CFTR and Ca2+ Signaling in Cystic Fibrosis

Among the diverse physiological functions exerted by calcium signaling in living cells, its role in the regulation of protein biogenesis and trafficking remains incompletely understood. In cystic fibrosis (CF) disease the most common CF transmembrane conductance regulator (CFTR) mutation, F508del-CFTR generates a misprocessed protein that is abnormally retained in the endoplasmic reticulum (ER) compartment, rapidly degraded by the ubiquitin/proteasome pathway and hence absent at the plasma membrane of CF epithelial cells. Recent studies have demonstrated that intracellular calcium signals consequent to activation of apical G-protein-coupled receptors by different agonists are increased in CF airway epithelia. Moreover, the regulation of various intracellular calcium storage compartments, such as ER is also abnormal in CF cells. Although the molecular mechanism at the origin of this increase remains puzzling in epithelial cells, the F508del-CFTR mutation is proposed to be the onset of abnormal Ca2+ influx linking the calcium signaling to CFTR pathobiology. This article reviews the relationships between CFTR and calcium signaling in the context of the genetic disease CF

Mécanismes et correction pharmacologique de l'adressage de la protéine mutée F508del-CFTR

La délétion sur le gène CFTR (Cystic Fibrosis Transmembrane conductance Regulator) d une phénylalanine en position 508 est à l origine d une activation et d un adressage défectueux de la protéine traduite. L objectif de ce travail a été de découvrir des molécules corrigeant l adressage du F508del-CFTR. Le miglustat, en inhibant la déglucosylation du F508del-CFTR prévient son interaction avec la calnexine et permet son adressage à la membrane plasmique. De même, la modulation du taux de calcium réticulaire, en prévenant l interaction F508del-CFTR/calnexine, permet la correction du F508del-CFTR. Enfin, nous avons démontré que les sels de benzo[c]quinolizinium, en se fixant sur la glycine 622 du F508del-CFTR inhibent sa dégradation et permettent son adressage. La recherche des mécanismes impliqués dans cette correction pharmacologique a ainsi permis de révéler de nouvelles cibles potentielles dans la mise en place d un traitement de la mucoviscidose.Most patients suffering from Cystic Fibrosis have the F508del mutation on at least one CFTR (Cystic Fibrosis Transmembrane conductance Regulator) allele which results in misfolding and gating flaw of CFTR. The aim of this work was focused on identifying compounds able to rescue the trafficking of F508del-CFTR. By inhibiting the deglucosylation of F508del-CFTR, miglustat, an a,1-2 glucosidase inhibitor, rescues the F508del-CFTR protein to the cell surface preventing its interaction with calnexin. Moreover, the modulation of Ca2+ level in the endoplasmic reticulum corrects F508del-CFTR trafficking preventing its interaction with calnexin. Finally, we identified a novel mechanism for correction of F508del-CFTR by the benzo[c]quinolizinium potentiators interacting with CFTR via the amino acid G622 to protect the channel from proteasome degradation. Research on the mechanisms of biosynthesis, trafficking and degradation of F508del-CFTR protein reveals new strategies for CF drug targets.POITIERS-BU Sciences (861942102) / SudocSudocFranceF

Implication des protéines chaperonnes dans la rétention réticulaire de la protéine F508del-CFTR

La mucoviscidose est une maladie génétique ayant pour origine des mutations de la protéine CFTR (cystic fibrosis transmembrane conductance regulator). La plus fréquente de ces mutations correspond à la délétion d'une phénylalanine en position 508 (F508del) et se traduit par un défaut d'adressage de la protéine mutée qui est alors retenue par le système de contrôle qualité du réticulum endoplasmique (ERCQ) et plus particulièrement par le cycle de la calnexine. Ce cycle compte plusieurs acteurs principaux, des protéines chaperonnes, des co-chaperonnes et des enzymes. L'objectif de cette étude est donc de démontrer l'implication et le rôle de ces partenaires avec la protéine naissante dans la rétention de la protéine F508del-CFTR. Pour cela, une stratégie de siRNA de ces protéines a été mise en place. Dans une première étude, nous avons pu mettre en évidence l'implication de protéines impliquées dans l'entrée du cycle de la calnexine. Et dans une deuxième étude, nous avons démontré l'importance de protéines impliquées dans la sortie du cycle de la calnexine. Nos travaux démontrent donc l'implication du cycle de la calnexine dans la rétention de la protéine F508del-CFTR ainsi que l'importance de l'interaction entre la calnexine et la protéine F508del-CFTR. De façon intéressante, ces études ont permis d'identifier d'éventuelles nouvelles cibles thérapeutiques pour le traitement de la mucoviscidose.Cystic fibrosis is a genetic disorder characterized by mutations on the CFTR (cystic fibrosis transmembrane conductance regulator). The most common mutation is a deletion of a phenylalanine at position 508, F508del, results in trafficking default of CFTR protein that is retained in the endoplasmic reticulum quality control (ERQC) and especially by the calnexin cycle. This cycle has several main proteins, the chaperones, co-chaperones and enzymes. The aim of this work is to demonstrate the involvement and role of these partners with the nascent protein in the retention of F508del-CFTR protein. For this, an siRNA strategy of these proteins was established. In a first study, we demonstrated the implication of proteins involved in the entrance of calnexin cycle. And in a second study, we demonstrated the importance of proteins involved in the release calnexin cycle. Our studies highlight the implication of calnexine cycle in the F508del-CFTR protein. Interestingly, these studies have identified potential new therapeutic targets for the treatment of cystic fibrosis.POITIERS-BU Sciences (861942102) / SudocSudocFranceF

Approches pharmacologiques de CFTR et de CaCC dans la mucoviscidose

La mucoviscidose résulte de la mutation du gène codant pour la protéine CFTR (Cystic Fibrosis Transmembrane conductance Regulator) induisant un défaut de sécrétion des ions chlorure à la membrane apicale des épithéliums respiratoires, digestifs et reproducteurs. L'objectif de ce travail était de caractériser des molécules capables de restaurer une sécrétion chlorure dans des modèles mucoviscidosiques in vitro, ex vivo et in vivo. Dans un premier temps, nous avons identifié deux composés de la famille des adduits méthylglyoxalalpha-aminoazahétérocycle (ie le GPact-11a et le GPact-26a) comme activateurs non-toxiques et hydrosolubles des CFTR sauvages et F508del. Dans une seconde partie de l'étude, nous avons identifié un nouvel activateur des canaux TRPC6 : le guanabenz. Nous avons mis en évidence que le guanabenz induit l'activation des canaux chlorures calcium-dépendant (CaCC) via une entrée de calcium extracellulaire par le canal TRPC6. La restauration de la sécrétion chlorure grâce à l'activation du F508del-CFTR ou du CaCC représente une approche pharmacologique efficace et mesurable pour le traitement de la mucoviscidose. De plus, l'identification d'une nouvelle voie d'activation du CaCC via TRPC6 représente une avancée scientifique pouvant se répercuter sur d'autres pathologies.Cystic Fibrosis (CF) is a genetic disorder characterized by mutations on the CFTR (Cystic Fibrosis Tran membrane conductance Regulator) protein resulting in a failure of CFTR dependent chloride conductance in epithelial cells. The aim of this work was focused on identifying compounds able to restore chloride secretion in Cystic Fibrosis model using in vitro, ex vivo and in vivo techniques. Firstly, we identified two methylglyoxal-alpha-aminoazaheterocycle adducts (GPact-11a and GPact-26a) as new water-soluble and non-toxic wt- and F508del-CFTR activators. In a second part of this study, we identified guanabenz as a new TRPC6 channel activator. We demonstrated that guanabenz activates CaCC by an entry of extracellular calcium through TRPC6 channel. Restoration of chloride secretion by the activation of F508del-CFTR or CaCC represents an effective and measurable pharmacological approach for Cystic Fibrosis treatments. Furthermore, identification of TRPC6 as a new target to activate CACC represents a scientific breakthrough with several therapeutic applications.POITIERS-BU Sciences (861942102) / SudocSudocFranceF

DECREASING PROTEASOME ACTIVITY USING THE PHARMACOLOGICAL CHAPERONE MPB RESCUE F508DEL-CFTR PROCESSING

The most common mutation F508del causing CF results in misfolding of the protein leading to its substantial degradation via the ubiquitin-26S proteasome. As this mutation induces also a gating defect of CFTR, a possible response to treat CF patients includes the discovery of molecules able to rescue the defective trafficking and the gating of F508del-CFTR. In previous studies, we reported that benzo[c]quinolizinium (MPB) derivatives act as potentiators of CFTR activity (Marving-Mounir et al., J Med Chem., 2004) and could rescue F508del-CFTR processing (Dormer et al., JCS., 2001). Moreover, itwas shown that MPBs inhibit the degradation of F508del-CFTR cytoplasmic domain (Stratford et al., BBRC, 2003). The purpose of this studywas i) to confirm the modulator and the corrector effects of severalMPBcompounds on the human airway epithelial CF cell line JME/CF15 (F508del/F508del), ii) to study the implication of MPB correction on the endoplasmic reticulum quality control (ERQC) and iii) to identify the molecular mechanism ofMPBeffect. Firstly, we compared using iodide efflux, the ability of different MPB compounds to activate F508del-CFTR. We show, for example, that MPB104 activates F508del-CFTR, in low-temperature corrected cells, with an EC50 of 6 μM. To investigate whether MPBs are able to correct the abnormal F508del-CFTR trafficking, we used western-blot analysis, confocal immunofluorescence and patch-clamp recording in MPB104-treated cells (2h, 100μM). We show that this compound allows the recovery of a mature and functional F508del-CFTR protein to the cell surface. To further study the rescue pathway of F508del-CFTR in MPB-corrected cells, we considered the effects of MPBs in competition with several inhibitors of the ERQC and of the degradation pathway. A significant potentiation of the corrector effect of MPBs with several ERQC inhibitors was observed suggesting a synergic effect between MPBs and these inhibitors. On contrary, only a small potentiation was observed with a co-treatment by MPBs and MG132 (a proteasome inhibitor) arguing an effect of MPBs on the degradation pathway. Co-immunoprecipitation experiments between F508del-CFTR and either calnexin, HSP70 or HSP90 show that MPB seems to have no effect on F508del-CFTR interactions with these chaperones. Measurements of the proteasome activity point to a MPB inhibitory effect on the proteasome in cell lysates but not on purified proteasome enzyme. These results suggest an effect of MPB compounds in early degradation steps and not a direct effect on proteasome. Our hypothesis is that the mechanism whereby MPBs rescue F508del-CFTR processing is through direct interaction with the F508del- CFTR preventing its recognition by the proteasome. In conclusion, we found that MPB compounds are able to correct the two F508del-CFTR defects (i.e. trafficking and gating). The mechanism of action of these drugs involves the prevention of the ubiquitin-proteasome- mediated degradation of F508del-CFTR. This study provides evidence for a novel cellular mechanism, via the decrease of the degradation pathway, to correct F508del-CFTR trafficking. Supported by VLM, MucoVie66 and CNRS

The Glycine Residues G551 and G1349 within the ATP-Binding Cassette Signature Motifs Play Critical Roles in the Activation and Inhibition of Cystic Fibrosis Transmembrane Conductance Regulator Channels by Phloxine B

The cystic fibrosis transmembrane conductance regulator (CFTR) protein contains a canonical ATP-binding cassette (ABC) signature motif, LSGGQ, in nucleotide binding domain 1 (NBD1) and a degenerate LSHGH in NBD2. Here, we studied the contribution of the conserved residues G551 and G1349 to the pharmacological modulation of CFTR chloride channels by phloxine B using iodide efflux and whole-cell patch clamp experiments performed on the following green fluorescent protein (GFP)-tagged CFTR: wild-type, delF508, G551D, G1349D, and G551D/G1349D double mutant. We found that phloxine B stimulates and inhibits channel activity of wild-type CFTR (Ks = 3.2 ± 1.6 μM, Ki = 38 ± 1.4 μM) and delF508 CFTR (Ks = 3 ± 1.8 μM, Ki = 33 ± 1 μM). However, CFTR channels with the LSGDQ mutated motif (mutation G551D) are activated (Ks = 2 ± 1.13 μM) but not inhibited by phloxine B. Conversely, CFTR channels with the LSHDH mutated motif (mutation G1349D) are inhibited (Ki = 40 ± 1.01 μM) but not activated by phloxine B. Finally, the double mutant G551D/G1349D CFTR failed to respond not only to phloxine B stimulation but also to phloxine B inhibition, confirming the importance of both amino acid locations. Similar results were obtained with genistein, and kinetic parameters were determined to compare the pharmacological effects of both agents. These data show that G551 and G1349 control the inhibition and activation of CFTR by these agents, suggesting functional nonequivalence of the signature motifs of NBD in the ABC transporter CFTR

Bronchorelaxation of the human bronchi by CFTR activators

International audienceThe airway functions are profoundly affected in many diseases including asthma, COPD and cystic fibrosis (CF). CF the most common lethal autosomal recessive genetic disease is caused by mutations of the CFTR (Cystic Fibrosis transmembrane Conductance Regulator) gene, which normally encodes a multifunctional and integral membrane cAMP regulated and ATP gated Cl(-) channel expressed in airway epithelial cells. Using human lung tissues obtained from patients undergoing surgery for lung cancer, we demonstrated that CFTR participates in bronchorelaxation. Using human bronchial smooth muscle cells (HBSMC), we applied iodide influx assay to analyze the CFTR-dependent ionic transport and immunofluorescence technique to localize CFTR proteins. Moreover, the relaxation was studied in isolated human bronchial segments after pre-contraction with carbachol to determine the implication of CFTR in bronchodilation. We found in HBSMC that the pharmacology and regulation of CFTR is similar to that of its epithelial counterpart both for activation (using forskolin/genistein or a benzo[c]quinolizinium derivative) and for inhibition (CFTR(inh)-172 and GPinh5a). With human bronchial rings, we observed that whatever the compound used including salbutamol, the activation of muscular CFTR leads to a bronchodilation after constriction with carbachol. Altogether, these observations revealed that CFTR in the human airways is expressed in bronchial smooth muscle cells and can be pharmacologically manipulated leading to the hypothesis that this ionic channel could contribute to bronchodilation in human