Chemical and biological folding contribute to temperature-sensitive DeltaF508 CFTR trafficking

Proteostasis (Balch WE, Morimoto RI, Dillin A, Kelly JW. Adapting proteostasis for disease intervention. Science 2008;319:916-919) refers to the biology that maintains the proteome in health and disease. Proteostasis is challenged by the most common mutant in cystic fibrosis, DeltaF508, a chloride channel [the cystic fibrosis transmembrane conductance regulator (CFTR)] that exhibits a temperature-sensitive phenotype for coupling to the coatomer complex II (COPII) transport machine for exit from the endoplasmic reticulum. Whether rescue of export of DeltaF508 CFTR at reduced temperature simply reflects energetic stabilization of the chemical fold defined by its primary sequence or requires a unique proteostasis environment is unknown. We now show that reduced temperature (30 degrees C) export of DeltaF508 does not occur in some cell types, despite efficient export of wild-type CFTR. We find that DeltaF508 export requires a local biological folding environment that is sensitive to heat/stress-inducible factors found in some cell types, suggesting that the energetic stabilization by reduced temperature is necessary, but not sufficient, for export of DeltaF508. Thus, the cell may require a proteostasis environment that is in part distinct from the wild-type pathway to restore DeltaF508 coupling to COPII. These results are discussed in the context of the energetics of the protein fold and the potential application of small molecules to achieve a proteostasis environment favoring export of a functional form of DeltaF508.     

A domain mimic increases DeltaF508 CFTR trafficking and restores cAMP-stimulated anion secretion in cystic fibrosis epithelia

The major disease-causing mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) is deletion of phenylalanine 508 (F508), which adversely affects processing and plasma membrane targeting of CFTR. Under conditions predicted to stabilize protein folding, F508 CFTR is capable of trafficking to the plasma membrane and retains cAMP-regulated anion channel activity. Overexpression is one factor that increases CFTR trafficking; therefore, we hypothesized that expression of a domain mimic of the first nucleotide-binding fold (NBF1) of CFTR, i.e., the site of F508, may be sufficient to overwhelm the quality control process or otherwise stabilize F508 CFTR and thereby restore cAMP-stimulated anion secretion. In epithelial cells expressing recombinant F508 human (h)CFTR, expression of wild-type NBF1 increased the amount of both core-glycosylated and mature protein to a greater extent than expression of F508 NBF1. Expression of wild-type NBF1 in the F508 hCFTR cells increased whole cell Cl^– current density to 50% of that in cells expressing wild-type hCFTR. Expression of NBF1 in polarized epithelial monolayers from a F508/F508 cystic fibrosis mouse (MGEF) restored cAMP-stimulated transepithelial anion secretion but not in monolayers from a CFTR-null mouse (MGEN). Restoration of anion secretion was sustained in NBF1-expressing MGEF for >30 passages, whereas MGEN corrected with hCFTR progressively lost anion secretion capability. We conclude that expression of a NBF1 domain mimic may be useful for correction of the F508 CFTR protein trafficking defect in cystic fibrosis epithelia. protein processing; mouse; retrovirus; gene therapy     

The DeltaF508 mutation disrupts packing of the transmembrane segments of the cystic fibrosis transmembrane conductance regulator

The most common mutation in cystic fibrosis (deletion of Phe-508 in the first nucleotide binding domain (DeltaF508)) in the cystic fibrosis transmembrane conductance regulator (CFTR) causes retention of the mutant protein in the endoplasmic reticulum. We previously showed that the DeltaF508 mutation causes the CFTR protein to be retained in the endoplasmic reticulum in an inactive and structurally altered state. Proper packing of the transmembrane (TM) segments is critical for function because the TM segments form the chloride channel. Here we tested whether the DeltaF508 mutation altered packing of the TM segments by disulfide cross-linking analysis between TM6 and TM12 in wild-type and DeltaF508 CFTRs. These TM segments were selected because TM6 appears to line the chloride channel, and cross-linking between these TM segments has been observed in the CFTR sister protein, the multidrug resistance P-glycoprotein. We first mapped potential contact points in wild-type CFTR by cysteine mutagenesis and thiol cross-linking analysis. Disulfide cross-linking was detected in CFTR mutants M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12), and W356C(TM6)/W1145C(TM12) in a wild-type background. The disulfide cross-linking occurs intramolecularly and was reducible by dithiothreitol. Introduction of the DeltaF508 mutation into these cysteine mutants, however, abolished cross-linking. The results suggest that the DeltaF508 mutation alters interactions between the TM domains. Therefore, a potential target to correct folding defects in the DeltaF508 mutant of CFTR is to identify compounds that promote correct folding of the TM domains.     

Oral DHA supplementation in DeltaF508 homozygous cystic fibrosis patients

AIM: The aim of this study was to evaluate whether the previously observed changes in the fatty acid profile, as a result of DHA supplementation, could be maintained during longer study trials and to observe its effect on the clinical outcome of cystic fibrosis (CF) patients. METHOD: A year-long double-blind placebo-controlled study was performed in DeltaF508 homozygous CF patients above the age of 6. Clinical data, including pulmonary function and number of infections, were collected. Blood for the determination of the fatty acid (FA) composition of serum phospholipid, vitamin E, liver enzymes, immunoglobulins, erythrocyte sedimentation rate and coagulation was drawn at the beginning and then every 6 months after the start of the study. RESULTS: Seventeen patients were included; one dropped out. The treatment group was supplemented with an algal DHA-rich oil and the control group with sunflower seed oil. There was no difference between the control and treatment groups for W/H%, caloric intake, FEV1% and FVC% at the start of the study and after 1 year of supplements. The phospholipid FA composition did not change in the control group. The treatment group had a significant increase in DHA and eicosapentaenoic acid (EPA) concentration. A concomitant decrease of dihomo-gammalinolenic acid, arachidonic acid, 22:5 n-6 and Mead acid was observed. The laboratory results showed no changes in vitamin E level, liver enzymes, albumin, erythrocyte sedimentation rate and IgG concentration in either the placebo or the intervention group. CONCLUSION: Although DHA-rich oil shifted the serum phospholipid FAs to a less pro-inflammatory profile, no conclusive clinical improvement could be observed so far.     

Enhanced cell-surface stability of rescued DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) by pharmacological chaperones

Misfolded proteins destined for the cell surface are recognized and degraded by the ERAD [ER (endoplasmic reticulum) associated degradation] pathway. TS (temperature-sensitive) mutants at the permissive temperature escape ERAD and reach the cell surface. In this present paper, we examined a TS mutant of the CFTR [CF (cystic fibrosis) transmembrane conductance regulator], CFTR DeltaF508, and analysed its cell-surface trafficking after rescue [rDeltaF508 (rescued DeltaF508) CFTR]. We show that rDeltaF508 CFTR endocytosis is 6-fold more rapid (approximately 30% per 2.5 min) than WT (wild-type, approximately 5% per 2.5 min) CFTR at 37 degrees C in polarized airway epithelial cells (CFBE41o-). We also investigated rDeltaF508 CFTR endocytosis under two further conditions: in culture at the permissive temperature (27 degrees C) and following treatment with pharmacological chaperones. At low temperature, rDeltaF508 CFTR endocytosis slowed to WT rates (20% per 10 min), indicating that the cell-surface trafficking defect of rDeltaF508 CFTR is TS. Furthermore, rDeltaF508 CFTR is stabilized at the lower temperature; its half-life increases from <2 h at 37 degrees C to >8 h at 27 degrees C. Pharmacological chaperone treatment at 37 degrees C corrected the rDeltaF508 CFTR internalization defect, slowing endocytosis from approximately 30% per 2.5 min to approximately 5% per 2.5 min, and doubled DeltaF508 surface half-life from 2 to 4 h. These effects are DeltaF508 CFTR-specific, as pharmacological chaperones did not affect WT CFTR or transferrin receptor internalization rates. The results indicate that small molecular correctors may reproduce the effect of incubation at the permissive temperature, not only by rescuing DeltaF508 CFTR from ERAD, but also by enhancing its cell-surface stability.     

Mild processing defect of porcine DeltaF508-CFTR suggests that DeltaF508 pigs may not develop cystic fibrosis disease

Recent efforts have made significant progress in generating transgenic pigs with the ΔF508-CFTR mutation to model the lung and pancreatic disease of human cystic fibrosis. However, species differences in the processing and function of human, pig and mouse ΔF508-CFTR reported recently raise concerns about the phenotypic consequence of the gene-targeted pig model. The purpose of the present study was to characterize the ΔF508 mutant of porcine CFTR to evaluate the severity of its processing defect. Biochemical and immunofluorescence analysis in transfected COS7 and FRT cells indicated that pig ΔF508-CFTR efficiently targets to the plasma membrane and is present mainly as the mature glycosylated protein. Functional characterization in stably transfected FRT cells by fluorometric and electrophysiological assays supported efficient plasma membrane targeting of pig ΔF508-CFTR. The mild cellular processing defect of pig ΔF508-CFTR suggests that its gene-targeted pig model may not develop the lung and pancreatic phenotypes seen in CF patients.     

Endocytic trafficking routes of wild type and DeltaF508 cystic fibrosis transmembrane conductance regulator

Intracellular trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) is a focus of attention because it is defective in most patients with cystic fibrosis. DeltaF508 CFTR, which does not mature conformationally, normally does not exit the endoplasmic reticulum, but if induced to do so at reduced temperature is short-lived at the surface. We used external epitope-tagged constructs to elucidate the itinerary and kinetics of wild type and DeltaF508 CFTR in the endocytic pathway and visualized movement of CFTR from the surface to intracellular compartments. Modulation of different endocytic steps with low temperature (16 degrees C) block, protease inhibitors, and overexpression of wild type and mutant Rab GTPases revealed that surface CFTR enters several different routes, including a Rab5-dependent initial step to early endosomes, then either Rab11-dependent recycling back to the surface or Rab7-regulated movement to late endosomes or alternatively Rab9-mediated transit to the trans-Golgi network. Without any of these modulations DeltaF508 CFTR rapidly disappears from and does not return to the cell surface, confirming that its altered structure is detected in the distal as well as proximal secretory pathway. Importantly, however, the mutant protein can be rescued at the plasma membrane by Rab11 overexpression, proteasome inhibitors, or inhibition of Rab5-dependent endocytosis.     

Defective formation of PKA/CnA-dependent annexin 2-S100A10/CFTR complex in DeltaF508 cystic fibrosis cells

Cystic fibrosis (CF) is characterised by impaired epithelial ion transport and is caused by mutations in the cystic fibrosis conductance regulator protein (CFTR), a cAMP/PKA and ATP-regulated chloride channel. We recently demonstrated a cAMP/PKA/calcineurin (CnA)-driven association between annexin 2 (anx 2), its cognate partner –S100A10 and cell surface CFTR. The complex is required for CFTR and outwardly rectifying chloride channel function in epithelia. Since the cAMP/PKA-induced Cl⁻ current is absent in CF epithelia, we hypothesized that the anx 2–S100A10/CFTR complex may be defective in CFBE41o cells expressing the commonest F508del-CFTR (ΔF-CFTR) mutation. Here, we demonstrate that, despite the presence of cell surface ΔF-CFTR, cAMP/PKA fails to induce anx 2–S100A10/CFTR complex formation in CFBE41o− cells homozygous for F508del-CFTR. Mechanistically, PKA-dependent serine phosphorylation of CnA, CnA–anx 2 complex formation and CnA-dependent dephosphorylation of anx 2 are all defective in CFBE41o− cells. Immunohistochemical analysis confirms an abnormal cellular distribution of anx 2 in human and CF mouse epithelia.

Thus, we demonstrate that cAMP/PKA/CnA signaling pathway is defective in CF cells and suggest that loss of anx 2–S100A10/CFTR complex formation may contribute to defective cAMP/PKA-dependent CFTR channel function.     

Beta-oestradiol rescues DeltaF508CFTR functional expression in human cystic fibrosis airway CFBE41o- cells through the up-regulation of NHERF1

Background information. CF (cystic fibrosis) is a disease caused by mutations within the CFTR (CF transmembrane conductance regulator) gene. The most common mutation, ΔF508 (deletion of Phe‐508), results in a protein that is defective in folding and trafficking to the cell surface but is functional if properly localized in the plasma membrane. We have recently demonstrated that overexpression of the PDZ protein NHERF1 (Na⁺/H⁺‐exchanger regulatory factor 1) in CF airway cells induced both a redistribution of ΔF508CFTR from the cytoplasm to the apical membrane and the PKA (protein kinase A)‐dependent activation of ΔF508CFTR‐dependent chloride secretion. In view of the potential importance of the targeted up‐regulation of NHERF1 in a therapeutic context, and since it has been demonstrated that oestrogen treatment increases endogenous NHERF1 expression, we tested the hypothesis that oestrogen treatment can increase NHERF1 expression in a human bronchiolar epithelial CF cell line, CFBE41o⁻, with subsequent rescue of apical ΔF508CFTR chloride transport activity. Results. We found that CFBE41o⁻ cells do express ERs (oestrogen receptors) in the nuclear fraction and that β‐oestradiol treatment was able to significantly rescue ΔF508CFTR‐dependent chloride secretion in CFBE41o⁻ cell monolayers with a peak between 6 and 12 h of treatment, demonstrating that the ΔF508CFTR translocated to the apical membrane can function as a cAMP‐responsive channel, with a significant increase in chloride secretion noted at 1 nM β‐oestradiol and a maximal effect observed at 10 nM. Importantly, knock‐down of NHERF1 expression by transfection with siRNA (small interfering RNA) for NHERF1 inhibited the β‐oestradiol‐dependent increase in ΔF508CFTR protein expression levels and completely prevented the β‐oestradiol‐dependent rescue of ΔF508CFTR transport activity. Conclusions. These results demonstrate that β‐oestradiol‐dependent up‐regulation of NHERF1 significantly increases ΔF508CFTR functional expression in CFBE41o⁻ cells.     

Pathophysiological basis of liver disease in cystic fibrosis employing a DeltaF508 mouse model

The molecular pathogenesis of cystic fibrosis (CF) liver disease is unknown. This study investigates its earliest pathophysiological manifestations employing a mouse model carrying DeltaF508, the commonest human CF mutation. We hypothesized that, if increased bile salt spillage into the colon occurs as in the human disease, then this should lead to a hydrophobic bile salt profile and to "hyperbilirubinbilia" because of induced enterohepatic cycling of unconjugated bilirubin. Hyperbilirubinbilia may then lead to an increased bile salt-to-phospholipid ratio in bile and, following hydrolysis, precipitation of divalent metal salts of unconjugated bilirubin. We document in CF mice elevated fecal bile acid excretion and biliary secretion of more hydrophobic bile salts compared with control wild-type mice. Biliary secretion rates of bilirubin monoglucuronosides, bile salts, phospholipids, and cholesterol are increased significantly with an augmented bile salt-to-phospholipid ratio. Quantitative histopathology of CF livers displays mild early cholangiopathy in approximately 53% of mice and multifocal divalent metal salt deposition in cholangiocytes. We conclude that increased fecal bile acid loss leads to more hydrophobic bile salts in hepatic bile and to hyperbilirubinbilia, a major contributor in augmenting the bile salt-to-phospholipid ratio and endogenous beta-glucuronidase hydrolysis of bilirubin glucuronosides. The confluence of these perturbations damages intrahepatic bile ducts and facilitates entrance of unconjugated bilirubin into cholangiocytes. This study of the earliest stages of CF liver disease provides a framework for investigating the molecular pathophysiology of more advanced disease in murine models and in humans with CF.     

The cystic fibrosis ΔF508 mutation in the French population

Summary French families (n = 129) with at least one cystic fibrosis (CF) affected child and 44 unrelated subjects from the general population were tested for the presence of the ΔF508 mutation by the polymerase chain reaction. The ΔF508/CF mutation ratio (CF: uncharacterised CF mutations) was tested in the CF families with and without meconium ileus. The association between ΔF508 and CF mutations and restriction fragment length polymorphism haplotypes (XV2c and KM19) has been estimated; these data suggest that the CF chromosomes include a panel of independent and probably different mutations.     

Beta-oestradiol rescues DeltaF508CFTR functional expression in human cystic fibrosis airway CFBE41o- cells through the up-regulation of NHERF1.

BACKGROUND INFORMATION: CF (cystic fibrosis) is a disease caused by mutations within the CFTR (CF transmembrane conductance regulator) gene. The most common mutation, DeltaF508 (deletion of Phe-508), results in a protein that is defective in folding and trafficking to the cell surface but is functional if properly localized in the plasma membrane. We have recently demonstrated that overexpression of the PDZ protein NHERF1 (Na(+)/H(+)-exchanger regulatory factor 1) in CF airway cells induced both a redistribution of DeltaF508CFTR from the cytoplasm to the apical membrane and the PKA (protein kinase A)-dependent activation of DeltaF508CFTR-dependent chloride secretion. In view of the potential importance of the targeted up-regulation of NHERF1 in a therapeutic context, and since it has been demonstrated that oestrogen treatment increases endogenous NHERF1 expression, we tested the hypothesis that oestrogen treatment can increase NHERF1 expression in a human bronchiolar epithelial CF cell line, CFBE41o(-), with subsequent rescue of apical DeltaF508CFTR chloride transport activity. RESULTS: We found that CFBE41o(-) cells do express ERs (oestrogen receptors) in the nuclear fraction and that beta-oestradiol treatment was able to significantly rescue DeltaF508CFTR-dependent chloride secretion in CFBE41o(-) cell monolayers with a peak between 6 and 12 h of treatment, demonstrating that the DeltaF508CFTR translocated to the apical membrane can function as a cAMP-responsive channel, with a significant increase in chloride secretion noted at 1 nM beta-oestradiol and a maximal effect observed at 10 nM. Importantly, knock-down of NHERF1 expression by transfection with siRNA (small interfering RNA) for NHERF1 inhibited the beta-oestradiol-dependent increase in DeltaF508CFTR protein expression levels and completely prevented the beta-oestradiol-dependent rescue of DeltaF508CFTR transport activity. CONCLUSIONS: These results demonstrate that beta-oestradiol-dependent up-regulation of NHERF1 significantly increases DeltaF508CFTR functional expression in CFBE41o(-) cells.     

Effects of cystic fibrosis transmembrane conductance regulator and DeltaF508CFTR on inflammatory response, ER stress, and Ca2+ of airway epithelia

We tested whether cystic fibrosis (CF) airway epithelia have larger innate immune responses than non-CF or cystic fibrosis transmembrane conductance regulator (CFTR)-corrected cells, perhaps resulting from ER stress due to retention of DeltaF508CFTR in the endoplasmic reticulum (ER) and activation of cytosolic Ca(2+) (Ca(i)) and nuclear factor (NF)-kappaB signaling. Adenovirus infections of a human CF (DeltaF508/DeltaF508) nasal cell line (CF15) provided isogenic comparisons of wild-type (wt) CFTR and DeltaF508CFTR. In the absence of bacteria, there were no or only small differences among CF15, CF15-lacZ (beta-galactosidase-expressing), CF15-wtCFTR (wtCFTR-corrected), and CF15-DeltaF508CFTR (to test ER retention of DeltaF508CFTR) cells in NF-kappaB activity, interleukin (IL)-8 secretion, Ca(i) responses, and ER stress. Non-CF and CF primary cultures of human bronchial epithelial cells (HBE) secreted IL-8 equivalently. Upon infection with Pseudomonas aeruginosa (PA) or flagellin (key activator for airway epithelia), CF15, CF15-lacZ, CF15-wtCFTR, and CF15DeltaF508CFTR cells exhibited equal PA binding, NF-kappaB activity, and IL-8 secretion; cells also responded similarly to flagellin when both CFTR (forskolin) and Ca(i) signaling (ATP) were activated. CF and non-CF HBE responded similarly to flagellin + ATP. Thapsigargin (Tg, releases ER Ca(2+)) increased flagellin-stimulated NF-kappaB and ER stress similarly in all cells. We conclude that ER stress, Ca(i), and NF-kappaB signaling and IL-8 secretion were unaffected by wt- or DeltaF508CFTR in control and during exposure to PA, flagellin, flagellin + ATP, or flagellin + ATP + forskolin. Tg, but not wt- or DeltaF508CFTR, triggered ER stress. Previous measurements showing hyperinflammatory responses in CF airway epithelia may have resulted from cell-specific, rather than CFTR- or DeltaF508CFTR-specific effects.     

Role of calnexin in the ER quality control and productive folding of CFTR; differential effect of calnexin knockout on wild-type and DeltaF508 CFTR

Cystic fibrosis (CF) is caused by the mutation in CF transmembrane conductance regulator (CFTR), a cAMP-dependent Cl(-) channel at the plasma membrane of epithelium. The most common mutant, DeltaF508 CFTR, has competent Cl(-) channel function, but fails to express at the plasma membrane since it is retained in the endoplasmic reticulum (ER) by the ER quality control system. Here, we show that calnexin (CNX) is not necessary for the ER retention of DeltaF508 CFTR. Our data show that CNX knockout (KO) does not affect the biosynthetic processing, cellular localization or the Cl(-) channel function of DeltaF508 CFTR. Importantly, cAMP-induced Cl(-) current in colonic epithelium from CNX KO/DeltaF508 CFTR mice was comparable with that of DeltaF508 CFTR mice, indicating that CNX KO failed to rescue the ER retention of DeltaF508 CFTR in vivo. Moreover, we show that CNX assures the efficient expression of WT CFTR, but not DeltaF508 CFTR, by inhibiting the proteasomal degradation, indicating that CNX might stimulate the productive folding of WT CFTR, but not DeltaF508 CFTR, which has folding defects.     

The ΔF508 mutation in cystic fibrosis patients of Southern Italy

Summary Fifty one independent cystic fibrosis (CF) families originating from a restricted area of Southern Italy (Campania) have been analyzed for KM19 and XV2c haplotypes and the ΔF508 mutation: 54% of the total CF chromosomes show the ΔF508 mutation. No significative correlations were obtained when clinical score, radiological score,Pseudomonas colonization, or clinical symptoms at presentation were matched with the presence or absence of the ΔF508 mutation.