A combination therapy for cystic fibrosis.

The most prevalent form of cystic fibrosis arises from an amino acid deletion in the cystic fibrosis transmembrane conductance regulator, CFTR. A recently approved treatment for individuals homozygous for this mutation combines a chemical corrector, which helps CFTR fold, and a potentiator that increases CFTR channel activity.

Separating the contributions of SLC26A9 and CFTR to anion secretion in primary human bronchial epithelia.

Aberrant anion secretion across the bronchial epithelium is associated with airway disease, most notably in cystic fibrosis. Although the cystic fibrosis transmembrane conductance regulator (CFTR) is recognized as the primary source of airway anion secretion, alternative anion transport mechanisms play a contributing role. An alternative anion transporter of growing interest is SLC26A9, a constitutively active chloride channel that has been shown to interact with CFTR and may also contribute to bicarbonate secretion. Interest in SLC26A9 has been fueled by genome-wide association studies that suggest it is a significant modifier of CF disease severity. Despite this growing evidence that SLC26A9 plays an important role in the airway, its presence and function in bronchial epithelia remain poorly understood, in part, because its activity is difficult to separate from the activity of CFTR. Here, we present results using primary human bronchial epithelia (HBE) from multiple patient sources to confirm that SLC26A9 mRNA is present in HBE and that its constitutive channel activity is unaffected by knockdown of CFTR. Furthermore, SLC26A9 and CFTR show differential responses to common inhibitors of anion secretion. Finally, we assess the impact of bicarbonate on the activity of SLC26A9 and CFTR. These results confirm that SLC26A9 is the primary source of constitutive anion secretion across HBE, and should inform future studies focused on activation of SLC26A9 as an alternative anion channel in CF. These results should provide a strong foundation to investigate how single-nucleotide polymorphisms in SLC26A9 modulate airway disease.

Improved correction of F508del-CFTR biogenesis with a folding facilitator and an inhibitor of protein ubiquitination.

A growing number of diseases are linked to the misfolding of integral membrane proteins, and many of these proteins are targeted for ubiquitin-proteasome-dependent degradation. One such substrate is a mutant form of the Cystic Fibrosis Transmembrane Conductance Regulator (F508del-CFTR). Protein folding "correctors" that repair the F508del-CFTR folding defect have entered the clinic, but they are unlikely to protect the entire protein from degradation. To increase the pool of F508del-CFTR protein that is available for correction by existing treatments, we determined a structure-activity relationship to improve the efficacy and reduce the toxicity of an inhibitor of the E1 ubiquitin activating enzyme that facilitates F508del-CFTR maturation. A resulting lead compound lacked measurable toxicity and improved the ability of an FDA-approved corrector to augment F508del-CFTR folding, transport the protein to the plasma membrane, and maintain its activity. These data support a proof-of-concept that modest inhibition of substrate ubiquitination improves the activity of small molecule correctors to treat CF and potentially other protein conformational disorders.

CK19 stabilizes CFTR at the cell surface by limiting its endocytic pathway degradation.

Protein interactions that stabilize the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) at the apical membranes of epithelial cells have not yet been fully elucidated. We identified keratin 19 (CK19 or K19) as a novel CFTR-interacting protein. CK19 overexpression stabilized both wild-type (WT)-CFTR and Lumacaftor (VX-809)-rescued F508del-CFTR (where F508del is the deletion of the phenylalanine residue at position 508) at the plasma membrane (PM), promoting Cl- secretion across human bronchial epithelial (HBE) cells. CK19 prevention of Rab7A-mediated lysosomal degradation was a key mechanism in apical CFTR stabilization. Unexpectedly, CK19 expression was decreased by ∼40% in primary HBE cells from homogenous F508del patients with CF relative to non-CF controls. CK19 also positively regulated multidrug resistance-associated protein 4 expression at the PM, suggesting that this keratin may regulate the apical expression of other ATP-binding cassette proteins as well as CFTR.-Hou, X., Wu, Q., Rajagopalan, C., Zhang, C., Bouhamdan, M., Wei, H., Chen, X., Zaman, K., Li, C., Sun, X., Chen, S., Frizzell, R. A., Sun, F. CK19 stabilizes CFTR at the cell surface by limiting its endocytic pathway degradation.

Silencing of the Hsp70-specific nucleotide-exchange factor BAG3 corrects the F508del-CFTR variant by restoring autophagy.

The protein chaperones heat shock protein 70 (Hsp70) and Hsp90 are required for de novo folding of proteins and protect against misfolding-related cellular stresses by directing misfolded or slowly folding proteins to the ubiquitin/proteasome system (UPS) or autophagy/lysosomal degradation pathways. Here, we examined the role of the Bcl2-associated athanogene (BAG) family of Hsp70-specific nucleotide-exchange factors in the biogenesis and functional correction of genetic variants of the cystic fibrosis transmembrane conductance regulator (CFTR) whose mutations cause cystic fibrosis (CF). We show that siRNA-mediated silencing of BAG1 and -3, two BAG members linked to the clearance of misfolded proteins via the UPS and autophagy pathways, respectively, leads to functional correction of F508del-CFTR and other disease-associated CFTR variants. BAG3 silencing was the most effective, leading to improved F508del-CFTR stability, trafficking, and restoration of cell-surface function, both alone and in combination with the FDA-approved CFTR corrector, VX-809. We also found that the BAG3 silencing-mediated correction of F508del-CFTR restores the autophagy pathway, which is defective in F508del-CFTR-expressing cells, likely because of the maladaptive stress response in CF pathophysiology. These results highlight the potential therapeutic benefits of targeting the cellular chaperone system to improve the functional folding of CFTR variants contributing to CF and possibly other protein-misfolding-associated diseases.

Comparative analyses of long non-coding RNA profiles in vivo in cystic fibrosis lung airway and parenchyma tissues.

BACKGROUND: Recent advances in the functional analyses of endogenous non-coding RNA (ncRNA) molecules, including long non-coding RNAs (LncRNAs), have provided a new perspective on the crucial roles of RNA in gene regulation. Consequently, LncRNA deregulation is a key factor in various diseases, including pulmonary disorders like Cystic Fibrosis (CF). CF is the most common life limiting recessive disease in the U.S., and is due to mutations in the CFTR gene. CF mutations, of which the most common is F508del-CFTR, prevents correct folding, trafficking and function of the mutant CFTR protein and is further manifested by the hyper-expression of pro-inflammatory cytokines and chemokines into the airway lumen leading to bronchiectasis and culminating in lung destruction. METHODS: Here we report a distinct LncRNA signature and corresponding mRNAs that distinguishes CF lung (airway and parenchyma) tissues from matched non-CF controls (n = 4 each group), generated by microarray specific for LncRNAs which includes corresponding mRNA expressions. In silico analyses of the cellular processes that are impacted by these LncRNAs was performed using Gene Ontology (GO). A selected subset of LncRNAs were validated by quantitative real-time PCR. RESULTS: We have identified 636 LncRNAs differentially expressed in CF airway epithelium and 1974 in CF lung parenchyma compared to matched non-CF controls (fold change ≥2, p < 0.05), majority of which (> 50%) are intergenic. Interestingly, 15 of these differentially expressed LncRNAs and 9 coding mRNAs are common to airway and parenchyma tissues. GO analyses indicates that signaling pathways and cell membrane functions are significantly affected by the alteration in LncRNA expressions in CF lung tissues. Seven of the differentially expressed LncRNAs, exhibit similar expression trends in CFBE41o- compared to control cells. CONCLUSION: Understanding the mechanisms by which these LncRNAs regulate CF disease phenotype will help develop novel therapeutic targets for CF and related pulmonary diseases, such as COPD and Asthma.

Non-native Conformers of Cystic Fibrosis Transmembrane Conductance Regulator NBD1 Are Recognized by Hsp27 and Conjugated to SUMO-2 for Degradation.

A newly identified pathway for selective degradation of the common mutant of the cystic fibrosis transmembrane conductance regulator (CFTR), F508del, is initiated by binding of the small heat shock protein, Hsp27. Hsp27 collaborates with Ubc9, the E2 enzyme for protein SUMOylation, to selectively degrade F508del CFTR via the SUMO-targeted ubiquitin E3 ligase, RNF4 (RING finger protein 4) (1). Here, we ask what properties of CFTR are sensed by the Hsp27-Ubc9 pathway by examining the ability of NBD1 (locus of the F508del mutation) to mimic the disposal of full-length (FL) CFTR. Similar to FL CFTR, F508del NBD1 expression was reduced 50-60% by Hsp27; it interacted preferentially with the mutant and was modified primarily by SUMO-2. Mutation of the consensus SUMOylation site, Lys(447), obviated Hsp27-mediated F508del NBD1 SUMOylation and degradation. As for FL CFTR and NBD1 in vivo, SUMO modification using purified components in vitro was greater for F508del NBD1 versus WT and for the SUMO-2 paralog. Several findings indicated that Hsp27-Ubc9 targets the SUMOylation of a transitional, non-native conformation of F508del NBD1: (a) its modification decreased as [ATP] increased, reflecting stabilization of the nucleotide-binding domain by ligand binding; (b) a temperature-induced increase in intrinsic fluorescence, which reflects formation of a transitional NBD1 conformation, was followed by its SUMO modification; and (c) introduction of solubilizing or revertant mutations to stabilize F508del NBD1 reduced its SUMO modification. These findings indicate that the Hsp27-Ubc9 pathway recognizes a non-native conformation of mutant NBD1, which leads to its SUMO-2 conjugation and degradation by the ubiquitin-proteasome system.

VAMP-associated Proteins (VAP) as Receptors That Couple Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Proteostasis with Lipid Homeostasis.

Unesterified cholesterol accumulates in late endosomes in cells expressing the misfolded cystic fibrosis transmembrane conductance regulator (CFTR). CFTR misfolding in the endoplasmic reticulum (ER) or general activation of ER stress led to dynein-mediated clustering of cholesterol-loaded late endosomes at the Golgi region, a process regulated by ER-localized VAMP-associated proteins (VAPs). We hypothesized that VAPs serve as intracellular receptors that couple lipid homeostasis through interactions with two phenylalanines in an acidic track (FFAT) binding signals (found in lipid sorting and sensing proteins, LSS) with proteostasis regulation. VAPB inhibited the degradation of ΔF508-CFTR. The activity was mapped to the ligand-binding major sperm protein (MSP) domain, which was sufficient in regulating CFTR biogenesis. We identified mutations in an unstructured loop within the MSP that uncoupled VAPB-regulated CFTR biogenesis from basic interactions with FFAT. Using this information, we defined functional and physical interactions between VAPB and proteostasis regulators (ligands), including the unfolded protein response sensor ATF6 and the ER degradation cluster that included FAF1, VCP, BAP31, and Derlin-1. VAPB inhibited the degradation of ΔF508-CFTR in the ER through interactions with the RMA1-Derlin-BAP31-VCP pathway. Analysis of pseudoligands containing tandem FFAT signals supports a competitive model for VAP interactions that direct CFTR biogenesis. The results suggest a model in which VAP-ligand binding couples proteostasis and lipid homeostasis leading to observed phenotypes of lipid abnormalities in protein folding diseases.

The CFTR trafficking mutation F508del inhibits the constitutive activity of SLC26A9.

Several members of the SLC26A family of anion transporters associate with CFTR, forming complexes in which CFTR and SLC26A functions are reciprocally regulated. These associations are thought to be facilitated by PDZ scaffolding interactions. CFTR has been shown to be positively regulated by NHERF-1, and negatively regulated by CAL in airway epithelia. However, it is unclear which PDZ-domain protein(s) interact with SLC26A9, a SLC26A family member found in airway epithelia. We have previously shown that primary, human bronchial epithelia (HBE) from non-CF donors exhibit constitutive anion secretion attributable to SLC26A9. However, constitutive anion secretion is absent in HBE from CF donors. We examined whether changes in SLC26A9 constitutive activity could be attributed to a loss of CFTR trafficking, and what role PDZ interactions played. HEK293 coexpressing SLC26A9 with the trafficking mutant F508del CFTR exhibited a significant reduction in constitutive current compared with cells coexpressing SLC26A9 and wt CFTR. We found that SLC26A9 exhibits complex glycosylation when coexpressed with F508del CFTR, but its expression at the plasma membrane is decreased. SLC26A9 interacted with both NHERF-1 and CAL, and its interaction with both significantly increased with coexpression of wt CFTR. However, coexpression with F508del CFTR only increased SLC26A9's interaction with CAL. Mutation of SLC26A9's PDZ motif decreased this association with CAL, and restored its constitutive activity. Correcting aberrant F508del CFTR trafficking in CF HBE with corrector VX-809 also restored SLC26A9 activity. We conclude that when SLC26A9 is coexpressed with F508del CFTR, its trafficking defect leads to a PDZ motif-sensitive intracellular retention of SLC26A9.

Simple image-based no-wash method for quantitative detection of surface expressed CFTR.

Cystic fibrosis (CF) is the most common lethal genetic disease among Caucasians. It is caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene, which encodes an apical membrane anion channel that is required for regulating the volume and composition of epithelial secretions. The most common CFTR mutation, present on at least one allele in >90% of CF patients, deletes phenylalanine at position 508 (F508del), which causes the protein to misfold. Endoplasmic reticulum (ER) quality control elicits the degradation of mutant CFTR, compromising its trafficking to the epithelial cell apical membrane. The absence of functional CFTR leads to depletion of airway surface liquid, impaired clearance of mucus and bacteria from the lung, and predisposes to recurrent infections. Ultimately, respiratory failure results from inflammation and bronchiectasis. Although high throughput screening has identified small molecules that can restore the anion transport function of F508del CFTR, they correct less than 15% of WT CFTR activity, yielding insufficient clinical benefit. To date, most primary CF drug discovery assays have employed measurements of CFTR's anion transport function, a method that depends on the recruitment of a functional CFTR to the cell surface, involves multiple wash steps, and relies on a signal that saturates rapidly. Screening efforts have also included assays for detection of extracellularly HA-tagged or HRP-tagged CFTR, which require multiple washing steps. We have recently developed tools and cell lines that report the correction of mutant CFTR trafficking by currently available small molecules, and have extended this assay to the 96-well format. This new and simple no-wash assay of F508del CFTR at the cell surface may permit the discovery of more efficacious drugs, and hopefully thereby prevent the catastrophic effects of this disease. In addition, the modular design of this platform should make it useful for other diseases where loss-of-function results from folding and/or trafficking defects in membrane proteins.

Divergent signaling via SUMO modification: potential for CFTR modulation.

The cystic fibrosis transmembrane conductance regulator (CFTR) is generally responsible for the cAMP/PKA regulated anion conductance at the apical membranes of secretory epithelial cells. Mutations in CFTR underlie cystic fibrosis (CF), in which the most common variant, F508del, causes protein misfolding and its proteasome-mediated degradation. A new pathway that contributes to mutant CFTR degradation is mediated by the small heat shock protein, Hsp27, which cooperates with Ubc9, the E2 enzyme for SUMOylation, to selectively conjugate mutant CFTR with SUMO-2/3. This SUMO paralog can form polychains, which are recognized by the ubiquitin E3 enzyme, RNF4, leading to CFTR ubiquitylation and recognition by the proteasome. We found also that F508del CFTR could be modified by SUMO-1, a paralog that does not support SUMO polychain formation. The use of different SUMO paralogs to modify and target a single substrate for divergent purposes is not uncommon. In this short review we discuss the possibility that conjugation with SUMO-1 could protect mutant CFTR from disposal by RNF4 and similar ubiquitin ligases. We hypothesize that such a pathway could contribute to therapeutic efforts to stabilize immature mutant CFTR and thereby enhance the action of therapeutics that correct CFTR trafficking to the apical membranes.

Functional and molecular identification of a TASK-1 potassium channel regulating chloride secretion through CFTR channels in the shark rectal gland: implications for cystic fibrosis.

In the shark rectal gland (SRG), apical chloride secretion through CFTR channels is electrically coupled to a basolateral K+ conductance whose type and molecular identity are unknown. We performed studies in the perfused SRG with 17 K+ channel inhibitors to begin this search. Maximal chloride secretion was markedly inhibited by low-perfusate pH, bupivicaine, anandamide, zinc, quinidine, and quinine, consistent with the properties of an acid-sensitive, four-transmembrane, two-pore-domain K+ channel (4TM-K2P). Using PCR with degenerate primers to this family, we identified a TASK-1 fragment in shark rectal gland, brain, gill, and kidney. Using 5' and 3' rapid amplification of cDNA ends PCR and genomic walking, we cloned the full-length shark gene (1,282 bp), whose open reading frame encodes a protein of 375 amino acids that was 80% identical to the human TASK-1 protein. We expressed shark and human TASK-1 cRNA in Xenopus oocytes and characterized these channels using two-electrode voltage clamping. Both channels had identical current-voltage relationships (outward rectifying) and a reversal potential of -90 mV. Both were inhibited by quinine, bupivicaine, and acidic pH. The pKa for current inhibition was 7.75 for shark TASK-1 vs. 7.37 for human TASK-1, values similar to the arterial pH for each species. We identified this protein in SRG by Western blot and confocal immunofluorescent microscopy and detected the protein in SRG and human airway cells. Shark TASK-1 is the major K+ channel coupled to chloride secretion in the SRG, is the oldest 4TM 2P family member identified, and is the first TASK-1 channel identified to play a role in setting the driving force for chloride secretion in epithelia. The detection of this potassium channel in mammalian lung tissue has implications for human biology and disease.

Regulatory R region of the CFTR chloride channel is a dynamic integrator of phospho-dependent intra- and intermolecular interactions.

Intrinsically disordered proteins play crucial roles in regulatory processes and often function as protein interaction hubs. Here, we present a detailed characterization of a full-length disordered hub protein region involved in multiple dynamic complexes. We performed NMR, CD, and fluorescence binding studies on the nonphosphorylated and highly PKA-phosphorylated human cystic fibrosis transmembrane conductance regulator (CFTR) regulatory region, a ∼200-residue disordered segment involved in phosphorylation-dependent regulation of channel trafficking and gating. Our data provide evidence for dynamic, phosphorylation-dependent, multisite interactions of various segments of the regulatory region for its intra- and intermolecular partners, including the CFTR nucleotide binding domains 1 and 2, a 42-residue peptide from the C terminus of CFTR, the SLC26A3 sulphate transporter and antisigma factor antagonist (STAS) domain, and 14-3-3ß. Because of its large number of binding partners, multivalent binding of individually weak sites facilitates rapid exchange between free and bound states to allow the regulatory region to engage with different partners and generate a graded or rheostat-like response to phosphorylation. Our results enrich the understanding of how disordered binding segments interact with multiple targets. We present structural models consistent with our data that illustrate this dynamic aspect of phospho-regulation of CFTR by the disordered regulatory region.

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 Ser(422). SGK1[Ser(P)(422)] and C4-CER coincidently bind PDK1 and permit PDK1 to autophosphorylate at Ser(241). Then PDK1[Ser(P)(241)] phosphorylates SGK1[Ser(P)(422)] at Thr(256) to generate fully activated SGK1[Ser(422), 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 (t½ ∼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.

Regulated recycling of mutant CFTR is partially restored by pharmacological treatment.

Efficient trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) to and from the cell surface is essential for maintaining channel density at the plasma membrane (PM) and ensuring proper physiological activity. The most common mutation, F508del, exhibits reduced surface expression and impaired function despite treatment with currently available pharmacological small molecules, called correctors. To gain more detailed insight into whether CFTR enters compartments that allow corrector stabilization in the cell periphery, we investigated the peripheral trafficking itineraries and kinetics of wild type (WT) and F508del in living cells using high-speed fluorescence microscopy together with fluorogen activating protein detection. We directly visualized internalization and accumulation of CFTR WT from the PM to a perinuclear compartment that colocalized with the endosomal recycling compartment (ERC) markers Rab11 and EHD1, reaching steady-state distribution by 25 minutes. Stimulation by protein kinase A (PKA) depleted this intracellular pool and redistributed CFTR channels to the cell surface, elicited by reduced endocytosis and active translocation to the PM. Corrector or temperature rescue of F508del also resulted in targeting to the ERC and exhibited subsequent PKA-stimulated trafficking to the PM. Corrector treatment (24 hours) led to persistent residence of F508del in the ERC, while thermally destabilized F508del was targeted to lysosomal compartments by 3 hours. Acute addition of individual correctors, C4 or C18, acted on peripheral trafficking steps to partially block lysosomal targeting of thermally destabilized F508del. Taken together, corrector treatment redirects F508del trafficking from a degradative pathway to a regulated recycling route, and proteins that mediate this process become potential targets for improving the efficacy of current and future correctors.

Human heat shock protein 105/110 kDa (Hsp105/110) regulates biogenesis and quality control of misfolded cystic fibrosis transmembrane conductance regulator at multiple levels.

Heat shock protein 105/110-kDa (Hsp105/110), a member of the Hsp70 super family of molecular chaperones, serves as a nucleotide exchange factor for Hsc70, independently prevents the aggregation of misfolded proteins, and functionally relates to Hsp90. We investigated the roles of human Hsp105α, the constitutively expressed isoform, in the biogenesis and quality control of the cystic fibrosis transmembrane conductance regulator (CFTR). In the endoplasmic reticulum (ER), Hsp105 facilitates CFTR quality control at an early stage in its biosynthesis but promotes CFTR post-translational folding. Deletion of Phe-508 (ΔF508), the most prevalent mutation causing cystic fibrosis, interferes with de novo folding of CFTR, impairing its export from the ER and accelerating its clearance in the ER and post-Golgi compartments. We show that Hsp105 preferentially associates with and stabilizes ΔF508 CFTR at both levels. Introduction of the Hsp105 substrate binding domain potently increases the steady state level of ΔF508 CFTR by reducing its early-stage degradation. This in turn dramatically enhances ΔF508 CFTR cell surface functional expression in cystic fibrosis airway epithelial cells. Although other Hsc70 nucleotide exchange factors such as HspBP1 and BAG-2 inhibit CFTR post-translational degradation in the ER through cochaperone CHIP, Hsp105 has a primary role promoting CFTR quality control at an earlier stage. The Hsp105-mediated multilevel regulation of ΔF508 CFTR folding and quality control provides new opportunities to understand how chaperone machinery regulates the homeostasis and functional expression of misfolded proteins in the cell. Future studies in this direction will inform therapeutics development for cystic fibrosis and other protein misfolding diseases.

Rab11b regulates the trafficking and recycling of the epithelial sodium channel (ENaC).

Expression of the epithelial sodium channel (ENaC) at the apical membrane of cortical collecting duct (CCD) principal cells is modulated by regulated trafficking mediated by vesicle insertion and retrieval. Small GTPases are known to facilitate vesicle trafficking, recycling, and membrane fusion events; however, little is known about the specific Rab family members that modify ENaC surface density. Using a mouse CCD cell line that endogenously expresses ENaC (mpkCCD), the channel was localized to both Rab11a- and Rab11b-positive endosomes by immunoisolation and confocal fluorescent microscopy. Expression of a dominant negative (DN) form of Rab11a or Rab11b significantly reduced the basal and cAMP-stimulated ENaC-dependent sodium (Na(+)) transport. The greatest reduction in Na(+) transport was observed with the expression of DN-Rab11b. Furthermore, small interfering RNA-mediated knockdown of each Rab11 isoform demonstrated the requirement for Rab11b in ENaC surface expression. These data indicate that Rab11b, and to a lesser extent Rab11a, is involved in establishing the constitutive and cAMP-stimulated Na(+) transport in mpkCCD cells.

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.

Reduced histone deacetylase 7 activity restores function to misfolded CFTR in cystic fibrosis.

Chemical modulation of histone deacetylase (HDAC) activity by HDAC inhibitors (HDACi) is an increasingly important approach for modifying the etiology of human disease. Loss-of-function diseases arise as a consequence of protein misfolding and degradation, which lead to system failures. The DeltaF508 mutation in cystic fibrosis transmembrane conductance regulator (CFTR) results in the absence of the cell surface chloride channel and a loss of airway hydration, leading to the premature lung failure and reduced lifespan responsible for cystic fibrosis. We now show that the HDACi suberoylanilide hydroxamic acid (SAHA) restores surface channel activity in human primary airway epithelia to levels that are 28% of those of wild-type CFTR. Biological silencing of all known class I and II HDACs reveals that HDAC7 plays a central role in restoration of DeltaF508 function. We suggest that the tunable capacity of HDACs can be manipulated by chemical biology to counter the onset of cystic fibrosis and other human misfolding disorders.

Rescue of CF airway epithelial cell function in vitro by a CFTR potentiator, VX-770.

Cystic fibrosis (CF) is a fatal genetic disease caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR), a protein kinase A (PKA)-activated epithelial anion channel involved in salt and fluid transport in multiple organs, including the lung. Most CF mutations either reduce the number of CFTR channels at the cell surface (e.g., synthesis or processing mutations) or impair channel function (e.g., gating or conductance mutations) or both. There are currently no approved therapies that target CFTR. Here we describe the in vitro pharmacology of VX-770, an orally bioavailable CFTR potentiator in clinical development for the treatment of CF. In recombinant cells VX-770 increased CFTR channel open probability (P(o)) in both the F508del processing mutation and the G551D gating mutation. VX-770 also increased Cl(-) secretion in cultured human CF bronchial epithelia (HBE) carrying the G551D gating mutation on one allele and the F508del processing mutation on the other allele by approximately 10-fold, to approximately 50% of that observed in HBE isolated from individuals without CF. Furthermore, VX-770 reduced excessive Na(+) and fluid absorption to prevent dehydration of the apical surface and increased cilia beating in these epithelial cultures. These results support the hypothesis that pharmacological agents that restore or increase CFTR function can rescue epithelial cell function in human CF airway.