Syntaxin 8 and the Endoplasmic Reticulum Processing of ΔF508-CFTR

Background/Aims: Cystic fibrosis (CF) is a lethal recessive disorder caused by mutations in the CF transmembrane conductance regulator (CFTR). ΔF508, the most common mutation, is a misfolded protein that is retained in the endoplasmic reticulum and degraded, precluding delivery to the cell surface [1]. Methods: Here we use a combination of western blotting, immunoprecipitation, and short circuit current techniques combined with confocal microscopy to address whether the SNARE attachment protein, STX8 plays a role in ΔF508’s processing and movement out of the ER. Results: Although the SNARE protein STX8 is thought to be functionally related and primarily localized to early endosomes, we show that silencing of STX8, particularly in the presence of the Vertex corrector molecule C18, rescues ΔF508-CFTR, allowing it to reach the cell surface and increasing CFTR-dependent chloride currents by approximately 2.5-fold over control values. STX8 silencing reduced the binding of quality control protein, Hsp 27, a protein that targets ΔF508-CFTR for sumoylation and subsequent degradation, to ΔF508-CFTR. STX8 silencing increased the levels of Hsp 60 a protein involving in early events in protein folding. Conclusion: STX8 knockdown creates an environment favorable for mature ΔF508 to reach the cell surface. The data also suggest that when present at normal levels, STX8 functions as part of the cell’s quality control mechanism

Impact of the F508del mutation on ovine CFTR, a Cl- channel with enhanced conductance and ATP-dependent gating

Cross-species comparative studies are a powerful approach to understand the epithelial Cl- channel cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in the genetic disease cystic fibrosis (CF). Here, we investigate the single-channel behaviour of ovine CFTR and the impact of the most common CF mutation, F508del-CFTR, using excised inside-out membrane patches from transiently transfected CHO cells. Like human CFTR, ovine CFTR formed a weakly inwardly rectifying Cl- channel regulated by PKA-dependent phosphorylation, inhibited by the open-channel blocker glibenclamide. However, for three reasons, ovine CFTR was noticeably more active than human CFTR. First, single-channel conductance was increased. Second, open probability was augmented because the frequency and duration of channel openings were increased. Third, with enhanced affinity and efficacy, ATP more strongly stimulated ovine CFTR channel gating. Consistent with these data, the CFTR modulator phloxine B failed to potentiate ovine CFTR Cl- currents. Like its impact on human CFTR, the F508del mutation caused a temperature-sensitive folding defect, which disrupted ovine CFTR protein processing and reduced membrane stability. However, the F508del mutation had reduced impact on ovine CFTR channel gating in contrast to its marked effects on human CFTR. We conclude that ovine CFTR forms a regulated Cl- channel with enhanced conductance and ATP-dependent channel gating. This phylogenetic analysis of CFTR structure and function demonstrates that subtle changes in structure have pronounced effects on channel function and the consequences of the CF mutation F508del. This article is protected by copyright. All rights reserved

Pharmacological correction of the function and the maturation of F508del-CFTR channel

La mucoviscidose est une maladie génétique autosomale récessive induite par des mutations du gène codant pour le canal chlorure CFTR (Cystic Fibrosis Transmembrane Conductance Regulator). Parmi les mutations les plus fréquentes, la délétion d’une phénylalanine en position 508 conduit à un défaut d’adressage de la protéine consécutif à sa rétention dans le réticulum endoplasmique. Bien que les mécanismes d’action soient encore largement méconnus, de nombreuses petites molécules appelées correcteurs (VX809, SAHA, Corr4a, iminosucres…) ont été identifiées pour corriger individuellement le défaut d’adressage et ainsi restaurer une partie de l’activité de F508del-CFTR. Dans une première partie, nous avons montré qu’en combinant de manière appropriée ces correcteurs, il est possible d’optimiser la restauration fonctionnelle de F508del-CFTR. Cependant, parmi ces composés, aucun de ne présentant de réels bénéfices cliniques. Ainsi, nous avons exploité les modèles de structure 3D de la protéine WT- et F508del-CFTR afin d’identifier un site d’intérêt (cavité F508del-CFTR) pour la conception de composés actifs et en particulier de correcteurs. Cette stratégie a conduit (1) à la conception de nouvelles molécules originales (2) à la synthèse de ces composés (3) à leur test sur la restauration fonctionnelle de F508del-CFTR. Les résultats obtenus par les techniques de Western-Blot, d’efflux d’iodure et d’électrophysiologie ont permis de montrer qu’il est possible, sur la base du modèle de structure 3D de la protéine et d’expériences d’amarrage moléculaire, de créer des molécules capables de restaurer les défauts d’adressage et de fonctionnalité de F508del-CFTR.Cystic fibrosis (CF) is an autosomal and recessive disease due to mutations in the gene encoding a chloride ion channel CFTR (Cystic Fibrosis Transmembrane Conductance Regulator). Deletion of phenylalamine at position 508 leads to the most common mutation, resulting from a mistrafficking of the CFTR protein and its retention in the Endoplasmic Reticulum (ER), abnormal gating of CFTR channel and endocytosis. Numerous small chemicals called CFTR correctors (VX809, SAHA, Corr-4a, iminosugars…) have been shown individually - albeit partially – to restore F508del-CFTR defective trafficking and functionally rescue chloride transport default. In previous studies, we showed that optimal combination of these correctors should lead to a more complete rescue of F508del-CFTR activity. Nevertheless, none of these correctors used show a good clinical issue. In a second set of experiments, based on models refinement of the open and closed forms of CFTR, we used molecular docking and virtual screening to identify new active molecules able to bind and interact with identified F508del-CFTR pocket. This strategy led to (1) design novel tailored correctors following molecular docking prediction, allow to bind specifically in F508del-CFTR pockets (2) synthetize these putative idealized compounds and (3) test them on the functional rescue of F508del-CFTR. Results showed, especially during Western-Blot combined to iodide efflux and patch-clamp experiments in whole cell configuration on F508del-CFTR cell that based on 3D structure model of entire CFTR protein, it is possible to develop new tailored correctors to target the root cause of CF