The Calpain,Caspase 12, Caspase 3 Cascade Leading to Apoptosis Is Altered in F508del-CFTR Expressing Cells

In cystic fibrosis (CF), the most frequent mutant variant of the cystic fibrosis transmembrane conductance regulator (CFTR), F508del-CFTR protein, is misfolded and retained in the endoplasmic reticulum (ER). We previously showed that the unfolded protein response (UPR) may be triggered in CF. Since prolonged UPR activation leads to apoptosis via the calcium-calpain-caspase-12-caspase-3 cascade and because apoptosis is altered in CF, our aim was to compare the ER stress-induced apoptosis pathway between wild type (Wt) and F508del-CFTR expressing cells. Here we show that the calcium-calpain-caspase-12-caspase-3 cascade is altered in F508del-CFTR expressing cells. We propose that this alteration is involved in the altered apoptosis triggering observed in CF.     

Deciphering the role of protein kinase CK2 in the maturation/stability of F508del-CFTR

F508del-CFTR, the most common mutation in cystic fibrosis (CF) patients, impairs CFTR trafficking to plasma membrane leading to its premature proteasomal degradation. Several post-translational modifications have been identified on CFTR with multiple roles in stability, localization and channel function, and the possibility to control the enzymes responsible of these modifications has been long considered a potential therapeutic strategy. Protein kinase CK2 has been previously suggested as an important player in regulating CFTR functions and it has been proposed as a pharmacological target in a combinatory therapy to treat CF patients. However, the real implication of CK2 in F508del-CFTR proteostasis, and in particular the hypothesis that its inhibition could be important in CF therapies, is still elusive. Here, by using immortalized cell lines, primary human cells, and knockout cell lines deprived of CK2 subunits, we do not disclose any direct correlation between F508del-CFTR proteostasis and CK2 expression/activity. Rather, our data indicate that the CK2α′ catalytic subunit should be preserved rather than inhibited for F508del rescue by the correctors of class-1, such as VX-809, disclosing new important features in CF therapeutic approaches.     

Rescue of F508del-CFTR by RXR motif inactivation triggers proteome modulation associated with the unfolded protein response

F508del-CFTR, the most common mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, disrupts intracellular trafficking leading to cystic fibrosis (CF). The trafficking defect of F508del-CFTR can be rescued by simultaneous inactivation of its four RXR motifs (4RK). Proteins involved in the F508del-CFTR trafficking defect and/or rescue are therefore potential CF therapeutic targets. We sought to identify these proteins by investigating differential proteome modulation in BHK cells over-expressing wt-CFTR, F508del-CFTR or the revertant F508del/4RK-CFTR. By 2-dimensional electrophoresis-based proteomics and western blot approaches we demonstrated that over-expression of F508del/4RK-CFTR modulates the expression of a large number of proteins, many of which are reported interactors of CFTR and/or 14-3-3 with potential roles in CFTR trafficking. GRP78/BiP, a marker of ER stress and unfolded protein response (UPR), is up-regulated in cells over-expressing either F508del-CFTR or F598del/4RK-CFTR. However, over-expression of F508del/4RK-CFTR induces the up-regulation of many other UPR-associated proteins (e.g. GRP94, PDI, GRP75/mortalin) and, interestingly, the down-regulation of proteasome components associated with CFTR degradation, such as the proteasome activator PA28 (PSME2) and COP9 signalosome (COPS5/CSN5). Moreover, the F508del-CFTR-induced proteostasis imbalance, which involves some heat shock chaperones (e.g. HSP72/Hpa2), ER-EF-hand Ca²⁺-binding proteins (calumenin) and the proteasome activator PA28 (PSME2), tends to be ‘restored’, i.e., in BHK cells over-expressing F508del/4RK-CFTR those proteins tend to have expression levels similar to the wild-type ones. These findings indicate that a particular cellular environment orchestrated by the UPR contributes to and/or is compatible with F508del/4RK-CFTR rescue.     

F508del-CFTR increases intracellular Ca(2+) signaling that causes enhanced calcium-dependent Cl(-) conductance in cystic fibrosis

In many cells, increase in intracellular calcium ([Ca(2+)](i)) activates a Ca(2+)-dependent chloride (Cl(-)) conductance (CaCC). CaCC is enhanced in cystic fibrosis (CF) epithelial cells lacking Cl(-) transport by the CF transmembrane conductance regulator (CFTR). Here, we show that in freshly isolated nasal epithelial cells of F508del-homozygous CF patients, expression of TMEM16A and bestrophin 1 was unchanged. However, calcium signaling was strongly enhanced after induction of expression of F508del-CFTR, which is unable to exit the endoplasmic reticulum (ER). Since receptor-mediated [Ca(2+)](i) increase is Cl(-) dependent, we suggested that F508del-CFTR may function as an ER chloride counter-ion channel for Ca(2+). This was confirmed by expression of the double mutant F508del/G551D-CFTR, which remained in the ER but had no effects on [Ca(2+)](i). Moreover, F508del-CFTR could serve as a scavenger for inositol-1,4,5-trisphosphate [IP3] receptor binding protein released with IP(3) (IRBIT). Our data may explain how ER-localized F508del-CFTR controls intracellular Ca(2+) signaling.     

Maintaining low Ca2+ level in the endoplasmic reticulum restores abnormal endogenous F508del-CFTR trafficking in airway epithelial cells

The most common mutation in cystic fibrosis, F508del, results in cystic fibrosis transmembrane conductance regulator protein (CFTR) that is retained in the endoplasmic reticulum (ER). Retention is dependent on chaperone proteins, many of which, like calnexin, require calcium for optimal activity. Here, we show that a limited and a maintained ER calcium level is sufficient to inhibit the F508del-CFTR/calnexin interaction and to restore the cAMP-dependent CFTR chloride transport, thus showing the correction of abnormal trafficking. We used Western blot analysis, iodide efflux and calcium measurement techniques applied to the human airway epithelial cystic fibrosis cell line CF15 (F508del/F508del). The inhibition of ER calcium pump, with thapsigargin, curcumin, 2,5-di(t-butyl)hydroquinone or cyclopiazonic acid, maintains a threshold levels of calcium that is correlated to the recovery of endogenous F508del-CFTR transport activity. In particular, cyclopiazonic acid restores a 2-aminoethyoxydiphenyl borate-sensitive F508del-CFTR trafficking with an EC50 of 915 nm. By contrast, the 1,4,5-trisphosphate or IP3 receptor activators, i.e., ATP and histamine, while transiently emptying the ER intracellular calcium store, did not affect the trafficking of F508del-CFTR. Our data suggest that decreasing the ER calcium level is not sufficient to restore the defective trafficking of F508del-CFTR, whereas decreasing and also maintaining low ER calcium level allow correction of defective biosynthetic pathway of endogenous F508del-CFTR in human airway epithelial cells.     

The ER‐resident ubiquitin‐specific protease 19 participates in the UPR and rescues ERAD substrates

Ubiquitination regulates membrane events such as endocytosis, membrane trafficking and endoplasmic‐reticulum‐associated degradation (ERAD). Although the involvement of membrane‐associated ubiquitin‐conjugating enzymes and ligases in these processes is well documented, their regulation by ubiquitin deconjugases is less well understood. By screening a database of human deubiquitinating enzymes (DUBs), we have identified a putative transmembrane domain in ubiquitin‐specific protease (USP)19. We show that USP19 is a tail‐anchored ubiquitin‐specific protease localized to the ER and is a target of the unfolded protein response. USP19 rescues the ERAD substrates cystic fibrosis transmembrane conductance regulator (CFTR)ΔF508 and T‐cell receptor‐α (TCRα) from proteasomal degradation. A catalytically inactive USP19 was still able to partly rescue TCRα but not CFTRΔF508, suggesting that USP19 might also exert a non‐catalytic function on specific ERAD substrates. Thus, USP19 is the first example of a membrane‐anchored DUB involved in the turnover of ERAD substrates.     

VIP-dependent increase in F508del-CFTR membrane localization is mediated by PKCε

The most common cystic fibrosis causing mutation F508del induces early degradation and reduced trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels to the apical membrane of epithelial cells. In the human nasal epithelial cells JME/CF15, we previously reported that vasoactive intestinal peptide (VIP) exposure corrects trafficking and membrane insertion of functional F508del-CFTR channels at 37°C. Correction of trafficking was PKA dependent, whereas enhanced membrane localization involved PKC. In the present study, we have identified PKCε as the isoform involved in VIP-dependent F508del-CFTR membrane insertion. Iodide effluxes were used to monitor the presence of VIP-rescued functional F508del-CFTR channels at the surface of JME/CF15 cells maintained at 37°C. Iodide efflux peaks measured in response to stimulation with forskolin were insensitive to PKC α, β, γ, δ, ζ inhibitors. In contrast, efflux peaks were completely inhibited by pretreatment with the PKCε inhibitor peptide EAVSLKPT with an IC(50) of 4.9 μM or by PKCε small interfering RNA (siRNA). Immunostaining and confocal microscopy confirmed that membrane localization of F508del-CFTR induced by VIP was abolished in the presence of EAVSLKPT but not with other isoform inhibitors. In recombinant baby hamster kidney cells, endogenously expressing PKCε but no VIP receptor, wild-type, and F508del-CFTR sensitivity to cpt-cAMP stimulation was increased by PMA treatment. Biotinylation assays and immunoblots confirmed that PMA (0.5-2 h) induced a greater than threefold increase in membrane CFTR, whereas forskolin had no effect. The PMA effect was abolished by specifically inhibiting PKCε (EAVSLKPT IC(50) = 5.7 μM) but not other PKC isoforms. Taken together, these results indicate that stimulating PKCε by VIP or PMA increases membrane insertion and activity of WT- and F508del-CFTR.     

A Highlights from MBoC Selection: Small heat shock proteins target mutant cystic fibrosis transmembrane conductance regulator for degradation via a small ubiquitin-like modifier–dependent pathway

Small heat shock proteins (sHsps) bind destabilized proteins during cell stress and disease, but their physiological functions are less clear. We evaluated the impact of Hsp27, an sHsp expressed in airway epithelial cells, on the common protein misfolding mutant that is responsible for most cystic fibrosis. F508del cystic fibrosis transmembrane conductance regulator (CFTR), a well-studied protein that is subject to cytosolic quality control, selectively associated with Hsp27, whose overexpression preferentially targeted mutant CFTR to proteasomal degradation. Hsp27 interacted physically with Ubc9, the small ubiquitin-like modifier (SUMO) E2 conjugating enzyme, implying that F508del SUMOylation leads to its sHsp-mediated degradation. Enhancing or disabling the SUMO pathway increased or blocked Hsp27’s ability to degrade mutant CFTR. Hsp27 promoted selective SUMOylation of F508del NBD1 in vitro and of full-length F508del CFTR in vivo, which preferred endogenous SUMO-2/3 paralogues that form poly-chains. The SUMO-targeted ubiquitin ligase (STUbL) RNF4 recognizes poly-SUMO chains to facilitate nuclear protein degradation. RNF4 overexpression elicited F508del degradation, whereas Hsp27 knockdown blocked RNF4’s impact on mutant CFTR. Similarly, the ability of Hsp27 to degrade F508del CFTR was lost during overexpression of dominant-negative RNF4. These findings link sHsp-mediated F508del CFTR degradation to its SUMOylation and to STUbL-mediated targeting to the ubiquitin–proteasome system and thereby implicate this pathway in the disposal of an integral membrane protein.     

Calpain digestion and HSP90-based chaperone protection modulate the level of plasma membrane F508del-CFTR

We are here showing that peripheral mononuclear blood cells (PBMC) from cystic fibrosis (CF) patients contain almost undetectable amounts of mature 170 kDa CF-transmembrane conductance regulator (CFTR) and a highly represented 100 kDa form. This CFTR protein, resembling the form produced by calpain digestion and present, although in lower amounts, also in normal PBMC, is localized in cytoplasmic internal vesicles. These observations are thus revealing that the calpain-mediated proteolysis is largely increased in cells from CF patients. To characterize the process leading to the accumulation of such split CFTR, FRT cells expressing the F508del-CFTR mutated channel protein and human leukaemic T cell line (JA3), expressing wild type CFTR were used. In in vitro experiments, the sensitivity of the mutated channel to the protease is identical to that of the wild type, whereas in Ca²⁺-loaded cells F508del-CFTR is more susceptible to digestion. Inhibition of intracellular calpain activity prevents CFTR degradation and leads to a 10-fold increase in the level of F508del-CFTR at the plasma membrane, further indicating the involvement of calpain activity in the maintenance of very low levels of mature channel form. The higher sensitivity to calpain of the mutated 170 kDa CFTR results from a reduced affinity for HSP90 causing a lower degree of protection from calpain digestion. The recovery of HSP90 binding capacity in F508del-CFTR, following digestion, explains the large accumulation of the 100 kDa CFTR form in circulating PBMC from CF patients.     

Pharmacological rescue of the mutant cystic fibrosis transmembrane conductance regulator (CFTR) detected by use of a novel fluorescence platform

Numerous human diseases arise because of defects in protein folding, leading to their degradation in the endoplasmic reticulum. Among them is cystic fibrosis (CF), caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR ), an epithelial anion channel. The most common mutation, F508del, disrupts CFTR folding, which blocks its trafficking to the plasma membrane. We developed a fluorescence detection platform using fluorogen-activating proteins (FAPs) to directly detect FAP-CFTR trafficking to the cell surface using a cell-impermeant probe. By using this approach, we determined the efficacy of new corrector compounds, both alone and in combination, to rescue F508del-CFTR to the plasma membrane. Combinations of correctors produced additive or synergistic effects, improving the density of mutant CFTR at the cell surface up to ninefold over a single-compound treatment. The results correlated closely with assays of stimulated anion transport performed in polarized human bronchial epithelia that endogenously express F508del-CFTR. These findings indicate that the FAP-tagged constructs faithfully report mutant CFTR correction activity and that this approach should be useful as a screening assay in diseases that impair protein trafficking to the cell surface.     

Insights into the mechanisms underlying CFTR channel activity, the molecular basis for cystic fibrosis and strategies for therapy

Mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) cause CF (cystic fibrosis), a fatal genetic disease commonly leading to airway obstruction with recurrent airway inflammation and infection. Pulmonary obstruction in CF has been linked to the loss of CFTR function as a regulated Cl- channel on the lumen-facing membrane of the epithelium lining the airways. We have learned much about the molecular basis for nucleotide- and phosphorylation-dependent regulation of channel activity of the normal (wild-type) version of the CFTR protein through electrophysiological studies. The major CF-causing mutation, F508del-CFTR, causes the protein to misfold and be retained in the ER (endoplasmic reticulum). Importantly, recent studies in cell culture have shown that retention in the ER can be 'corrected' through the application of certain small-molecule modulators and, once at the surface, the altered channel function of the major mutant can be 'potentiated', pharmacologically. Importantly, two such small molecules, a 'corrector' (VX-809) and a 'potentiator' (VX-770) compound are undergoing clinical trial for the treatment of CF. In this chapter, we describe recent discoveries regarding the wild-type CFTR and F508del-CFTR protein, in the context of molecular models based on X-ray structures of prokaryotic ABC (ATP-binding cassette) proteins. Finally, we discuss the promise of small-molecule modulators to probe the relationship between structure and function in the wild-type protein, the molecular defects caused by the most common mutation and the structural changes required to correct these defects.     

Removal of multiple arginine-framed trafficking signals overcomes misprocessing of delta F508 CFTR present in most patients with cystic fibrosis.

Many cystic fibrosis transmembrane conductance regulator (CFTR) mutants are recognized as aberrant by the quality control apparatus at the endoplasmic reticulum (ER) and are targeted for degradation. The mechanism whereby nascent chains are distinguished as either competent or incompetent for ER export has not been elucidated. Here we show that export-incompetent chains display multiple arginine-framed tripeptide sequences like the one recently identified in ATP-sensitive K+ channels. Replacement of arginine residues at positions R29, R516, R555, and R766 with lysine residues to inactivate four of these motifs simultaneously causes delta F508 CFTR, present in approximately 90% of CF patients, to escape ER quality control and function at the cell surface. Interference with recognition of these signals may be helpful in the management of CF.     

FK506 binding protein 8 peptidylprolyl isomerase activity manages a late stage of cystic fibrosis transmembrane conductance regulator (CFTR) folding and stability

Cystic fibrosis (CF) is caused by mutations in the apical chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) with 90% of patients carrying at least one deletion of the F508 (ΔF508) allele. This mutant form of CFTR is characterized by a folding and trafficking defect that prevents exit from the endoplasmic reticulum. We previously reported that ΔF508 CFTR can be recovered in a complex with Hsp90 and its co-chaperones as an on-pathway folding intermediate, suggesting that Δ508 CF disease arises due to a failure of the proteostasis network (PN), which manages protein folding and degradation in the cell. We have now examined the role of FK506-binding protein 8 (FKBP8), a component of the CFTR interactome, during the biogenesis of wild-type and ΔF508 CFTR. FKBP8 is a member of the peptidylprolyl isomerase family that mediates the cis/trans interconversion of peptidyl prolyl bonds. Our results suggest that FKBP8 is a key PN factor required at a post-Hsp90 step in CFTR biogenesis. In addition, changes in its expression level or alteration of its activity by a peptidylprolyl isomerase inhibitor alter CFTR stability and transport. We propose that CF is caused by the sequential failure of the prevailing PN pathway to stabilize ΔF508-CFTR for endoplasmic reticulum export, a pathway that can be therapeutically managed.     

VX-809 corrects folding defects in cystic fibrosis transmembrane conductance regulator protein through action on membrane-spanning domain 1

Cystic fibrosis (CF) is a fatal genetic disorder associated with defective hydration of lung airways due to the loss of chloride transport through the CF transmembrane conductance regulator protein (CFTR). CFTR contains two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a regulatory domain, and its channel assembly requires multiple interdomain contacts. The most common CF-causing mutation, F508del, occurs in NBD1 and results in misfolding and premature degradation of F508del-CFTR. VX-809 is an investigational CFTR corrector that partially restores CFTR function in people who are homozygous for F508del-CFTR. To identify the folding defect(s) in F508del-CFTR that must be repaired to treat CF, we explored the mechanism of VX-809 action. VX-809 stabilized an N-terminal domain in CFTR that contains only MSD1 and efficaciously restored function to CFTR forms that have missense mutations in MSD1. The action of VX-809 on MSD1 appears to suppress folding defects in F508del-CFTR by enhancing interactions among the NBD1, MSD1, and MSD2 domains. The ability of VX-809 to correct F508del-CFTR is enhanced when combined with mutations that improve F508del-NBD1 interaction with MSD2. These data suggest that the use of VX-809 in combination with an additional CFTR corrector that suppresses folding defects downstream of MSD1 may further enhance CFTR function in people with F508del-CFTR.     

Most F508del-CFTR is targeted to degradation at an early folding checkpoint and independently of calnexin

Biosynthesis and folding of multidomain transmembrane proteins is a complex process. Structural fidelity is monitored by endoplasmic reticulum (ER) quality control involving the molecular chaperone calnexin. Retained misfolded proteins undergo ER-associated degradation (ERAD) through the ubiquitin-proteasome pathway. Our data show that the major degradation pathway of the cystic fibrosis transmembrane conductance regulator (CFTR) with F508del (the most frequent mutation found in patients with the genetic disease cystic fibrosis) from the ER is independent of calnexin. Moreover, our results demonstrate that inhibition of mannose-processing enzymes, unlike most substrate glycoproteins, does not stabilize F508del-CFTR, although wild-type (wt) CFTR is drastically stabilized under the same conditions. Together, our data support a novel model by which wt and F508del-CFTR undergo ERAD from two distinct checkpoints, the mutant being disposed of independently of N-glycosidic residues and calnexin, probably by the Hsc70/Hsp70 machinery, and wt CFTR undergoing glycan-mediated ERAD.     

Altered biogenesis of deltaF508-CFTR following treatment with doxorubicin.

Cystic fibrosis (CF) is caused by mutations to the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The most common of these mutations is deletion of a phenylalanine residue at position 508 (Delta F508), which accounts for approximately 70% of all CF alleles. This mutation interferes with the biogenesis and maturation of Delta F508-CFTR to the plasma membrane. However, Delta F508-CFTR can partially function upon proper localization. Thus, pharmacological correction of Delta F508-CFTR maturation holds promise in CF therapy. Our previous studies indicate that a single non-cytotoxic dose of the anthracycline doxorubicin (Dox) significantly increase Delta F508-CFTR-associated chloride secretion in MDCK cells by increasing the expression of this protein at the apical plasma membrane. We report here that Dox alters the biogenesis of Delta F508-CFTR. Treatment with Dox increases the resistance of Delta F508-CFTR to trypsin digestion, possibly by expediting protein folding. Further, treatment with Dox reduces the amount of polyubiquitinated Delta F508-CFTR in cells and prolongs the half-life of this protein. Concomitantly, treatment with Dox decreases the association of Delta F508-CFTR with HSP70 but does not alter the expression of major HSP70 family members. Based on these results, we propose that Dox expedites the folding and maturation of Delta F508-CFTR by acting as a pharmacological chaperone, which consequently promotes the functional expression of this protein in MDCK cells.     

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.     

Establishment and characterization of a novel polarized MDCK epithelial cellular model for CFTR studies.

F508del is the most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that is responsible for the genetic disease Cystic Fibrosis (CF). It results in a major failure of CFTR to traffic to the apical membrane of epithelial cells, where it should function as a chloride (Cl-) channel. Most studies on localization, processing and cellular trafficking of wild-type (wt) and F508del-CFTR have been performed in non-epithelial cells. Notwithstanding, polarized epithelial cells possess distinctly organized and regulated membrane trafficking pathways. We have used Madin-Darby canine kidney (MDCK) type II cells (proximal tubular cells which do not express endogenous CFTR) to generate novel epithelial, polarized cellular models stably expressing wt- or F508del-CFTR through transduction with recombinant lentiviral vectors. Characterization of these cell lines shows that wt-CFTR is correctly processed and apically localized, producing a cAMP-activated Cl- conductance. In contrast, F508del-CFTR is mostly detected in itsimmature form, localized intracellularly and producing only residual Cl- conductance. These novel cell lines constitute bona fide models and significantly improved resources to investigate the molecular mechanisms of polarized membrane traffic of wt- and F508del-CFTR in the same cellular background. They are also useful to identify/validate novel therapeutic compounds for CF.     

Activation of the JNK pathway by nanosecond pulsed electric fields

12-O-tetradecanoyl phorbol-13-acetate-induced sequence 7/interferon related development regulator 1 (Tis7/IFRD1) has been recently identified as a modifier gene in lung inflammatory disease severity in patients with cystic fibrosis (CF), based upon its capacity to regulate inflammatory activities in neutrophils. In CF patients, the F508del mutation in the Cftr gene encoding a chloride channel, the CF transmembrane conductance regulator (CFTR) in airway epithelial cells results in an exaggerated inflammatory response of these cells. At present, it is unknown whether the Tis7/IFRD1 gene product is expressed in airway epithelial cells. We therefore investigated the possibility there is an intrinsic alteration in Tis7/IFRD1 protein level in cells lacking CFTR function in tracheal homogenates of F508del-CFTR mice and in a F508del-CFTR human bronchial epithelial cell line (CFBE41o⁻ cells). When Tis7/IFRD1 protein was detectable, trachea from F508del-CFTR mice showed a reduction in the level of Tis7/IFRD1 protein compared to wild-type control littermates. A significant reduction of IFRD1 protein level was found in CFBE41o⁻ cells compared to normal bronchial epithelial cells 16HBE14o⁻. Surprisingly, messenger RNA level of IFRD1 in CFBE41o⁻ cells was found elevated. Treating CFBE41o⁻ cells with the antioxidant glutathione rescued the IFRD1 protein level closer to control level and also reduced the pro-inflammatory cytokine IL-8 release. This work provides evidence for the first time of reduced level of IFRD1 protein in murine and human F508del-CFTR airway epithelial cell models, possibly mediated in response to oxidative stress which might contribute to the exaggerated inflammatory airway response observed in CF patients homozygous for the F508del mutation.     

F508del CFTR with two altered RXR motifs escapes from ER quality control but its channel activity is thermally sensitive

Most cystic fibrosis (CF) patients carry the F508del mutation in the CFTR chloride channel protein resulting in its misassembly, retention in the endoplasmic reticulum (ER), and proteasomal degradation. Therefore, characterization of the retention and attempts to rescue the mutant CFTR are a major focus of CF research. Earlier, we had shown that four arginine-framed tripeptide (AFT) signals in CFTR participate in the quality control. Now we have mutated these four AFTs in all possible combinations and found that simultaneous inactivation of two of them (R29K and R555K) is necessary and sufficient to overcome F508del CFTR retention. Immunofluorescence staining of BHK cells expressing this variant indicates that it matures and is routed to the plasma membrane. Acquisition of at least some wild-type structure was detected in the pattern of proteolytic digestion fragments. Functional activity at the cell surface was evident in chloride efflux assays. However, single channel activity of the rescued mutant measured in planar lipid bilayers diminished as temperature was increased from 30 to 37 degrees C. These findings support the idea that absence of Phe 508 causes not only a kinetic folding defect but also steady-state structural instability. Therefore effective molecular therapies developed to alleviate disease caused by F508del and probably other misprocessing mutants will require overcoming both their kinetic and steady-state impacts.