importance of H3, H4, and H5 helices for achieving normal CFTR processing.…”

Section: Discussionmentioning

confidence: 84%

“…Moreover, the orientation of loop residues 509–511 is also changed in ∆F508 NBD1 (18, 26). Alterations around this loop, such as the ∆I507 mutation (28–30) or mutations of the Y512 residue (31), all impair CFTR processing. Moreover, structural differences between crystal structures of WT and ∆F508 NBD1 (18) might be underestimated because solubilizing mutations in these constructs partially correct ∆F508‐CFTR processing and gating defects (32).…”

mentioning

confidence: 99%

A defective flexible loop contributes to the processing and gating defects of the predominant cystic fibrosis‐causing mutation

et al. 2019

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People with the genetic disease cystic fibrosis (CF) often carry a deletion mutation ∆F508 on the gene encoding the CF transmembrane conductance regulator (CFTR) Cl− channel. This mutation greatly reduces the CFTR maturation process and slows the channel opening rate. Here, we investigate whether residues near F508 contribute to these defects in ∆F508‐CFTR. Most deletion mutations, but not alanine substitutions, of individual residues from positions 503 to 513 impaired CFTR maturation. Interestingly, only protein processing of ∆Y512‐CFTR, like that of ∆F508‐CFTR, was greatly improved by low‐temperature culture at 27°C or small‐molecule corrector C18. The 2 mutant Cl− channels were equally slow to open, suggesting that they may share common structural flaws. Studies on the H3‐H4 loop that links residues F508 and Y512 demonstrate that G509A/V510G mutations, moving G5091 position backward in the loop, markedly enhanced ∆F508‐CFTR maturation and opening rate while promoting protein stability and persistence of the H3 helix in ∆F508 nucleotide‐binding domain 1. Moreover, V510A/S511A mutations noticeably increased ∆Y512‐CFTR maturation at 27°C and its opening rate. Thus, loop abnormalities may contribute to ∆F508‐ and ∆Y512‐CFTR defects. Importantly, correcting defects from G509 displacement in ∆F508‐CFTR may offer a new avenue for drug discovery and CF treatments.—Chen, X., Zhu, S., Zhenin, M., Xu, W., Bose, S. J., Wong, M. P.‐F., Leung, G. P. H., Senderowitz, H., Chen, J.‐H. A defective flexible loop contributes to the processing and gating defects of the predominant cystic fibrosis‐causing mutation. FASEB J. 33, 5126–5142 (2019). http://www.fasebj.org

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

Cited by 31 publications

References 41 publications

Phenotyping of Rare CFTR Mutations Reveals Distinct Trafficking and Functional Defects

Phenotyping of Rare CFTR Mutations Reveals Distinct Trafficking and Functional Defects

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

A defective flexible loop contributes to the processing and gating defects of the predominant cystic fibrosis‐causing mutation

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