The peripheral protein quality control (PPQC) checkpoint removes improperly folded proteins from the plasma membrane through a mechanism involving the E3 ubiquitin ligase CHIP (carboxyl terminus of Hsc70 interacting protein). PPQC limits the efficacy of some cystic fibrosis (CF) drugs, such as VX-809, that improve trafficking to the plasma membrane of misfolded mutants of the CF transmembrane conductance regulator (CFTR), including F508del-CFTR, which retains partial functionality. We investigated the PPQC checkpoint in lung epithelial cells with F508del-CFTR that were exposed to VX-809. The conformation of the scaffold protein NHERF1 (Na(+)/H(+) exchange regulatory factor 1) determined whether the PPQC recognized "rescued" F508del-CFTR (the portion that reached the cell surface in VX-809-treated cells). Activation of the cytoskeletal regulator Rac1 promoted an interaction between the actin-binding adaptor protein ezrin and NHERF1, triggering exposure of the second PDZ domain of NHERF1, which interacted with rescued F508del-CFTR. Because binding of F508del-CFTR to the second PDZ of NHERF1 precluded the recruitment of CHIP, the coexposure of airway cells to Rac1 activator nearly tripled the efficacy of VX-809. Interference with the NHERF1-ezrin interaction prevented the increase of efficacy of VX-809 by Rac1 activation, but the actin-binding domain of ezrin was not required for the increase in efficacy. Thus, rather than mainly directing anchoring of F508del-CFTR to the actin cytoskeleton, induction of ezrin activation by Rac1 signaling triggered a conformational change in NHERF1, which was then able to bind and stabilize misfolded CFTR at the plasma membrane. These insights into the cell surface stabilization of CFTR provide new targets to improve treatment of CF.

Cystic fibrosis (CF), the most common inherited disease in Caucasians, is caused by mutations in the CFTR chloride channel, the most frequent of which is Phe508del. Phe508del causes not only intracellular retention and premature degradation of the mutant CFTR protein, but also defective channel gating and decreased half-life when experimentally rescued to the plasma membrane (PM). Despite recent successes in the functional rescue of several CFTR mutations with small-molecule drugs, the folding-corrector/gating-potentiator drug combinations approved for Phe508del-CFTR homozygous patients have shown only modest benefit. Several factors have been shown to contribute to this outcome, including an unexpected intensification of corrector-rescued Phe508del-CFTR PM instability after persistent co-treatment with potentiator drugs. We have previously shown that acute co-treatment with hepatocyte growth factor (HGF) can significantly enhance the chemical correction of Phe508del-CFTR. HGF coaxes the anchoring of rescued channels to the actin cytoskeleton via induction of RAC1 GTPase signalling. Here, we demonstrate that a prolonged, 15-day HGF treatment also significantly improves the functional rescue of Phe508del-CFTR by the VX-809 corrector/VX-770 potentiator combination, in polarized bronchial epithelial monolayers. Importantly, we found that HGF treatment also prevented VX-770-mediated destabilization of rescued Phe508del-CFTR and enabled further potentiation of the rescued channels. Most strikingly, prolonged HGF treatment prevented previously unrecognized epithelial dedifferentiation effects of sustained exposure to VX-809. This was observed in epithelium-like monolayers from both lung and intestinal origin, representing the two systems most affected by adverse symptoms in patients treated with VX-809 or the VX-809/VX-770 combination. Taken together, our findings strongly suggest that co-administration of HGF with corrector/potentiator drugs could be beneficial for CF patients.

Edited by Roger J. Colbran Cystic fibrosis (CF) is a genetic disease caused by mutations in the gene encoding CF transmembrane conductance regulator (CFTR), a chloride channel normally expressed at the surface of epithelial cells. The most frequent mutation, resulting in Phe-508 deletion, causes CFTR misfolding and its premature degradation. Low temperature or pharmacological correctors can partly rescue the Phe508del-CFTR processing defect and enhance trafficking of this channel variant to the plasma membrane (PM). Nevertheless, the rescued channels have an increased endocytosis rate, being quickly removed from the PM by the peripheral protein quality-control pathway. We previously reported that rescued Phe508del-CFTR (rPhe508del) can be retained at the cell surface by stimulating signaling pathways that coax the adaptor molecule ezrin (EZR) to tether rPhe508del-Na ؉ /H ؉-exchange regulatory factor-1 complexes to the actin cytoskeleton, thereby averting the rapid internalization of this channel variant. However, the molecular basis for why rPhe508del fails to recruit active EZR to the PM remains elusive. Here, using a proteomics approach, we characterized and compared the core components of wt-CFTR-or rPhe508del-containing macromolecular complexes at the surface of human bronchial epithelial cells. We identified calpain 1 (CAPN1) as an exclusive rPhe508del interactor that prevents active EZR recruitment, impairs rPhe508del anchoring to actin, and reduces its stability in the PM. We show that either CAPN1 down-regulation or its chemical inhibition dramatically improves the functional rescue of Phe508del-CFTR in airway cells. These observations suggest that CAPN1 constitutes an appealing target for pharmacological intervention, as part of CF combination therapies restoring Phe508del-CFTR function. The cystic fibrosis (CF) 2 transmembrane conductance regulator (CFTR) is a multispanning cAMP-regulated chloride channel primarily localized to the apical membrane of polarized epithelial cells (1). Mutations in the CFTR gene cause the complex inherited disorder CF (1, 2). The most common CFTR mutation is the deletion of phenylalanine 508 (Phe508del), with ϳ85% of all CF patients having at least one copy of the Phe508del mutant (Welcome to CFTR2 website, https://www.cftr2.org/) 3 (4). This mutation is mainly characterized by protein misfolding and premature degradation by the endoplasmic reticulum (ER) quality control, preventing the mutant protein from trafficking to the cell surface (5-7). Although Phe508del ER retention is not complete, the very small fraction of channels that reach the cell surface possesses only partial activity because of a gating defect (5, 8, 9) and show a severely decreased plasma membrane (PM) halflife, because of accelerated endocytosis and lysosomal degradation (10, 11). At present, there are two approved corrector drugs that target the molecular defects in Phe508del (12-14). The first to be used in the clinic is the drug Orkambi, which combines "corrector" VX-809 (lumacaftor) and "potentiato...

Rac1 is a member of the Rho family of small GTPases that not only regulates signaling pathways involved in cell adhesion and migration but also regulates gene transcription. Here we show that the transcriptional repressor BCL-6 is regulated by Rac1 signaling. Transfection of active Rac1 mutants into colorectal DLD-1 cells led to increased expression of a BCL-6-controlled luciferase reporter construct. Conversely, inhibition of endogenous Rac1 activation by the Rac1 inhibitor NSC23766 decreased reporter activity. Moreover, BCL-6 lost its typical localization to nuclear dots upon activation of Rac1 and became predominantly soluble in a non-chromatin-bound cell fraction. Rac1 signaling also regulated the expression of endogenous BCL-6-regulated genes, including the p50 precursor NF-B1/p105 and the cell adhesion molecule CD44. Interestingly, these effects were not stimulated by the alternative splice variant Rac1b. The mechanism of BCL-6 inhibition does not involve formation of a stable Rac1/BCL-6 complex and is independent of Rac-induced reactive oxygen species production or Jun NH 2 -terminal kinase activation. We show that PAK1 mediates inhibition downstream of Rac and can directly phosphorylate BCL-6. Together, these data provide substantial evidence that Rac1 signaling inhibits the transcriptional repressor BCL-6 in colorectal cells and reveal a novel pathway that links Rac1 signaling to the regulation of gene transcription.