Wild-type Cystic Fibrosis Transmembrane Conductance Regulator Research Articles

Involvement of the Cdc42 pathway in CFTR post-translational turnover and in its plasma membrane stability in airway epithelial cells.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is expressed on the apical plasma membrane (PM) of epithelial cells. The most common deleterious allele encodes a trafficking-defective mutant protein undergoing endoplasmic reticulum-associated degradation (ERAD) and presenting lower PM stability. In this study, we investigated the involvement of the Cdc42 pathway in CFTR turnover and trafficking in a human bronchiolar epithelial cell line (CFBE41o-) expressing wild-type CFTR. Cdc42 is a small GTPase of the Rho family that fulfils numerous cell functions, one of which is endocytosis and recycling process via actin cytoskeleton remodelling. When we treated cells with chemical inhibitors such as ML141 against Cdc42 and wiskostatin against the downstream effector N-WASP, we observed that CFTR channel activity was inhibited, in correlation with a decrease in CFTR amount at the cell surface and an increase in dynamin-dependent CFTR endocytosis. Anchoring of CFTR to the cortical cytoskeleton was then presumably impaired by actin disorganization. When we performed siRNA-mediated depletion of Cdc42, actin polymerization was not impacted, but we observed actin-independent consequences upon CFTR. Total and PM CFTR amounts were increased, resulting in greater activation of CFTR. Pulse-chase experiments showed that while CFTR degradation was slowed, CFTR maturation through the Golgi apparatus remained unaffected. In addition, we observed increased stability of CFTR in PM and reduction of its endocytosis. This study highlights the involvement of the Cdc42 pathway at several levels of CFTR biogenesis and trafficking: (i) Cdc42 is implicated in the first steps of CFTR biosynthesis and processing; (ii) it contributes to the stability of CFTR in PM via its anchoring to cortical actin; (iii) it promotes CFTR endocytosis and presumably its sorting toward lysosomal degradation.

Functional reconstitution and channel activity measurements of purified wildtype and mutant CFTR protein.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

PharmGKB summary: very important pharmacogene information for CFTR.

The cystic fibrosis transmembrane conductance regulator (CFTR, ATP-binding cassette sub-family C, member 7, ABCC7) protein is 1480 amino acids in length. It is encoded by a single large gene with 27 exons spanning around 250kbp on chromosome 7q31.2, identified in the search to find the gene underlying cystic fibrosis (CF) disease [1–4]. The protein structure is made up of two units, each with six transmembrane helices and an intracellular nucleotide-binding domain (NBD) that can interact with adenosine triphosphate (ATP). A regulatory “R” domain connects the two units and contains sites for protein kinase phosphorylation [1]. The structure creates a channel in the plasma membrane through which anions can flow, and the gate is thought to be opened and closed by ATP binding and hydrolysis (NBDs) and phosphorylation mechanisms (R domain) which alter the protein’s conformation [1, 5, 6]. CFTR is expressed predominantly in epithelial tissues, but is also found in other cell types such as smooth muscle, cardiac myocytes, macrophages, and erythrocytes [1]. CFTR is multi-functional. It is an anion channel that transports chloride (Cl−) and bicarbonate. It is also involved in the regulation of a range of transporters including the epithelial sodium channel (ENaC encoded by SCNN1A, SCNN1B, SCNN1D and SCNN1G) and outwardly rectifying chloride channels (ORCC) [1, 7–10]. In addition, CFTR has been proposed to be a hub for signaling pathways and may regulate a variety of other physiological processes including exocytosis and endocytosis, ATP export, proinflammatory cytokine expression and intracellular pH [1, 10]. Defective CFTR therefore results in widespread cellular homeostasis dysfunction [10]. CF is an autosomal recessive disease resulting from a defect-causing variant on each CFTR allele. More than 1800 variants in the CFTR gene have been reported [11]. Despite a large collection of variants, there is a gap in our knowledge regarding which cause CF disease. To address this, the Clinical and Functional Translation of CFTR project was established to collect information regarding the functional consequences and resulting phenotypes associated with CFTR variants [12, 13]. Data for 39,696 subjects from 25 CF patient registries or specialty clinics were collected for the database, and an initial set of 159 CFTR variants (those with a frequency of ≥0.01% in the CFTR2 database) was evaluated for whether they cause CF disease by both clinical phenotype and functional analysis [12]. A variant was defined clinically as causing CF if mean sweat chloride concentration was ≥60mM for at least three individuals with the variant or >90mM if only 2 individuals with the variant were available; 140 variants met the clinical criteria to be CF-causing. The variants were sorted by their predicted functional effect, and 77 were investigated further using in vitro assays appropriate to the genetic variant (<10% of wild-type CFTR function was considered disease-causing); 133 variants were deemed CF-causing by functional criteria. In total, 127 variants met both the clinical and functional criteria, and were defined as CF-causing. Penetrance analysis in fathers with CF children was carried out on the variants that did not meet both/either criteria and 12 variants were deemed non-CF causing, with the remaining 20 variants indeterminate [12, 13]. CF is a disease that predominantly affects the lungs but has a diverse array of phenotypes due to the expression of CFTR in different tissues, its wide-ranging physiological role, and its involvement in many signaling pathways [1, 7, 10]. Progressive lung disease, pancreatic dysfunction, infertility in males and elevated sweat electrolytes characterize a “classical” CF diagnosis [14]. CF is also associated with a reduced life expectancy (early adulthood) and an increased risk of cancer [1, 10]. There is, however, wide variability in clinical presentation, severity and the rate of disease progression between patients, which can be influenced by the underlying CFTR genotype as well as other genetic modifiers and environmental factors [1, 7, 10, 14–17]. The incidence of CF is thought to be around 70,000 cases worldwide [18], though it may be largely under-diagnosed in parts of Asia, Africa and Latin America [14, 19]. Genetic testing is now a routine part of CF diagnosis in many countries. A recommended panel for genetic screening for determining prenatal and preconception carrier status of CF in the US includes 23 CFTR variants, designed to cover variants with a frequency of at least 0.1% in CF patients that are associated with classical CF disease, for a pan-ethnic US population [20–22]. The WHO recommends sequencing of the complete CFTR gene in CF patients from populations where CF is likely under-diagnosed in order to establish panels of population-specific variants known to cause disease [14].

Synonymous Codon Usage Affects the Expression of Wild Type and F508del CFTR

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel composed of 1480 amino acids. The major mutation responsible for cystic fibrosis results in loss of amino acid residue, F508 (F508del). Loss of F508 in CFTR alters the folding pathway resulting in endoplasmic-reticulum-associated degradation. This study investigates the role of synonymous codon in the expression of CFTR and CFTR F508del in human HEK293 cells. DNA encoding the open reading frame (ORF) for CFTR containing synonymous codon replacements was expressed using a heterologous vector integrated into the genome. The results indicate that the codon usage greatly affects the expression of CFTR. While the promoter strength driving expression of the ORFs was largely unchanged and the mRNA half-lives were unchanged, the steady-state levels of the mRNA varied by as much as 30-fold. Experiments support that this apparent inconsistency is attributed to nonsense mediated decay independent of exon junction complex. The ratio of CFTR/mRNA indicates that mRNA containing native codons was more efficient in expressing mature CFTR as compared to mRNA containing synonymous high-expression codons. However, when F508del CFTR was expressed after codon optimization, a greater percentage of the protein escaped endoplasmic-reticulum-associated degradation resulting in considerable levels of mature F508del CFTR on the plasma membrane, which showed channel activity. These results indicate that codon usage has an effect on mRNA levels and protein expression, for CFTR, and likely on chaperone-assisted folding pathway, for F508del CFTR.

On the interactions between nucleotide binding domains and membrane spanning domains in cystic fibrosis transmembrane regulator: A molecular dynamic study.

The Cystic Fibrosis Transmembrane Regulator (CFTR) is a membrane protein whose mutations cause cystic fibrosis, a lethal genetic disease. We performed a molecular dynamic (MD) study of the properties of the nucleotide binding domains (NBD) whose conformational changes, upon ATP binding, are the direct responsible of the gating mechanisms of CFTR. This study was done for the wild type (WT) CFTR and for the two most common mutations, ΔF508, that produces a traffic defect of the protein, and the mutation G551D, that causes a gating defect on CFTR. Using an homology model of the open channel conformation of the CFTR we thus introduced the mutations to the structure. Although the overall structures of the G551D and ΔF508 are quite well conserved, the NBD1-NBD2 interactions are severely modified in both mutants. NBD1 and NBD2 are indeed destabilized with a higher internal energy (Ei) in the ΔF508-CFTR. Differently, Ei does not change in the NBDs of G551D but, while the number of close contacts between NBD1 and NBD2 in ΔF508 is increased, a significant reduction of close contacts is found in the G551D mutated form. Hydrogen bonds formation between NBDs of the two mutated forms is also altered and it is slightly increased for the ΔF508, while are severely reduced in G551D. A consequent modification of the NBDs-ICLs interactions between residues involved in the transduction of the ATP binding and the channel gating is also registered. Indeed, while a major interaction is noticed between NBDs interface and ICL2 and ICL4 in the WT, this interaction is somehow altered in both mutated forms plausibly with effect on channel gating. Thus, single point mutations of the CFTR protein can reasonably results in channel gating defects due to alteration of the interaction mechanisms between the NBDs and NBDs-ICLs interfaces upon ATP-binding process.

Investigating the Effect of PKA Phosphorylation on Intramolecular Interactions in Purified Full Length Wildtype CFTR

Cystic fibrosis transmembrane conductance regulator (CFTR) is an unique anion channel of the ATP-Binding Cassette (ABC) superfamily. CFTR consists of two nucleotide binding domains (NBDs), two membrane spanning domains (MSDs) and a regulatory (R) domain. There are also alpha-helical extensions and intracellular loops (ICLs) which couple the NBDs and MSDs via “coupling helices”. The regulation CFTR channel activity involves protein kinase A (PKA) phosphorylation at the R domain. However, not much is known about the effect of phosphorylation on the intramolecular interactions of full length CFTR. We propose that phosphorylation modifies the interactions of full length CFTR, especially at the ICL4:NBD1 interface. Previous studies of a similar ABC transporter, BtuCD, have shown that intrinsic tryptophan fluorescence can detect urea sensitive changes in the coupling between the MSDs and NBDs of full length BtuCD. Thus, we decided monitor intrinsic tryptophan fluorescence to study the effect of phosphorylation on the purified full length Wt CFTR. Interestingly, we found that the urea sensitive changes in the intrinsic tryptophan fluorescence of full length CFTR were modified upon PKA phosphorylation. We were interested to determine whether this result was due to phosphorylation modifying the ICL4:NBD1 interface. In order to specifically study that interface, cysteine crosslinking studies were employed using a short cell permeable cysteine crosslinker on a cys-less CFTR mutant with two cysteines, V510C (NBD1) and A106C7C (ICL4), transfected in HEK293 cells. Phosphorylation induced by cAMP agonists in cells resulted in a significantly increased the crosslinking of cysteines at the ICL4:NBD1 interface compared to the inhibition of phosphorylation with adenyl cyclase inhibitors. This suggests that phosphorylation modifies the ICL4 and NBD1 interface. These studies further our understanding of the molecular mechanisms underlying phosphorylation dependent gating of CFTR.

Covering the Cover

Covering the Cover

Modulation of CFTR gating by permeant ions

Cystic fibrosis transmembrane conductance regulator (CFTR) is unique among ion channels in that after its phosphorylation by protein kinase A (PKA), its ATP-dependent gating violates microscopic reversibility caused by the intimate involvement of ATP hydrolysis in controlling channel closure. Recent studies suggest a gating model featuring an energetic coupling between opening and closing of the gate in CFTR's transmembrane domains and association and dissociation of its two nucleotide-binding domains (NBDs). We found that permeant ions such as nitrate can increase the open probability (Po) of wild-type (WT) CFTR by increasing the opening rate and decreasing the closing rate. Nearly identical effects were seen with a construct in which activity does not require phosphorylation of the regulatory domain, indicating that nitrate primarily affects ATP-dependent gating steps rather than PKA-dependent phosphorylation. Surprisingly, the effects of nitrate on CFTR gating are remarkably similar to those of VX-770 (N-(2,4-Di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide), a potent CFTR potentiator used in clinics. These include effects on single-channel kinetics of WT CFTR, deceleration of the nonhydrolytic closing rate, and potentiation of the Po of the disease-associated mutant G551D. In addition, both VX-770 and nitrate increased the activity of a CFTR construct lacking NBD2 (ΔNBD2), indicating that these gating effects are independent of NBD dimerization. Nonetheless, whereas VX-770 is equally effective when applied from either side of the membrane, nitrate potentiates gating mainly from the cytoplasmic side, implicating a common mechanism for gating modulation mediated through two separate sites of action.

P204 Immune Responses To Single And Repeated Administration Of Pgm169/gl67a: The Uk Cf Gene Therapy Consortium Clinical Trials

Although most CF patients express CFTR protein (albeit mutant) and should therefore not recognise the wild-type CFTR protein as foreign, there is an inherent risk of activation of T-cells against the recombinant wild-type protein after gene therapy. In addition, we have previously shown that approximately 10% of CF and non-CF subjects carry self-reactive CFTR-specific T-cells ( Calcedo et al , Hum Gene Ther Clin Dev 2013 ). The reason for this is unknown and it is also unclear whether being positive for self-reactive T-cells affects disease severity or increases the risk of further T-cell activation after gene therapy. As part of the UKCFGTC Phase I/IIa Pilot study [in which patients received a single dose (5, 10 or 20 mls) of the non-viral formulation pGM169/GL67A] peripheral blood mononuclear cells (PBMC) were collected prior to dosing and approximately 4 weeks after nebulisation of 5 ml (n = 2), 10 ml (n = 6) or 20 ml (n = 17) of pGM169/GL67A. IFN-g ELISPOT to detect CFTR-specific T-cells in PBMC was performed. CFTR-specific T-cells were detectable in one patient pre- and post-dosing. In the remaining 18 patients we did not detect CFTR-specific T-cells. In addition we quantified anti-DNA antibodies (anti-nuclear and anti-cytoplasmic) in blood samples taken pre- and approximately 4 weeks post-dosing (n = 7 (5 ml), n = 10 (10 ml) and n = 17 (20 ml). We did not observe any evidence for induction of anti-DNA antibodies after a single dose of pGM169/GL67A. The UKCFGTC has now completed a Phase IIb multi-dose clinical trial in May 2014 (ClinicalTrials.gov identification number – NCT01621867). CF patients received 12 monthly doses of pGM169/GL67A (115 completed nine or more doses), or placebo by aerosol. PBMC were collected on two occasions prior to dose 1 to establish baseline levels for CFTR-specific T-cells, approximately 4 weeks after Dose 4 or 5, and 2 to 4 weeks after Dose 12 and the ELISPOT was performed. In addition anti-DNA antibodies were quantified. The Phase IIb trial will be unblinded in Summer 2014 to allow data analysis and all data will be presented at the conference. Funded by the NIHR/EME Programme and the Cystic Fibrosis Trust.

Targeted proteomic quantitation of the absolute expression and turnover of cystic fibrosis transmembrane conductance regulator in the apical plasma membrane.

Deficient chloride transport through cystic fibrosis (CF) transmembrane conductance regulator (CFTR) causes lethal complications in CF patients. CF is the most common autosomal recessive genetic disease, which is caused by mutations in the CFTR gene; thus, CFTR mutants can serve as primary targets for drugs to modulate and rescue the ion channel’s function. The first step of drug modulation is to increase the expression of CFTR in the apical plasma membrane (PM); thus, accurate measurement of CFTR in the PM is desired. This work reports a tandem enrichment strategy to prepare PM CFTR and uses a stable isotope labeled CFTR sample as the quantitation reference to measure the absolute amount of apical PM expression of CFTR in CFBE 41o- cells. It was found that CFBE 41o- cells expressing wild-type CFTR (wtCFTR), when cultured on plates, had 2.9 ng of the protein in the apical PM per million cells; this represented 10% of the total CFTR found in the cells. When these cells were polarized on filters, the apical PM expression of CFTR increased to 14%. Turnover of CFTR in the apical PM of baby hamster kidney cells overexpressing wtCFTR (BHK-wtCFTR) was also quantified by targeted proteomics based on multiple reaction monitoring mass spectrometry; wtCFTR had a half-life of 29.0 ± 2.5 h in the apical PM. This represents the first direct measurement of CFTR turnover using stable isotopes. The absolute quantitation and turnover measurements of CFTR in the apical PM can significantly facilitate understanding the disease mechanism of CF and thus the development of new disease-modifying drugs. Absolute CFTR quantitation allows for direct result comparisons among analyses, analysts, and laboratories and will greatly amplify the overall outcome of CF research and therapy.

Functional and pharmacological induced structural changes of the cystic fibrosis transmembrane conductance regulator in the membrane solved using SAXS.

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a membrane-integral protein that belongs to the ATP-binding cassette superfamily. Mutations in the CFTR gene cause cystic fibrosis in which salt, water, and protein transports are defective in various tissues. To investigate the conformation of the CFTR in the membrane, we applied the small-angle x-ray scattering (SAXS) technique on microsomal membranes extracted from NIH/3T3 cells permanentely transfected with wild-type (WT) CFTR and with CFTR carrying the ΔF508 mutation. The electronic density profile of the membranes was calculated from the SAXS data, assuming the lipid bilayer electronic density to be composed by a series of Gaussian shells. The data indicate that membranes in the microsome vesicles, that contain mostly endoplasmic reticulum membranes, are oriented in the outside-out conformation. Phosphorylation does not change significantly the electronic density profile, while dephosphorylation produces a significant modification in the inner side of the profile. Thus, we conclude that the CFTR and its associated protein complex in microsomes are mostly phosphorylated. The electronic density profile of the ΔF508-CFTR microsomes is completely different from WT, suggesting a different assemblage of the proteins in the membranes. Low-temperature treatment of cells rescues the ΔF508-CFTR protein, resulting in a conformation that resembles the WT. Differently, treatment with the corrector VX-809 modifies the electronic profile of ΔF508-CFTR membrane, but does not recover completely the WT conformation. To our knowledge, this is the first report of a directphysical measurement of the structure of membranes containing CFTR in its native environment and in different functional and pharmacological conditions.

Chemical rescue of ΔF508-CFTR in C127 epithelial cells reverses aberrant extracellular pH acidification to wild-type alkalization as monitored by microphysiometry

Cystic fibrosis (CF) is caused by mutations in the gene for CFTR, a cAMP-activated anion channel expressed in apical membranes of wet epithelia. Since CFTR is permeable to HCO3−, and may regulate bicarbonate exchangers, it is not surprising evidence of changes in extracellular pH (pHo) have been found in CF. Previously we have shown that tracking pHo can be used to differentiate cells expressing wild-type CFTR from controls in mouse mammary epithelial (C127) and fibroblast (NIH/3T3) cell lines. In this study we characterized forskolin-stimulated extracellular acidification rates in epithelia where chemical correction of mutant ΔF508-CFTR converted an aberrant response in acidification (10%+ increase) to wild-type (25%+ decrease). Thus treatment with corrector (10% glycerol) and the resulting increased expression of ΔF508-CFTR at the surface was detected by microphysiometry as a significant reversal from acidification to alkalization of pHo. These results suggest that CFTR activation as well as correction can be detected by carefully monitoring pHo and support findings in the field that extracellular pH acidification may impact the function of airway surface liquid in CF.

Biophysical characterisation of calumenin as a charged F508del-CFTR folding modulator.

The cystic fibrosis transmembrane regulator (CFTR) is a cyclic-AMP dependent chloride channel expressed at the apical surface of epithelial cells lining various organs such as the respiratory tract. Defective processing and functioning of this protein caused by mutations in the CFTR gene results in loss of ionic balance, defective mucus clearance, increased proliferation of biofilms and inflammation of human airways observed in cystic fibrosis (CF) patients. The process by which CFTR folds and matures under the influence of various chaperones in the secretory pathway remains incompletely understood. Recently, calumenin, a secretory protein, belonging to the CREC family of low affinity calcium binding proteins has been identified as a putative CFTR chaperone whose biophysical properties and functions remain uncharacterized. We compared hydropathy, instability, charge, unfoldability, disorder and aggregation propensity of calumenin and other CREC family members with CFTR associated chaperones and calcium binding proteins, wild-type and mutant CFTR proteins and intrinsically disordered proteins (IDPs). We observed that calumenin, along with other CREC proteins, was significantly more charged and less folded compared to CFTR associated chaperones. Moreover like IDPs, calumenin and other CREC proteins were found to be less hydrophobic and aggregation prone. Phylogenetic analysis revealed a close link between calumenin and other CREC proteins indicating how evolution might have shaped their similar biophysical properties. Experimentally, calumenin was observed to significantly reduce F508del-CFTR aggregation in a manner similar to AavLEA1, a well-characterized IDP. Fluorescence microscopy based imaging analysis also revealed altered trafficking of calumenin in bronchial cells expressing F508del-CFTR, indicating its direct role in the pathophysiology of CF. In conclusion, calumenin is characterized as a charged protein exhibiting close similarity with IDPs and is hypothesized to regulate F508del-CFTR folding by electrostatic effects. This work provides useful insights for designing optimized synthetic structural correctors of CFTR mutant proteins in the future.

Sinupret activates CFTR and TMEM16A-dependent transepithelial chloride transport and improves indicators of mucociliary clearance.

IntroductionWe have previously demonstrated that Sinupret, an established treatment prescribed widely in Europe for respiratory ailments including rhinosinusitis, promotes transepithelial chloride (Cl−) secretion in vitro and in vivo. The present study was designed to evaluate other indicators of mucociliary clearance (MCC) including ciliary beat frequency (CBF) and airway surface liquid (ASL) depth, but also investigate the mechanisms that underlie activity of this bioflavonoid.MethodsPrimary murine nasal septal epithelial (MNSE) [wild type (WT) and transgenic CFTR−/−], human sinonasal epithelial (HSNE), WT CFTR-expressing CFBE and TMEM16A-expressing HEK cultures were utilized for the present experiments. CBF and ASL depth measurements were performed. Mechanisms underlying transepithelial Cl− transport were determined using pharmacologic manipulation in Ussing chambers, Fura-2 intracellular calcium [Ca2+]i imaging, cAMP signaling, regulatory domain (R-D) phosphorylation of CFTR, and excised inside out and whole cell patch clamp analysis.ResultsSinupret-mediated Cl− secretion [ΔISC(µA/cm2)] was pronounced in WT MNSE (20.7+/−0.9 vs. 5.6+/−0.9(control), p<0.05), CFTR−/− MNSE (10.1+/−1.0 vs. 0.9+/−0.3(control), p<0.05) and HSNE (20.7+/−0.3 vs. 6.4+/−0.9(control), p<0.05). The formulation activated Ca2+ signaling and TMEM16A channels, but also increased CFTR channel open probability (Po) without stimulating PKA-dependent pathways responsible for phosphorylation of the CFTR R-domain and resultant Cl− secretion. Sinupret also enhanced CBF and ASL depth.ConclusionSinupret stimulates CBF, promotes transepithelial Cl− secretion, and increases ASL depth in a manner likely to enhance MCC. Our findings suggest that direct stimulation of CFTR, together with activation of Ca2+-dependent TMEM16A secretion account for the majority of anion transport attributable to Sinupret. These studies provide further rationale for using robust Cl− secretagogue based therapies as an emerging treatment modality for common respiratory diseases of MCC including acute and chronic bronchitis and CRS.

Interference with ubiquitination in CFTR modifies stability of core glycosylated and cell surface pools.

It is recognized that both wild-type and mutant CFTR proteins undergo ubiquitination at multiple lysines in the proteins and in one or more subcellular locations. We hypothesized that ubiquitin is added to specific sites in wild-type CFTR to stabilize it and at other sites to signal for proteolysis. Mass spectrometric analysis of wild-type CFTR identified ubiquitinated lysines 68, 710, 716, 1041, and 1080. We demonstrate that the ubiquitinated K710, K716, and K1041 residues stabilize wild-type CFTR, protecting it from proteolysis. The polyubiquitin linkage is predominantly K63. N-tail mutants, K14R and K68R, lead to increased mature band CCFTR, which can be augmented by proteasomal (but not lysosomal) inhibition, allowing trafficking to the surface. The amount of CFTR in the K1041R mutant was drastically reduced and consisted of bands A/B, suggesting that the site in transmembrane 10 (TM10) was critical to further processing beyond the proteasome. The K1218R mutant increases total and cell surface CFTR, which is further accumulated by proteasomal and lysosomal inhibition. Thus, ubiquitination at residue 1218 may destabilize wild-type CFTR in both the endoplasmic reticulum (ER) and recycling pools. Small molecules targeting the region of residue 1218 to block ubiquitination or to preserving structure at residues 710 to 716 might be protein sparing for some forms of cystic fibrosis.

Stabilizing rescued surface-localized δf508 CFTR by potentiation of its interaction with Na(+)/H(+) exchanger regulatory factor 1.

Cystic fibrosis (CF) is a recessive genetic disease caused by mutations in CFTR, a plasma-membrane-localized anion channel. The most common mutation in CFTR, deletion of phenylalanine at residue 508 (ΔF508), causes misfolding of CFTR resulting in little or no protein at the plasma membrane. The CFTR corrector VX-809 shows promise for treating CF patients homozygous for ΔF508. Here, we demonstrate the significance of protein–protein interactions in enhancing the stability of the ΔF508 CFTR mutant channel protein at the plasma membrane. We determined that VX-809 prolongs the stability of ΔF508 CFTR at the plasma membrane. Using competition-based assays, we demonstrated that ΔF508 CFTR interacts poorly with Na+/H+ exchanger regulatory factor 1 (NHERF1) compared to wild-type CFTR, and VX-809 significantly increased this binding affinity. We conclude that stabilized CFTR–NHERF1 interaction is a determinant of the functional efficiency of rescued ΔF508 CFTR. Our results demonstrate the importance of macromolecular-complex formation in stabilizing rescued mutant CFTR at the plasma membrane and suggest this to be foundational for the development of a new generation of effective CFTR-corrector-based therapeutics.

Oxidative stress modulates the expression of genes involved in cell survival in ΔF508 cystic fibrosis airway epithelial cells.

Although cystic fibrosis (CF) pathophysiology is explained by a defect in CF transmembrane conductance regulator (CFTR) protein, the broad spectrum of disease severity is the consequence of environmental and genetic factors. Among them, oxidative stress has been demonstrated to play an important role in the evolution of this disease, with susceptibility to oxidative damage, decline of pulmonary function, and impaired lung antioxidant defense. Although oxidative stress has been implicated in the regulation of inflammation, its molecular outcomes in CF cells remain to be evaluated. To address the question, we compared the gene expression profile in NuLi-1 cells with wild-type CFTR and CuFi-1 cells homozygous for ΔF508 mutation cultured at air-liquid interface. We analyzed the transcriptomic response of these cell lines with microarray technology, under basal culture conditions and after 24 h oxidative stress induced by 15 μM 2,3-dimethoxy-1,4-naphtoquinone. In the absence of oxidative conditions, CuFi-1 gene profiling showed typical dysregulated inflammatory responses compared with NuLi-1. In the presence of oxidative conditions, the transcriptome of CuFi-1 cells reflected apoptotic transcript modulation. These results were confirmed in the CFBE41o- and corrCFBE41o- cell lines as well as in primary culture of human CF airway epithelial cells. Altogether, our data point to the influence of oxidative stress on cell survival functions in CF and identify several genes that could be implicated in the inflammation response observed in CF patients.

WS13.2 Change in FEV1% predicted in one year in patients with nonsense mutations and patients homozygous for F508del

WS13.1 Patients with mutations that permit 3% or more of wild-type CFTR function are associated with higher FEV1 P.R. Sosnay1, K.S. Raraigh1, F. Vangoor2, H. Yu2, C.M. Penland3, M. Corey4, M.H. Lewis1, C. Castellani5, J. Rommens6, G.R. Cutting1, CFTR2 Study Group. 1Johns Hopkins University, Baltimore, United States; 2Vertex Pharmaceuticals Incorporated, San Diego, United States; 3Cystic Fibrosis Foundation, Bethesda, United States; 4The Hospital for Sick Children, Toronto, Canada; 5Cystic Fibrosis Center, Azienda Ospedaliera Universitaria Integrata, Verona, Italy; 6University of Toronto, Toronto, Canada

WS13.1 Patients with mutations that permit 3% or more of wild-type CFTR function are associated with higher FEV1

WS13.1 Patients with mutations that permit 3% or more of wild-type CFTR function are associated with higher FEV1 P.R. Sosnay1, K.S. Raraigh1, F. Vangoor2, H. Yu2, C.M. Penland3, M. Corey4, M.H. Lewis1, C. Castellani5, J. Rommens6, G.R. Cutting1, CFTR2 Study Group. 1Johns Hopkins University, Baltimore, United States; 2Vertex Pharmaceuticals Incorporated, San Diego, United States; 3Cystic Fibrosis Foundation, Bethesda, United States; 4The Hospital for Sick Children, Toronto, Canada; 5Cystic Fibrosis Center, Azienda Ospedaliera Universitaria Integrata, Verona, Italy; 6University of Toronto, Toronto, Canada