Ubiquitination and Degradation of CFTR by the E3 Ubiquitin Ligase MARCH2 through Its Association with Adaptor Proteins CAL and STX6

Golgi-localized cystic fibrosis transmembrane conductance regulator (CFTR)-associated ligand (CAL) and syntaxin 6 (STX6) regulate the abundance of mature, post-ER CFTR by forming a CAL/STX6/CFTR complex (CAL complex) that promotes CFTR degradation in lysosomes. However, the molecular mechanism underlying this degradation is unknown. Here we investigated the interaction of a Golgi-localized, membrane-associated RING-CH E3 ubiquitin ligase, MARCH2, with the CAL complex and the consequent binding, ubiquitination, and degradation of mature CFTR. We found that MARCH2 not only co-immunoprecipitated and co-localized with CAL and STX6, but its binding to CAL was also enhanced by STX6, suggesting a synergistic interaction. In vivo ubiquitination assays demonstrated the ubiquitination of CFTR by MARCH2, and overexpression of MARCH2, like that of CAL and STX6, led to a dose-dependent degradation of mature CFTR that was blocked by bafilomycin A1 treatment. A catalytically dead MARCH2 RING mutant was unable to promote CFTR degradation. In addition, MARCH2 had no effect on a CFTR mutant lacking the PDZ motif, suggesting that binding to the PDZ domain of CAL is required for MARCH2-mediated degradation of CFTR. Indeed, silencing of endogenous CAL ablated the effect of MARCH2 on CFTR. Consistent with its Golgi localization, MARCH2 had no effect on ER-localized ΔF508-CFTR. Finally, siRNA-mediated silencing of endogenous MARCH2 in the CF epithelial cell line CFBE-CFTR increased the abundance of mature CFTR. Taken together, these data suggest that the recruitment of the E3 ubiquitin ligase MARCH2 to the CAL complex and subsequent ubiquitination of CFTR are responsible for the CAL-mediated lysosomal degradation of mature CFTR.     

Targeting autophagy as a novel strategy for facilitating the therapeutic action of potentiators on ΔF508 cystic fibrosis transmembrane conductance regulator

Channel activators (potentiators) of cystic fibrosis (CF) transmembrane conductance regulator (CFTR), can be used for the treatment of the small subset of CF patients that carry plasma membrane-resident CFTR mutants. However, approximately 90% of CF patients carry the misfolded ΔF508-CFTR and are poorly responsive to potentiators, because ΔF508-CFTR is intrinsically unstable at the plasma membrane (PM) even if rescued by pharmacological correctors. We have demonstrated that human and mouse CF airways are autophagy deficient due to functional sequestration of BECN1 and that the tissue transglutaminase-2 inhibitor, cystamine, or antioxidants restore BECN1-dependent autophagy and reduce SQSTM1/p62 levels, thus favoring ΔF508-CFTR trafficking to the epithelial surface. Here, we investigated whether these treatments could facilitate the beneficial action of potentiators on ΔF508-CFTR homozygous airways. Cystamine or the superoxide dismutase (SOD)/catalase-mimetic EUK-134 stabilized ΔF508-CFTR at the plasma membrane of airway epithelial cells and sustained the expression of CFTR at the epithelial surface well beyond drug withdrawal, overexpressing BECN1 and depleting SQSTM1. This facilitates the beneficial action of potentiators in controlling inflammation in ex vivo ΔF508-CFTR homozygous human nasal biopsies and in vivo in mouse ΔF508-CFTR lungs. Direct depletion of Sqstm1 by shRNAs in vivo in ΔF508-CFTR mice synergized with potentiators in sustaining surface CFTR expression and suppressing inflammation. Cystamine pre-treatment restored ΔF508-CFTR response to the CFTR potentiators genistein, Vrx-532 or Vrx-770 in freshly isolated brushed nasal epithelial cells from ΔF508-CFTR homozygous patients. These findings delineate a novel therapeutic strategy for the treatment of CF patients with the ΔF508-CFTR mutation in which patients are first treated with cystamine and subsequently pulsed with CFTR potentiators.     

Potentiation of ΔF508- and G551D-CFTR-Mediated Cl- Current by Novel Hydroxypyrazolines

The most common mutation of CFTR, affecting approximately 90% of CF patients, is a deletion of phenylalanine at position 508 (F508del, ΔF508). Misfolding of ΔF508-CFTR impairs both its trafficking to the plasma membrane and its chloride channel activity. To identify small molecules that can restore channel activity of ΔF508-CFTR, we synthesized and evaluated eighteen novel hydroxypyrazoline analogues as CFTR potentiators. To elucidate potentiation activities of hydroxypyrazolines for ΔF508-CFTR, CFTR activity was measured using a halide-sensitive YFP assay, Ussing chamber assay and patch-clamp technique. Compounds 7p, 7q and 7r exhibited excellent potentiation with EC₅₀ value <10 μM. Among the compounds, 7q (a novel CFTR potentiator, CP7q) showed the highest potentiation activity with EC₅₀ values of 0.88 ± 0.11 and 4.45 ± 0.31 μM for wild-type and ΔF508-CFTR, respectively. In addition, CP7q significantly potentiated chloride conductance of G551D-CFTR, a CFTR gating mutant; its maximal potentiation activity was 1.9 fold higher than the well-known CFTR potentiator genistein. Combination treatment with CP7q and VX-809, a corrector of ΔF508-CFTR, significantly enhanced functional rescue of ΔF508-CFTR compared with VX-809 alone. CP7q did not alter the cytosolic cAMP level and showed no cytotoxicity at the concentration showing maximum efficacy. The hydroxypyrazolines may be potential development candidates for drug therapy of cystic fibrosis.     

Insulin-Like Growth Factor 1 (IGF-1) Enhances the Protein Expression of CFTR

Low levels of insulin-like growth factor 1 (IGF-1) have been observed in the serum of cystic fibrosis (CF) patients. However, the effects of low serum IGF-1 on the cystic fibrosis transmembrane conductance regulator (CFTR), whose defective function is the primary cause of cystic fibrosis, have not been studied. Here, we show in human cells that IGF-1 increases the steady-state levels of mature wildtype CFTR in a CFTR-associated ligand (CAL)- and TC10-dependent manner; moreover, IGF-1 increases CFTR-mediated chloride transport. Using an acceptor photobleaching fluorescence resonance energy transfer (FRET) assay, we have confirmed the binding of CAL and CFTR in the Golgi. We also show that CAL overexpression inhibits forskolin-induced increases in the cell-surface expression of CFTR. We found that IGF-1 activates TC10, and active TC10 alters the functional association between CAL and CFTR. Furthermore, IGF-1 and active TC10 can reverse the CAL-mediated reduction in the cell-surface expression of CFTR. IGF-1 does not increase the expression of ΔF508 CFTR, whose processing is arrested in the ER. This finding is consistent with our observation that IGF-1 alters the functional interaction of CAL and CFTR in the Golgi. However, when ΔF508 CFTR is rescued with low temperature or the corrector VRT-325 and proceeds to the Golgi, IGF-1 can increase the expression of the rescued ΔF508 CFTR. Our data support a model indicating that CAL-CFTR binding in the Golgi inhibits CFTR trafficking to the cell surface, leading CFTR to the degradation pathway instead. IGF-1-activated TC10 changes the interaction of CFTR and CAL, allowing CFTR to progress to the plasma membrane. These findings offer a potential strategy using a combinational treatment of IGF-1 and correctors to increase the post-Golgi expression of CFTR in cystic fibrosis patients bearing the ΔF508 mutation.     

Thermally unstable gating of the most common cystic fibrosis mutant channel (ΔF508): "rescue" by suppressor mutations in nucleotide binding domain 1 and by constitutive mutations in the cytosolic loops

Most cystic fibrosis (CF) cases are caused by the ΔF508 mutation in the CF transmembrane conductance regulator (CFTR), which disrupts both the processing and gating of this chloride channel. The cell surface expression of ΔF508-CFTR can be "rescued" by culturing cells at 26-28 °C and treating cells with small molecule correctors or intragenic suppressor mutations. Here, we determined whether these various rescue protocols induce a ΔF508-CFTR conformation that is thermally stable in excised membrane patches. We also tested the impact of constitutive cytosolic loop mutations that increase ATP-independent channel activity (K978C and K190C/K978C) on ΔF508-CFTR function. Low temperature-rescued ΔF508-CFTR channels irreversibly inactivated with a time constant of 5-6 min when excised patches were warmed from 22 °C to 36.5 °C. A panel of CFTR correctors and potentiators that increased ΔF508-CFTR maturation or channel activity failed to prevent this inactivation. Conversely, three suppressor mutations in the first nucleotide binding domain rescued ΔF508-CFTR maturation and stabilized channel activity at 36.5 °C. The constitutive loop mutations increased ATP-independent activity of low temperature-rescued ΔF508-CFTR but did not enhance protein maturation. Importantly, the ATP-independent activities of these ΔF508-CFTR constructs were stable at 36.5 °C, whereas their ATP-dependent activities were not. Single channel recordings of this thermally stable ATP-independent activity revealed dynamic gating and unitary currents of normal amplitudes. We conclude that: (i) ΔF508-CFTR gating is highly unstable at physiologic temperature; (ii) most rescue protocols do not prevent this thermal instability; and (iii) ATP-independent gating and the pore are spared from ΔF508-induced thermal instability, a finding that may inform alternative treatment strategies.     

LMTK2-mediated Phosphorylation Regulates CFTR Endocytosis in Human Airway Epithelial Cells

Cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl⁻-selective ion channel expressed in fluid-transporting epithelia. Lemur tyrosine kinase 2 (LMTK2) is a transmembrane protein with serine and threonine but not tyrosine kinase activity. Previous work identified CFTR as an in vitro substrate of LMTK2, suggesting a functional link. Here we demonstrate that LMTK2 co-immunoprecipitates with CFTR and phosphorylates CFTR-Ser⁷³⁷ in human airway epithelial cells. LMTK2 knockdown or expression of inactive LMTK2 kinase domain increases cell surface density of CFTR by attenuating its endocytosis in human airway epithelial cells. Moreover, LMTK2 knockdown increases Cl⁻ secretion mediated by the wild-type and rescued ΔF508-CFTR. Compared with the wild-type CFTR, the phosphorylation-deficient mutant CFTR-S737A shows increased cell surface density and decreased endocytosis. These results demonstrate a novel mechanism of the phospho-dependent inhibitory effect of CFTR-Ser⁷³⁷ mediated by LMTK2 via endocytosis and inhibition of the cell surface density of CFTR Cl⁻ channels. These data indicate that targeting LMTK2 may increase the cell surface density of CFTR Cl⁻ channels and improve stability of pharmacologically rescued ΔF508-CFTR in patients with cystic fibrosis.     

Cross-linking of F508-CFTR promotes its trafficking to the plasma membrane

CFTR is a cAMP-activated chloride channel responsible for agonist stimulated chloride and fluid transport across epithelial surfaces.¹ Mutations in the CFTR gene lead to cystic fibrosis (CF) which affects the function of secretory organs like the intestine, the pancreas, the airways and the sweat glands. Most of the morbidity and mortality in CF has been linked to a decrease in airway function.² The ΔF508 mutation is the most common CF-related mutation in the Caucasian population and represents 90% of CF alleles. Homozygote carriers of this mutation present with a severe CF phenotype.³ The ΔF508 mutation causes misfolding of the nascent CFTR polypeptide, which leads to inefficient export from the endoplasmic reticulum (ER) and rapid degradation by the proteasome.⁴Key words: cystic fibrosis, endoplasmic reticulum, oligomer, processing mutation, curcuminGiven the frequency of the ΔF508 processing mutation and the severity of its corresponding phenotype, much research has focused on identifying compounds that restore the trafficking and function of this mutant at the plasma membrane. Several synthetic ‘correctors’ of ΔF508 mis-processing and ‘potentiators’ of mutant channel activity have been identified.^5,6 Natural compounds such as curcumin also have generated interest. Curcumin is an organic phenolic compound abundant in turmeric, an Indian spice extracted from the rhizome of Curcuma longa.⁷ Earlier studies performed using ΔF508/ΔF508 mouse models and human airway epithelial cell lines suggested that curcumin may act as a ΔF508-CFTR trafficking corrector.⁸ Also, we and others showed that curcumin stimulates CFTR channel activity in excised membrane patches.^9,10 This stimulation occurs in the absence of ATP binding, which is normally required for channel opening.¹⁰ Binding sites of correctors and potentiators within the CFTR polypeptide as well as the molecular mechanisms underlying the rescue of CFTR trafficking and function remain to be elucidated. In our attempt to understand how curcumin could circumvent the normally critical step of ATP binding to promote CFTR channel activity we investigated the effect of curcumin on CFTR conformation by using biochemical assays. We showed that curcumin caused dimerization of several CFTR channel constructs (including ΔF508-CFTR) in a dose- and time-dependent manner both in microsomes and within intact cells. This effect of curcumin on CFTR oligomerization is attributable to its reactive β-diketone groups, which may undergo an oxidation reaction with CFTR nucleophilic amino acid residues.¹¹ Importantly, CFTR channel activation by curcumin is unrelated to its cross-linking effect. We identified cyclic derivatives of curcumin that lack this cross-linking activity but still promote CFTR channel function.¹¹Here we examined the possibility that the cross-linking of ΔF508-CFTR channels by curcumin promotes the delivery of this ER processing mutant to the cell surface. We were motivated to test this possibility for three reasons: (i) our previous evidence that curcumin-induced dimers of wild-type CFTR polypeptides were detected at the cell surface where they remained over an hour after the removal of curcumin;¹¹ (ii) the very efficient cross-linking of the immature (ER) forms of wild-type CFTR and the ΔF508-CFTR mutant that we observed earlier¹¹ and (iii) prior evidence from our group that the ER export and cell surface delivery of ΔF508-CFTR polypeptides could be promoted by the co-expression of this mutant with certain CFTR fragments (trans-complementation).¹² The latter result might be due to the existence of ER retention ‘signals’ that are exposed on the ΔF508-CFTR polypeptide but become buried by interacting (complementing) fragments.Figure 1 provides evidence that ΔF508-CFTR oligomers that form in response to curcumin treatment do indeed appear at the surfaces of cultured airway epithelial cells (CF bronchial epithelial (CFBE) cells stably transfected with this CFTR mutant). Surface biotinylation assays were performed to detect the appearance of ΔF508-CFTR polypeptides at the cell surface. MESNA, a cell impermeant reducing agent that cleaves the biotin label, was used to verify the surface accessibility of the labeled ΔF508-CFTR polypeptides. ΔF508-CFTR polypeptides were precipititated with streptavidinagarose (surface pool) or with a CFTR monoclonal antibody (total pool). In the absence of curcumin treatment the great majority of the ΔF508-CFTR protein existed as the ER form (monomeric band B), as previously observed by many investigators (Fig. 1, lane 5). No band B was detected in the surface pool before or after curcumin treatment (Fig. 1, lanes 1, 2). As we reported earlier, treatment of the cells with 50 µM curcumin for 15 mins at 37°C cross-linked nearly all of the ΔF508-CFTR polypeptides into higher order complexes (e.g., dimers, termed band D here; lanes 6–8 in Fig. 1). Interestingly, these higher order forms of ΔF508-CFTR were readily apparent in the surface pool (Fig. 1, lane 2).Open in a separate windowFigure 1ΔF508-CFTR oligomers detected at the surfaces of airway epithelial cells after curcumin treatment. ΔF508-CFTR expressing CFBE cells were treated with curcumin (50 µM) for 15 min at 37°C. Cell surface proteins were then biotinylated (Sulfo-NHS-SS-Biotin, 1 mg/ml) for 30 min at 4°C followed by cell lysis with 1% Triton X-100. Surface proteins were isolated by streptavidin pulldown and ΔF508-CFTR was isolated from the total cell protein pool by immunoprecipitation with an anti-CFTR C-terminus antibody (clone 24-1, R&D systems). After SDS-PAGE the ΔF508-CFTR signal was detected by immunoblotting using the 24-1 antibody described above. (SP: streptavidin pulldown; IP: immunoprecipitation). As an additional control curcumin-treated cells were treated with the cell impermeant MESNA after biotinylation to strip the biotin off the cell surface proteins with which it had reacted.CFTR oligomers also can be generated by standard chemical cross-linkers such as DSS, as previously reported by others and confirmed by us.¹³ Figure 2 shows that oligomers of ΔF508-CFTR that are induced by DSS treatment also appear in the surface pool. These experiments were performed using transiently transfected HEK-293T cells with 30 µM curcumin as a positive control. Quantitative densitometry results are shown in Figure 3. By titrating the DSS concentration we observed a dose-dependent disappearance of the monomeric band B form, a corresponding increase in the band D (dimer) pool and the appearance of higher order oligomers (band E) which prevailed at higher DSS concentrations (see total cell pool data in right-hand). A small amount of the band D form was detected in the absence of DSS or curcumin treatment, which might represent some spontaneous cross-linking of ΔF508-CFTR polypeptides under these conditions. The DSS and curcumin-induced ΔF508-CFTR oligomers were readily detected in the surface pool. The densitometry analysis revealed that 20 ± 5% and 33 ± 19% of the total oligomer pool (combined bands D and E) was found in the surface pool after treatment with 0.1 mM DSS (n = 3) or 30 µM curcumin (n = 3), respectively, which corresponded to a 17 ± 7 and 26 ± 20 fold increase compared to the control condition (i.e., no DSS or no curcumin).Open in a separate windowFigure 2ΔF508-CFTR oligomers detected at the surfaces of HEK cells after DSS or curcumin treatment. ΔF508-CFTR expressing HEK cells were treated with the indicated concentrations of DSS or with 30 µM curcumin (*) for 15 min at 37°C. Cell surface proteins were then biotinylated and isolated by streptavidin pulldown as described above. ΔF508-CFTR was immunoprecipitated from the total cell protein pool with the 24-1 antibody and detected by immunoblotting as before (SP: streptavidin pulldown; IP: immunoprecipitation). Band B corresponds to ΔF508 monomer (ER form). Band D corresponds to ΔF508 dimer. Band E corresponds to a higher degree of ΔF508 oligomerization. Each panel corresponds to a different exposure of the same blot.Open in a separate windowFigure 3Dose-dependent expression of ΔF508-CFTR oligomers at the surfaces of HEK cells after DSS treatment. CFTR signals detected by the 24-1 antibody from three different experiments as the one described in Figure 2 were analyzed using the ImageJ software (from the National Institute of Health). (A) band B signal intensity is plotted as a function of the DSS concentrations. Signals analyzed correspond to ΔF508-CFTR band B immunoprecipitated by the 24-1 antibody. (B) band D plus band E signal intensities are plotted as a function of the DSS concentration. Signals analyzed correspond to the sum of ΔF508-CFTR band D and band E immunoprecipitated by the 24-1 antibody. (C) band D plus band E signal intensities at the cell surface are plotted as a function of the DSS concentration. Signals analyzed correspond to the sum of ΔF508-CFTR band D and band E isolated from the surfaces of ΔF508-CFTR expressing HEK cells by biotinylation and streptavidin pulldown. (D) the ratio between the amount of band E and D at the surfaces of ΔF508-CFTR expressing HEK cells is plotted as a function of the DSS concentration. Error bars are SEMs.Altogether these data indicate that the cross-linking of ΔF508-CFTR band B into oligomers by curcumin or DSS allows ΔF508-CFTR to traffic to the cell surface. This effect might be caused by the burial of ER retention motifs within the oligomer, which also could explain our previous trans-complementation results in which we observed that certain CFTR fragments promote the cell surface delivery of this processing mutant.¹² Although non-specific protein cross-linkers like DSS would not be therapeutically beneficial, more specific CFTR cross-linkers (perhaps curcumin?) may be worth considering for treating CF disease linked to ER processing mutations in CFTR. In this regard, we note that cross-linked CFTR polypeptides appear to retain chloride channel activity. Namely, in our prior excised patch clamp studies we observed stable CFTR channel activity when these patches were exposed to curcumin at doses and times that promote robust cross-linking of CFTR polypeptides.^10,11     

Reduced PDZ Interactions of Rescued ΔF508CFTR Increases Its Cell Surface Mobility

Deletion of phenylalanine 508 (ΔF508) in the cystic fibrosis transmembrane conductance regulator (CFTR) plasma membrane chloride channel is the most common cause of cystic fibrosis (CF). Though several maneuvers can rescue endoplasmic reticulum-retained ΔF508CFTR and promote its trafficking to the plasma membrane, rescued ΔF508CFTR remains susceptible to quality control mechanisms that lead to accelerated endocytosis, ubiquitination, and lysosomal degradation. To investigate the role of scaffold protein interactions in rescued ΔF508CFTR surface instability, the plasma membrane mobility of ΔF508CFTR was measured in live cells by quantum dot single particle tracking. Following rescue by low temperature, chemical correctors, thapsigargin, or overexpression of GRASP55, ΔF508CFTR diffusion was more rapid than that of wild-type CFTR because of reduced interactions with PDZ domain-containing scaffold proteins. Knock-down of the plasma membrane quality control proteins CHIP and Hsc70 partially restored ΔF508CFTR-scaffold association. Quantitative comparisons of CFTR cell surface diffusion and endocytosis kinetics suggested an association between reduced scaffold binding and CFTR internalization. Our surface diffusion measurements in live cells indicate defective scaffold interactions of rescued ΔF508CFTR at the cell surface, which may contribute to its defective peripheral processing.     

ΔF508 CFTR Surface Stability Is Regulated by DAB2 and CHIP-Mediated Ubiquitination in Post-Endocytic Compartments

The ΔF508 mutant form of the cystic fibrosis transmembrane conductance regulator (ΔF508 CFTR) that is normally degraded by the ER-associated degradative pathway can be rescued to the cell surface through low-temperature (27°C) culture or small molecular corrector treatment. However, it is unstable on the cell surface, and rapidly internalized and targeted to the lysosomal compartment for degradation. To understand the mechanism of this rapid turnover, we examined the role of two adaptor complexes (AP-2 and Dab2) and three E3 ubiquitin ligases (c-Cbl, CHIP, and Nedd4-2) on low-temperature rescued ΔF508 CFTR endocytosis and degradation in human airway epithelial cells. Our results demonstrate that siRNA depletion of either AP-2 or Dab2 inhibits ΔF508 CFTR endocytosis by 69% and 83%, respectively. AP-2 or Dab2 depletion also increases the rescued protein half-life of ΔF508 CFTR by ~18% and ~91%, respectively. In contrast, the depletion of each of the E3 ligases had no effect on ΔF508 CFTR endocytosis, whereas CHIP depletion significantly increased the surface half-life of ΔF508 CFTR. To determine where and when the ubiquitination occurs during ΔF508 CFTR turnover, we monitored the ubiquitination of rescued ΔF508 CFTR during the time course of CFTR endocytosis. Our results indicate that ubiquitination of the surface pool of ΔF508 CFTR begins to increase 15 min after internalization, suggesting that CFTR is ubiquitinated in a post-endocytic compartment. This post-endocytic ubiquination of ΔF508 CFTR could be blocked by either inhibiting endocytosis, by siRNA knockdown of CHIP, or by treating cells with the CFTR corrector, VX-809. Our results indicate that the post-endocytic ubiquitination of CFTR by CHIP is a critical step in the peripheral quality control of cell surface ΔF508 CFTR.     

Combination of Correctors Rescue ΔF508-CFTR by Reducing Its Association with Hsp40 and Hsp27

Correcting the processing of ΔF508-CFTR, the most common mutation in cystic fibrosis, is the major goal in the development of new therapies for this disease. Here, we determined whether ΔF508 could be rescued by a combination of small-molecule correctors, and identified the mechanism by which correctors rescue the trafficking mutant of cystic fibrosis transmembrane conductance regulator (CFTR). We transfected COS-7 cells with ΔF508, created HEK-293 stably expressing ΔF508, and utilized CFBE41o⁻ cell lines stably transduced with ΔF508. As shown previously, ΔF508 expressed less protein, was unstable at physiological temperature, and rapidly degraded. When the cells were treated with the combination C18 + C4 the mature C-band was expressed at the cell surface. After treatment with C18 + C4, we saw a lower rate of protein disappearance after translation was stopped with cycloheximide. To understand how this rescue occurs, we evaluated the change in the binding of proteins involved in endoplasmic reticulum-associated degradation, such as Hsp27 (HspB1) and Hsp40 (DnaJ). We saw a dramatic reduction in binding to heat shock proteins 27 and 40 following combined corrector therapy. siRNA experiments confirmed that a reduction in Hsp27 or Hsp40 rescued CFTR in the ΔF508 mutant, but the rescue was not additive or synergistic with C4 + 18 treatment, indicating these correctors shared a common pathway for rescue involving a network of endoplasmic reticulum-associated degradation proteins.     

RNA Interference Screen to Identify Kinases That Suppress Rescue of ΔF508-CFTR

Cystic Fibrosis (CF) is an autosomal recessive disorder caused by mutations in the gene encoding the Cystic fibrosis transmembrane conductance regulator (CFTR). ΔF508-CFTR, the most common disease-causing CF mutant, exhibits folding and trafficking defects and is retained in the endoplasmic reticulum, where it is targeted for proteasomal degradation. To identify signaling pathways involved in ΔF508-CFTR rescue, we screened a library of endoribonuclease-prepared short interfering RNAs (esiRNAs) that target ∼750 different kinases and associated signaling proteins. We identified 20 novel suppressors of ΔF508-CFTR maturation, including the FGFR1. These were subsequently validated by measuring channel activity by the YFP halide-sensitive assay following shRNA-mediated knockdown, immunoblotting for the mature (band C) ΔF508-CFTR and measuring the amount of surface ΔF508-CFTR by ELISA. The role of FGFR signaling on ΔF508-CFTR trafficking was further elucidated by knocking down FGFRs and their downstream signaling proteins: Erk1/2, Akt, PLCγ-1, and FRS2. Interestingly, inhibition of FGFR1 with SU5402 administered to intestinal organoids (mini-guts) generated from the ileum of ΔF508-CFTR homozygous mice resulted in a robust ΔF508-CFTR rescue. Moreover, combination of SU5402 and VX-809 treatments in cells led to an additive enhancement of ΔF508-CFTR rescue, suggesting these compounds operate by different mechanisms. Chaperone array analysis on human bronchial epithelial cells harvested from ΔF508/ΔF508-CFTR transplant patients treated with SU5402 identified altered expression of several chaperones, an effect validated by their overexpression or knockdown experiments. We propose that FGFR signaling regulates specific chaperones that control ΔF508-CFTR maturation, and suggest that FGFRs may serve as important targets for therapeutic intervention for the treatment of CF.Cystic fibrosis (CF)¹ is a pleiotropic disease caused by an abnormal ion transport in the secretory epithelia lining the tubular organs of the body such as lungs, intestines, pancreas, liver, and male reproductive tract. In the airways of CF patients, reduced Cl⁻ and bicarbonate secretion caused by lack of functional Cystic fibrosis transmembrane conductance regulator (CFTR) on the apical surface, and hyper-absorption of Na⁺ because of elevated activity of ENaC (1), lead to a dehydration of the airway surface liquid (ASL). This reduces the viscosity of the mucus layer and the deposited layer of thickened mucus creates an environment that promotes bacterial colonization, which eventually leads to chronic infection of the lungs and death (2, 3).CFTR is a transmembrane protein that functions as a cAMP-regulated, ATP-dependent Cl⁻ channel that also allows passage of bicarbonate through its pore (4, 5). It also possesses ATPase activity important for Cl⁻ conductance (6, 7). The CFTR structure is predicted to consist of five domains: two membrane spanning domains (MSD1, MSD2), each composed of six putative transmembrane helices, two nucleotide binding domains (NBD1, NBD2), and a unique regulatory (R) region (8).More than 1900 CFTR mutations have been identified to date (www.genet.sickkids.on.ca/cftr). The most common mutation is a deletion of phenylalanine at position 508 (ΔF508 or ΔF508-CFTR) in NBD1 (9). The ΔF508 mutation causes severe defects in the processing and function of CFTR. The protein exhibits impaired trafficking from the endoplasmic reticulum (ER) to the plasma membrane (PM), impaired intramolecular interactions between NBD1 and the transmembrane domain, and cell surface instability (10–15). Nevertheless, the ΔF508 defect can be corrected, because treating cells expressing ΔF508-CFTR with low temperature or chemical chaperones (e.g. glycerol) can restore some surface expression of the mutant (11, 16).Numerous small molecules that can at least partially correct (or potentiate) the ΔF508-CFTR defect have been identified to date (17–27), and some were already tested in clinical trials (e.g. sildenafil, VX-809/Lumacaftor), or have made it to the clinic (VX-770/Kalydeco/Ivacaftor) (http://www.cff.org/research/DrugDevelopmentPipeline/). However, the need to identify new ΔF508-CFTR correctors remains immense as the most promising corrector, VX-809, has proven ineffective in alleviating lung disease of CF patients when administered alone (27). Thus, our group developed a high-content technology aimed at identifying proteins and small molecules that correct the trafficking and functional defects of ΔF508-CFTR (28). We successfully used this approach to carry out three separate high-content screens: a protein overexpression screen (28), a small-molecule kinase inhibitor screen (29) and a kinome RNA interference (RNAi) screen, described here.     

Targeting CAL as a negative regulator of DeltaF508-CFTR cell-surface expression: an RNA interference and structure-based mutagenetic approach

PDZ domains are ubiquitous peptide-binding modules that mediate protein-protein interactions in a wide variety of intracellular trafficking and localization processes. These include the pathways that regulate the membrane trafficking and endocytic recycling of the cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel mutated in patients with cystic fibrosis. Correspondingly, a number of PDZ proteins have now been identified that directly or indirectly interact with the C terminus of CFTR. One of these is CAL, whose overexpression in heterologous cells directs the lysosomal degradation of WT-CFTR in a dose-dependent fashion and reduces the amount of CFTR found at the cell surface. Here, we show that RNA interference targeting endogenous CAL specifically increases cell-surface expression of the disease-associated DeltaF508-CFTR mutant and thus enhances transepithelial chloride currents in a polarized human patient bronchial epithelial cell line. We have reconstituted the CAL-CFTR interaction in vitro from purified components, demonstrating for the first time that the binding is direct and allowing us to characterize its components biochemically and biophysically. To test the hypothesis that inhibition of the binding site could also reverse CAL-mediated suppression of CFTR, a three-dimensional homology model of the CAL.CFTR complex was constructed and used to generate a CAL mutant whose binding pocket is correctly folded but has lost its ability to bind CFTR. Although produced at the same levels as wild-type protein, the mutant does not affect CFTR expression levels. Taken together, our data establish CAL as a candidate therapeutic target for correction of post-maturational trafficking defects in cystic fibrosis.     

Computational design of a PDZ domain peptide inhibitor that rescues CFTR activity

The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial chloride channel mutated in patients with cystic fibrosis (CF). The most prevalent CFTR mutation, ΔF508, blocks folding in the endoplasmic reticulum. Recent work has shown that some ΔF508-CFTR channel activity can be recovered by pharmaceutical modulators ("potentiators" and "correctors"), but ΔF508-CFTR can still be rapidly degraded via a lysosomal pathway involving the CFTR-associated ligand (CAL), which binds CFTR via a PDZ interaction domain. We present a study that goes from theory, to new structure-based computational design algorithms, to computational predictions, to biochemical testing and ultimately to epithelial-cell validation of novel, effective CAL PDZ inhibitors (called "stabilizers") that rescue ΔF508-CFTR activity. To design the "stabilizers", we extended our structural ensemble-based computational protein redesign algorithm K* to encompass protein-protein and protein-peptide interactions. The computational predictions achieved high accuracy: all of the top-predicted peptide inhibitors bound well to CAL. Furthermore, when compared to state-of-the-art CAL inhibitors, our design methodology achieved higher affinity and increased binding efficiency. The designed inhibitor with the highest affinity for CAL (kCAL01) binds six-fold more tightly than the previous best hexamer (iCAL35), and 170-fold more tightly than the CFTR C-terminus. We show that kCAL01 has physiological activity and can rescue chloride efflux in CF patient-derived airway epithelial cells. Since stabilizers address a different cellular CF defect from potentiators and correctors, our inhibitors provide an additional therapeutic pathway that can be used in conjunction with current methods.     

A Synonymous Single Nucleotide Polymorphism in ΔF508 CFTR Alters the Secondary Structure of the mRNA and the Expression of the Mutant Protein

Recent advances in our understanding of translational dynamics indicate that codon usage and mRNA secondary structure influence translation and protein folding. The most frequent cause of cystic fibrosis (CF) is the deletion of three nucleotides (CTT) from the cystic fibrosis transmembrane conductance regulator (CFTR) gene that includes the last cytosine (C) of isoleucine 507 (Ile507ATC) and the two thymidines (T) of phenylalanine 508 (Phe508TTT) codons. The consequences of the deletion are the loss of phenylalanine at the 508 position of the CFTR protein (ΔF508), a synonymous codon change for isoleucine 507 (Ile507ATT), and protein misfolding. Here we demonstrate that the ΔF508 mutation alters the secondary structure of the CFTR mRNA. Molecular modeling predicts and RNase assays support the presence of two enlarged single stranded loops in the ΔF508 CFTR mRNA in the vicinity of the mutation. The consequence of ΔF508 CFTR mRNA “misfolding” is decreased translational rate. A synonymous single nucleotide variant of the ΔF508 CFTR (Ile507ATC), that could exist naturally if Phe-508 was encoded by TTC, has wild type-like mRNA structure, and enhanced expression levels when compared with native ΔF508 CFTR. Because CFTR folding is predominantly cotranslational, changes in translational dynamics may promote ΔF508 CFTR misfolding. Therefore, we propose that mRNA “misfolding” contributes to ΔF508 CFTR protein misfolding and consequently to the severity of the human ΔF508 phenotype. Our studies suggest that in addition to modifier genes, SNPs may also contribute to the differences observed in the symptoms of various ΔF508 homozygous CF patients.     

Chemical chaperones correct the mutant phenotype of the ΔF508 cystic fibrosis transmembrane conductance regulator protein

Mutations in the cystic fibriosis transmembrane conductance regulator protein (CFTR) often result in a failure of the protein to be propely processed at the level of the endoplasmic reticulum (ER) and subsequently transported to the plasma membrane. The folding defect associated with the most common CFTR mutation (ΔF508) has been shown to be temperature sensitive. Incubation of cells expressing ΔF508 CFTR at lower growth temperatures results in the proper processing of a portion of the mutant CFTR protein. Under these conditions, the mutant protein can move to the plasma membrane where it functions, similar to the wild-type protein, in mediating cholride transport. We set out to identify other methods, which like temperature treatment, would rescue the folding defect associated with the ΔF508 CFTR mutation. Here we show that treatment of cells expressing the ΔF508 mutant with a number of low molecular weight compounds, all known to stabilize proteins in their native conformation, results in the correct processing of the mutant CFTR protein and its deposiotion at the plasma membrane. Such compounds included the cellular osmolytes glycerol and trimethylamine N-oxide, as well as deuterated water. Treatment of the ΔF508 CFTR-expressing cells with any one of these compounds, which we now refer to as ‘chemical chaperones’, restored the ability of the mutant cells to exhibit forskolin-dependent chloride transport, similar to that observed for the cells expressing the wild-type CFTR protein. We suggest that the use of ‘chemical chaperones’ may prove to be effective for the treatment of cystic fibriosis, as well as other genetic diseases whose underlying basis involoves defective protein folding and/or a failure in normal protein trafficking events.     

5’-adenosine monophosphate mediated cooling treatment enhances ΔF508-Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) stability in vivo

Background

Gene mutations that produce misprocessed proteins are linked to many human disorders. Interestingly, some misprocessed proteins retained their biological function when stabilized by low temperature treatment of cultured cells in vitro. Here we investigate whether low temperature treatment in vivo can rescue misfolded proteins by applying 5’-AMP mediated whole body cooling to a Cystic Fibrosis (CF) mouse model carrying a mutant cystic fibrosis transmembrane conductance regulator (CFTR) with a deletion of the phenylalanine residue in position 508 (ΔF508-CFTR). Low temperature treatment of cultured cells was previously shown to be able to alleviate the processing defect of ΔF508-CFTR, enhancing its plasma membrane localization and its function in mediating chloride ion transport.

Results

Here, we report that whole body cooling enhanced the retention of ΔF508-CFTR in intestinal epithelial cells. Functional analysis based on β-adrenergic dependent salivary secretion and post-natal mortality rate revealed a moderate but significant improvement in treated compared with untreated CF mice.

Conclusions

Our findings demonstrate that temperature sensitive processing of mutant proteins can be responsive to low temperature treatment in vivo.     

Depletion of the Ubiquitin-binding Adaptor Molecule SQSTM1/p62 from Macrophages Harboring cftr ΔF508 Mutation Improves the Delivery of Burkholderia cenocepacia to the Autophagic Machinery

Cystic fibrosis is the most common inherited lethal disease in Caucasians. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), of which the cftr ΔF508 mutation is the most common. ΔF508 macrophages are intrinsically defective in autophagy because of the sequestration of essential autophagy molecules within unprocessed CFTR aggregates. Defective autophagy allows Burkholderia cenocepacia (B. cepacia) to survive and replicate in ΔF508 macrophages. Infection by B. cepacia poses a great risk to cystic fibrosis patients because it causes accelerated lung inflammation and, in some cases, a lethal necrotizing pneumonia. Autophagy is a cell survival mechanism whereby an autophagosome engulfs non-functional organelles and delivers them to the lysosome for degradation. The ubiquitin binding adaptor protein SQSTM1/p62 is required for the delivery of several ubiquitinated cargos to the autophagosome. In WT macrophages, p62 depletion and overexpression lead to increased and decreased bacterial intracellular survival, respectively. In contrast, depletion of p62 in ΔF508 macrophages results in decreased bacterial survival, whereas overexpression of p62 leads to increased B. cepacia intracellular growth. Interestingly, the depletion of p62 from ΔF508 macrophages results in the release of the autophagy molecule beclin1 (BECN1) from the mutant CFTR aggregates and allows its redistribution and recruitment to the B. cepacia vacuole, mediating the acquisition of the autophagy marker LC3 and bacterial clearance via autophagy. These data demonstrate that p62 differentially dictates the fate of B. cepacia infection in WT and ΔF508 macrophages.     

Activation of 3-Phosphoinositide-dependent Kinase 1 (PDK1) and Serum- and Glucocorticoid-induced Protein Kinase 1 (SGK1) by Short-chain Sphingolipid C4-ceramide Rescues the Trafficking Defect of ΔF508-Cystic Fibrosis Transmembrane Conductance Regulator (ΔF508-CFTR)

Cystic fibrosis (CF) is due to a folding defect in the CF transmembrane conductance regulator (CFTR) protein. The most common mutation, ΔF508, prevents CFTR from trafficking to the apical plasma membrane. Here we show that activation of the PDK1/SGK1 signaling pathway with C4-ceramide (C4-CER), a non-toxic small molecule, functionally corrects the trafficking defect in both cultured CF cells and primary epithelial cell explants from CF patients. The mechanism of C4-CER action involves a series of mutual autophosphorylation and phosphorylation events between PDK1 and SGK1. Detailed mechanistic studies indicate that C4-CER initially induces autophosphorylation of SGK1 at Ser⁴²². SGK1[Ser(P)⁴²²] and C4-CER coincidently bind PDK1 and permit PDK1 to autophosphorylate at Ser²⁴¹. Then PDK1[Ser(P)²⁴¹] phosphorylates SGK1[Ser(P)⁴²²] at Thr²⁵⁶ to generate fully activated SGK1[Ser⁴²², Thr(P)²⁵⁶]. SGK1[Ser(P)⁴²²,Thr(P)²⁵⁶] phosphorylates and inactivates the E3 ubiquitin ligase Nedd4-2. ΔF508-CFTR is thus free to traffic to the plasma membrane. Importantly, C4-CER-mediated activation of both PDK1 and SGK1 is independent of the PI3K/Akt/mammalian target of rapamycin signaling pathway. Physiologically, C4-CER significantly increases maturation and stability of ΔF508-CFTR (t_½ ∼10 h), enhances cAMP-activated chloride secretion, and suppresses hypersecretion of interleukin-8 (IL-8). We suggest that candidate drugs for CF directed against the PDK1/SGK1 signaling pathway, such as C4-CER, provide a novel therapeutic strategy for a life-limiting disorder that affects one child, on average, each day.