Co-Translational Folding of the First Transmembrane Domain of ABC-Transporter CFTR is Supported by Assembly with the First Cytosolic Domain

Kleizen, Bertrand; Willigen, Marcel van; Mijnders, Marjolein; Peters, Florence; Grudniewska, Magda; Hillenaar, Tamara; Thomas, Ann; Versluis, Laurens; Peters, Kathryn W.; Frizzell, Raymond A.; Sluijs, Peter van der; Braakman, Ineke

doi:10.1016/j.jmb.2021.166955

Cited by 36 publications

(108 citation statements)

References 83 publications

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“…Consistent with the structure-activity relationship, these two analogous compounds bind to a common site in TMD1. The location of the binding site is entirely consistent with functional studies demonstrating that lumacaftor promotes folding of isolated TMD1 but does not affect other domains (Farinha et al, 2013;Kleizen et al, 2021;Laselva et al, 2018;Loo et al, 2013;Ren et al, 2013). Furthermore, binding of lumacaftor or tezacaftor did not alter the structure of CFTR, supporting the previous conclusion that lumacaftor stabilizes TMD1 in its native conformation (Loo et al, 2013).…”

Section: Discussionsupporting

confidence: 86%

“…Consistent with this analysis, Clarke and colleagues showed that lumacaftor increased the lifetime of TMD1 by about 5-fold (Loo et al, 2013). And most recently, Braakman’s laboratory demonstrated that the type I correctors lumacaftor and C18 act at an early folding stage, supporting the hypothesis that rescuing ΔF508 by lumacaftor arises from the increased stability of TMD1 (Kleizen et al, 2021).…”

Section: Discussionmentioning

confidence: 86%

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Mechanism of CFTR correction by type I folding correctors

Chen

Fiedorczuk

2021

Preprint

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Small molecule chaperones have been exploited as therapeutics for the hundreds of diseases caused by protein misfolding. The most successful examples are the CFTR correctors, which transformed cystic fibrosis therapy. These molecules revert folding defects of the ΔF508 mutant and are widely used to treat patients. However, their mechanism of action is unknown. Here we present cryo-electron microscopy structures of CFTR in complex with two FDA-approved correctors: lumacaftor and tezacaftor. Both drugs insert into a hydrophobic pocket in the first transmembrane domain (TMD1), linking together four helices that are thermodynamically unstable. Mutating residues at the binding site rendered ΔF508-CFTR insensitive to lumacaftor and tezacaftor, underscoring the functional significance of the structural discovery. These results support a mechanism in which the correctors stabilize TMD1 at an early stage of biogenesis, prevent its pre-mature degradation, and thereby allosterically rescue a large number of disease-causing mutations.

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Section: Discussionsupporting

confidence: 86%

Section: Discussionmentioning

confidence: 86%

Mechanism of CFTR correction by type I folding correctors

Chen

Fiedorczuk

2021

Preprint

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“…To identify mutations that exhibit preferential responsiveness to type I correctors, the ratio of the relative correction efficacy of VX-661 and VX-445 was calculated ( Figure 1 A). Mutations in the MSD1 and the proximal N-terminus were more responsive to VX-661 than to VX-445 ( Figure 1 A), consistent with their high responsiveness to, and the stabilization of the MSD1 and CFTR N-terminal half by VX-809 ( Figure 1 B) [ 38 , 39 , 40 , 41 ]. Mutations in the other CFTR domains showed partial selectivity for correction with VX-445, with the exception of the S549R in the NBD1, which could be nearly completely corrected by either corrector ( Figure 1 A and Supplementary Figure S1A ).…”

Section: Resultsmentioning

confidence: 61%

A Precision Medicine Approach to Optimize Modulator Therapy for Rare CFTR Folding Mutants

Veit

Velkov

et al. 2021

JPM

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Trikafta, a triple-combination drug, consisting of folding correctors VX-661 (tezacaftor), VX-445 (elexacaftor) and the gating potentiator VX-770 (ivacaftor) provided unprecedented clinical benefits for patients with the most common cystic fibrosis (CF) mutation, F508del. Trikafta indications were recently expanded to additional 177 mutations in the CF transmembrane conductance regulator (CFTR). To minimize life-long pharmacological and financial burden of drug administration, if possible, we determined the necessary and sufficient modulator combination that can achieve maximal benefit in preclinical setting for selected mutants. To this end, the biochemical and functional rescue of single corrector-responsive rare mutants were investigated in a bronchial epithelial cell line and patient-derived human primary nasal epithelia (HNE), respectively. The plasma membrane density of P67L-, L206W- or S549R-CFTR corrected by VX-661 or other type I correctors was moderately increased by VX-445. Short-circuit current measurements of HNE, however, uncovered that correction comparable to Trikafta was achieved for S549R-CFTR by VX-661 + VX-770 and for P67L- and L206W-CFTR by the VX-661 + VX-445 combination. Thus, introduction of a third modulator may not provide additional benefit for patients with a subset of rare CFTR missense mutations. These results also underscore that HNE, as a precision medicine model, enable the optimization of mutation-specific modulator combinations to maximize their efficacy and minimize life-long drug exposure of CF patients.

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“…The location of the CFTR2 mutants in different CFTR domains, together with the concept that the domains of CFTR fold cooperatively, suggests that these cooperative interactions require different pharmaco-chaperones to optimize their overall folding efficacy. It has also been suggested that these CFTR domain interactions occur posttranslationally ( Du and Lukacs, 2009 ; Kleizen et al, 2021 ) and are coupled to one another, consistent with the concept that folding defects arising from missense mutations in different CFTR domains would be distinct and require compounds that target different steps in the cooperative folding pathway of CFTR.…”

Section: Discussionmentioning

confidence: 68%

Cystic Fibrosis Transmembrane Conductance Regulator Folding Mutations Reveal Differences in Corrector Efficacy Linked to Increases in Immature Cystic Fibrosis Transmembrane Conductance Regulator Expression

2021

Self Cite

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Background: Most cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that lead to protein misfolding and degradation by the ubiquitin–proteasome system. Previous studies demonstrated that PIAS4 facilitates the modification of wild-type (WT) and F508del CFTR by small ubiquitin-like modifier (SUMO)-1, enhancing CFTR biogenesis by slowing immature CFTR degradation and producing increased immature CFTR band B.Methods: We evaluated two correction strategies using misfolding mutants, including the common variant, F508del. We examined the effects on mutant expression of co-expression with PIAS4 (E3 SUMO ligase), and/or the corrector, C18. To study the impact of these correction conditions, we transfected CFBE410- cells, a bronchial epithelial cell line, with a CFTR mutant plus: (1) empty vector, (2) empty vector plus overnight 5 μM C18, (3) PIAS4, and (4) PIAS4 plus C18. We assessed expression at steady state by immunoblot of CFTR band B, and if present, band C, and the corresponding C:B band ratio. The large PIAS4-induced increase in band B expression allowed us to ask whether C18 could act on the now abundant immature protein to enhance correction above the control level, as reported by the C:B ratio.Results: The data fell into three mutant CFTR categories as follows: (1) intransigent: no observable band C under any condition (i.e., C:B = 0); (2) throughput responsive: a C:B ratio less than control, but suggesting that the increased band C resulted from PIAS4-induced increases in band B production; and (3) folding responsive: a C:B ratio greater than control, reflecting C18-induced folding greater than that expected from increased throughput due to the PIAS4-induced band B level.Conclusion: These results suggest that the immature forms of CFTR folding intermediates occupy different loci within the energetic/kinetic folding landscape of CFTR. The evaluation of their properties could assist in the development of correctors that can target the more difficult-to-fold mutant conformations that occupy different sites within the CFTR folding pathway.

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Co-Translational Folding of the First Transmembrane Domain of ABC-Transporter CFTR is Supported by Assembly with the First Cytosolic Domain

Cited by 36 publications

References 83 publications

Mechanism of CFTR correction by type I folding correctors

Mechanism of CFTR correction by type I folding correctors

A Precision Medicine Approach to Optimize Modulator Therapy for Rare CFTR Folding Mutants

Cystic Fibrosis Transmembrane Conductance Regulator Folding Mutations Reveal Differences in Corrector Efficacy Linked to Increases in Immature Cystic Fibrosis Transmembrane Conductance Regulator Expression

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