Cystic fibrosis transmembrane regulator missing the first four transmembrane segments increases wild type and DeltaF508 processing

We previously generated an adenoassociated viral gene therapy vector, rAAV-Delta264 cystic fibrosis transmembrane conductance regulator (CFTR), missing the first four transmembrane domains of CFTR. When infected into monkey lungs, Delta264 CFTR increased the levels of endogenous wild type CFTR protein. To understand this process, we transfected Delta264 CFTR plasmid cDNA into COS7 cells, and we noted that protein expression from the truncation mutant is barely detectable when compared with wild type or DeltaF508 CFTR. Delta264 CFTR protein expression increases dramatically when cells are treated with proteasome inhibitors. Cycloheximide experiments show that Delta264 CFTR is degraded faster than DeltaF508 CFTR. VCP and HDAC6, two proteins involved in retrograde translocation from endoplasmic reticulum to cytosol for proteasomal and aggresomal degradation, coimmunoprecipitate with Delta264 CFTR. In cotransfection studies in COS7 cells and in transfection of Delta264 CFTR into cells stably expressing wild type and DeltaF508 CFTR, Delta264 CFTR increases wild type CFTR protein and increases levels of maturation of immature band B to mature band C of DeltaF508 CFTR. Thus the adenoassociated viral vector, rAAV-Delta264 CFTR, is a highly promising cystic fibrosis gene therapy vector because it increases the amount of mature band C protein both from wild type and DeltaF508 CFTR and associates with key elements in quality control mechanism of CFTR.     

Modulation of protein kinase CK2 activity by fragments of CFTR encompassing F508 may reflect functional links with cystic fibrosis pathogenesis

Deletion of F508 in the first nucleotide binding domain (NBD1) of cystic fibrosis transmembrane conductance regulator protein (CFTR) is the commonest cause of cystic fibrosis (CF). Functional interactions between CFTR and CK2, a highly pleiotropic protein kinase, have been recently described which are perturbed by the F508 deletion. Here we show that both NBD1 wild type and NBD1 DeltaF508 are phosphorylated in vitro by CK2 catalytic alpha-subunit but not by CK2 holoenzyme unless polylysine is added. MS analysis reveals that, in both NBD1 wild type and DeltaF508, the phosphorylated residues are S422 and S670, while phosphorylation of S511 could not be detected. Accordingly, peptides encompassing the 500-518 sequence of CFTR are not phosphorylated by CK2; rather they inhibit CK2alpha catalytic activity in a manner which is not competitive with respect to the specific CK2 peptide substrate. In contrast, 500-518 peptides promote the phosphorylation of NBD1 by CK2 holoenzyme overcoming inhibition by the beta-subunit. Such a stimulatory efficacy of the CFTR 500-518 peptide is dramatically enhanced by deletion of F508 and is abolished by deletion of the II507 doublet. Kinetics of NBD1 phosphorylation by CK2 holoenzyme, but not by CK2alpha, display a sigmoid shape denoting a positive cooperativity which is dramatically enhanced by the addition of the DeltaF508 CFTR peptide. SPR analysis shows that NBD1 DeltaF508 interacts more tightly than NBD1 wt with the alpha-subunit of CK2 and that CFTR peptides which are able to trigger NBD1 phosphorylation by CK2 holoenzyme also perturb the interaction between the alpha- and the beta-subunits of CK2.     

Genistein restores functional interactions between Delta F508-CFTR and ENaC in Xenopus oocytes.

The cystic fibrosis transmembrane conductance regulator (CFTR), in addition to its Cl(-) channel properties, has regulatory interactions with other epithelial ion channels including the epithelial Na(+) channel (ENaC). Both the open probability and surface expression of wild type CFTR Cl(-) channels are increased significantly when CFTR is co-expressed in Xenopus oocytes with alphabetagamma-ENaC, and conversely, the activity of ENaC is inhibited following wild type CFTR activation. Using the Xenopus oocyte expression system, a lack of functional regulatory interactions between DeltaF508-CFTR and ENaC was observed following activation of DeltaF508-CFTR by forskolin and isobutylmethylxanthine (IBMX). Whole cell currents in oocytes expressing ENaC alone decreased in response to genistein but increased in response to a combination of forskolin and IBMX followed by genistein. In contrast, ENaC currents in oocytes co-expressing ENaC and DeltaF508-CFTR remained stable following stimulation with forskolin/IBMX/genistein. Furthermore, co-expression of DeltaF508-CFTR with ENaC enhanced the forskolin/IBMX/genistein-mediated activation of DeltaF508-CFTR. Our data suggest that genistein restores regulatory interactions between DeltaF508-CFTR and ENaC and that combinations of protein repair agents, such as 4-phenylbutyrate and genistein, may be necessary to restore DeltaF508-CFTR function in vivo.     

The medium is the message: glycosphingolipids and their soluble analogues

We have made adamantylGSLs by substituting the fatty acids of primarily, globotriaosyl ceramide(Gb(3)) and sulfogalactosyl ceramide(SGC), with the rigid alpha-adamantane hydrocarbon frame. These analogues have proven to be remarkably water-soluble but retain the receptor function of the parent membrane GSL. AdaGb(3) prevents the binding of verotoxins to target cells but increased pathology in vivo, likely due to the partitioning into receptor negative target cells to provide pseudo-receptors. Preincubation of HIV with adaGb(3) prevents cellular infection in vitro and viral-host cell fusion. Cellular accumulation of Gb(3) reduces HIV susceptibility in vitro, whereas lack of Gb(3) promotes infection, suggesting that Gb(3) expression could be a novel risk factor for HIV susceptibility. AdaGb(3) has proven to be a new inhibitor for the MDR1 drug pump (P-glycoprotein) and can reverse drug resistance in cell culture. AdaSGC is bound by hsp70/hsc70 within the N-terminal ATPase domain and inhibits chaperone function. When added to cells transfected with the DeltaF508 CFTR mutant, adaSGC was able to decrease ER degradation of this mutant protein, an hsc70 dependent process. Our finding that DeltaF508 CFTR expressing cells show reduced SGC biosynthesis suggests that SGC could be an additional natural regulator of the hsp70 chaperone ATPase cycle.     

Diminished self-chaperoning activity of the DeltaF508 mutant of CFTR results in protein misfolding

The absence of a functional ATP Binding Cassette (ABC) protein called the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) from apical membranes of epithelial cells is responsible for cystic fibrosis (CF). Over 90% of CF patients carry at least one mutant allele with deletion of phenylalanine at position 508 located in the N-terminal nucleotide binding domain (NBD1). Biochemical and cell biological studies show that the DeltaF508 mutant exhibits inefficient biosynthetic maturation and susceptibility to degradation probably due to misfolding of NBD1 and the resultant misassembly of other domains. However, little is known about the direct effect of the Phe508 deletion on the NBD1 folding, which is essential for rational design strategies of cystic fibrosis treatment. Here we show that the deletion of Phe508 alters the folding dynamics and kinetics of NBD1, thus possibly affecting the assembly of the complete CFTR. Using molecular dynamics simulations, we find that meta-stable intermediate states appearing on wild type and mutant folding pathways are populated differently and that their kinetic accessibilities are distinct. The structural basis of the increased misfolding propensity of the DeltaF508 NBD1 mutant is the perturbation of interactions in residue pairs Q493/P574 and F575/F578 found in loop S7-H6. As a proof-of-principle that the S7-H6 loop conformation can modulate the folding kinetics of NBD1, we virtually design rescue mutations in the identified critical interactions to force the S7-H6 loop into the wild type conformation. Two redesigned NBD1-DeltaF508 variants exhibited significantly higher folding probabilities than the original NBD1-DeltaF508, thereby partially rescuing folding ability of the NBD1-DeltaF508 mutant. We propose that these observed defects in folding kinetics of mutant NBD1 may also be modulated by structures separate from the 508 site. The identified structural determinants of increased misfolding propensity of NBD1-DeltaF508 are essential information in correcting this pathogenic mutant.     

Diffusional mobility of the cystic fibrosis transmembrane conductance regulator mutant, delta F508-CFTR, in the endoplasmic reticulum measured by photobleaching of GFP-CFTR chimeras

Mutations in the cystic fibrosis transmembrane conductance regulator protein (CFTR) cause cystic fibrosis. The most common disease-causing mutation, DeltaF508, is retained in the endoplasmic reticulum (ER) and is unable to function as a plasma membrane chloride channel. To investigate whether the ER retention of DeltaF508-CFTR is caused by immobilization and/or aggregation, we have measured the diffusional mobility of green fluorescent protein (GFP) chimeras of wild type (wt)-CFTR and DeltaF508-CFTR by fluorescence recovery after photobleaching. GFP-labeled DeltaF508-CFTR was localized in the ER and wt-CFTR in the plasma membrane and intracellular membranes in transfected COS7 and Chinese hamster ovary K1 cells. Both chimeras localized to the ER after brefeldin A treatment. Spot photobleaching showed that CFTR diffusion (diffusion coefficient approximately 10(-9) cm(2)/s) was not significantly slowed by the DeltaF508 mutation and that nearly all wt-CFTR and DeltaF508-CFTR diffused throughout the ER without restriction. Stabilization of molecular chaperone interactions by ATP depletion produced remarkable DeltaF508-CFTR immobilization ( approximately 50%) and slowed diffusion (6.5 x 10(-10) cm(2)/s) but had little effect on wt-CFTR. Fluorescence depletion experiments revealed that the immobilized DeltaF508-CFTR in ATP-depleted cells remained in an ER pattern. The mobility of wt-CFTR and DeltaF508-CFTR was reduced by maneuvers that alter CFTR processing or interactions with molecular chaperones, including tunicamycin, geldanamycin, and lactacystin. Photobleaching of the fluorescent ER lipid diOC(4)(3) showed that neither ER restructuring nor fragmentation during these maneuvers was responsible for the slowing and immobilization of CFTR. These results suggest that (a) the ER retention of DeltaF508-CFTR is not due to restricted ER mobility, (b) the majority of DeltaF508-CFTR is not aggregated or bound to slowly moving membrane proteins, and (c) DeltaF508-CFTR may interact to a greater extent with molecular chaperones than does wt-CFTR.     

Monocytes recruited into the alveolar air space of mice show a monocytic phenotype but upregulate CD14

The DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) is a temperature-sensitive trafficking mutant that is detected as an immature 160-kDa form (band B) in gel electrophoresis. The goal of this study was to test the hypothesis that HSP70, a member of the 70-kDa heat shock protein family, promotes DeltaF508 CFTR processing to the mature 180-kDa form (band C). Both pharmacological and genetic techniques were used to induce HSP70. IB3-1 cells were treated with sodium 4-phenylbutyrate (4PBA) to promote maturation of DeltaF508 CFTR to band C. A dose-dependent increase in band C and total cellular HSP70 was observed. Under these conditions, HSP70-CFTR complexes were increased and 70-kDa heat shock cognate protein-CFTR complexes were decreased. Increased DeltaF508 CFTR maturation was also seen after transfection with an HSP70 expression plasmid and exposure to glutamine, an inducer of HSP70. With immunofluorescence techniques, the increased appearance of CFTR band C correlated with CFTR distribution beyond the perinuclear regions. These data suggest that induction of HSP70 promotes DeltaF508 CFTR maturation and trafficking.     

Conformational and temperature-sensitive stability defects of the delta F508 cystic fibrosis transmembrane conductance regulator in post-endoplasmic reticulum compartments

Deletion of phenylalanine at position 508 (DeltaF508) is the most common cystic fibrosis (CF)-associated mutation in the CF transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel. The consensus notion is that DeltaF508 imposes a temperature-sensitive folding defect and targets newly synthesized CFTR for degradation at endoplasmic reticulum (ER). A limited amount of CFTR activity, however, appears at the cell surface in the epithelia of homozygous DeltaF508 CFTR mice and patients, suggesting that the ER retention is not absolute in native tissues. To further elucidate the reasons behind the inability of DeltaF508 CFTR to accumulate at the plasma membrane, its stability was determined subsequent to escape from the ER, induced by reduced temperature and glycerol. Biochemical and functional measurements show that rescued DeltaF508 CFTR has a temperature-sensitive stability defect in post-ER compartments, including the cell surface. The more than 4-20-fold accelerated degradation rate between 37 and 40 degrees C is, most likely, due to decreased conformational stability of the rescued DeltaF508 CFTR, demonstrated by in situ protease susceptibility and SDS-resistant thermoaggregation assays. We propose that the decreased stability of the spontaneously or pharmacologically rescued mutant may contribute to its inability to accumulate at the cell surface. Thus, therapeutic efforts to correct the folding defect should be combined with stabilization of the native DeltaF508 CFTR.     

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.     

Increased functional cell surface expression of CFTR and DeltaF508-CFTR by the anthracycline doxorubicin

Cystic fibrosis (CF) is a disease that is caused by mutations within the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The most common mutation, DeltaF508, accounts for 70% of all CF alleles and results in a protein that is defective in folding and trafficking to the cell surface. However, DeltaF508-CFTR is functional when properly localized. We report that a single, noncytotoxic dose of the anthracycline doxorubicin (Dox, 0.25 microM) significantly increased total cellular CFTR protein expression, cell surface CFTR protein expression, and CFTR-associated chloride secretion in cultured T84 epithelial cells. Dox treatment also increased DeltaF508-CFTR cell surface expression and DeltaF508-CFTR-associated chloride secretion in stably transfected Madin-Darby canine kidney cells. These results suggest that anthracycline analogs may be useful for the clinical treatment of CF.     

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.     

Derlin-1 promotes the efficient degradation of the cystic fibrosis transmembrane conductance regulator (CFTR) and CFTR folding mutants

A complex involving Derlin-1 and p97 mediates the retrotranslocation and endoplasmic reticulum (ER)-associated degradation of misfolded proteins in yeast and is used by certain viruses to promote host cell protein degradation (Romisch, K. (2005) Annu. Rev. Cell Dev. Biol. 21, 435-456; Lilley, B. N., and Ploegh, H. L. (2004) Nature 429, 834-840; Ye, Y., Shibata, Y., Yun, C., Ron, D., and Rapoport, T. A. (2004) Nature 429, 841-847). We asked whether the components of this pathway are involved in the endoplasmic reticulum-associated degradation of the mammalian integral membrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), a substrate for the ubiquitin-proteasome system. We report that Derlin-1 and p97 formed complexes with CFTR in human airway epithelial cells. Derlin-1 interacted with nonubiquitylated CFTR, whereas p97 associated with ubiquitylated CFTR. Exogenous expression of Derlin-1 led to its co-localization with CFTR in the ER where it reduced wild type (WT) CFTR expression and efficiently degraded the disease-associated CFTR folding mutants, DeltaF508 and G85E (>90%). Consistent with this, Derlin-1 also reduced the amount of WT or DeltaF508 CFTR appearing in detergent-in-soluble aggregates. An approximately 70% knockdown of endogenous Derlin-1 by RNA interference increased the steady-state levels of WT and DeltaF508 CFTR by 10-15-fold, reflecting its significant role in CFTR degradation. Derlin-1 mediated the degradation of N-terminal CFTR fragments corresponding to the first transmembrane domain of CFTR, but CFTR fragments that incorporated additional domains were degraded less efficiently. These findings suggest that Derlin-1 recognizes misfolded, nonubiquitylated CFTR to initiate its dislocation and degradation early in the course of CFTR biogenesis, perhaps by detecting structural instability within the first transmembrane domain.     

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.     

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.     

The DeltaF508 cystic fibrosis mutation impairs domain-domain interactions and arrests post-translational folding of CFTR

Misfolding accounts for the endoplasmic reticulum-associated degradation of mutant cystic fibrosis transmembrane conductance regulators (CFTRs), including deletion of Phe508 (DeltaF508) in the nucleotide-binding domain 1 (NBD1). To study the role of Phe508, the de novo folding and stability of NBD1, NBD2 and CFTR were compared in conjunction with mutagenesis of Phe508. DeltaF508 and amino acid replacements that prevented CFTR folding disrupted the NBD2 fold and its native interaction with NBD1. DeltaF508 caused limited alteration in NBD1 conformation. Whereas nonpolar and some aliphatic residues were permissive, charged residues and glycine compromised the post-translational folding and stability of NBD2 and CFTR. The results suggest that hydrophobic side chain interactions of Phe508 are required for vectorial folding of NBD2 and the domain-domain assembly of CFTR, representing a combined co- and post-translational folding mechanism that may be used by other multidomain membrane proteins.     

The short apical membrane half-life of rescued {Delta}F508-cystic fibrosis transmembrane conductance regulator (CFTR) results from accelerated endocytosis of {Delta}F508-CFTR in polarized human airway epithelial cells

The most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in individuals with cystic fibrosis, DeltaF508, causes retention of DeltaF508-CFTR in the endoplasmic reticulum and leads to the absence of CFTR Cl(-) channels in the apical plasma membrane. Rescue of DeltaF508-CFTR by reduced temperature or chemical means reveals that the DeltaF508 mutation reduces the half-life of DeltaF508-CFTR in the apical plasma membrane. Because DeltaF508-CFTR retains some Cl(-) channel activity, increased expression of DeltaF508-CFTR in the apical membrane could serve as a potential therapeutic approach for cystic fibrosis. However, little is known about the mechanisms responsible for the short apical membrane half-life of DeltaF508-CFTR in polarized human airway epithelial cells. Accordingly, the goal of this study was to determine the cellular defects in the trafficking of rescued DeltaF508-CFTR that lead to the decreased apical membrane half-life of DeltaF508-CFTR in polarized human airway epithelial cells. We report that in polarized human airway epithelial cells (CFBE41o-) the DeltaF508 mutation increased endocytosis of CFTR from the apical membrane without causing a global endocytic defect or affecting the endocytic recycling of CFTR in the Rab11a-specific apical recycling compartment.     

Mutations in the nucleotide binding domain 1 signature motif region rescue processing and functional defects of cystic fibrosis transmembrane conductance regulator delta f508

The gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), an ATP binding cassette (ABC) transporter that functions as a phosphorylation- and nucleotide-regulated chloride channel, is mutated in cystic fibrosis (CF) patients. Deletion of a phenylalanine at amino acid position 508 (DeltaF508) in the first nucleotide binding domain (NBD1) is the most prevalent CF-causing mutation and results in defective protein processing and reduced CFTR function, leading to chloride impermeability in CF epithelia and heterologous systems. Using a STE6/CFTRDeltaF508 chimera system in yeast, we isolated two novel DeltaF508 revertant mutations, I539T and G550E, proximal to and within the conserved ABC signature motif of NBD1, respectively. Western blot and functional analysis in mammalian cells indicate that mutations I539T and G550E each partially rescue the CFTRDeltaF508 defect. Furthermore, a combination of both revertant mutations resulted in a 38-fold increase in CFTRDeltaF508-mediated chloride current, representing 29% of wild type channel activity. The G550E mutation increased the sensitivity of CFTRDeltaF508 and wild type CFTR to activation by cAMP agonists and blocked the enhancement of CFTRDeltaF508 channel activity by 2 mm 3-isobutyl-1-methylxanthine. The data show that the DeltaF508 defect can be significantly rescued by second-site mutations in the nucleotide binding domain 1 region, that includes the LSGGQ consensus motif.     

Protein kinase CK2, cystic fibrosis transmembrane conductance regulator, and the deltaF508 mutation: F508 deletion disrupts a kinase-binding site

Deletion of phenylalanine 508 (DeltaF508) from the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) is the most common mutation in cystic fibrosis. The F508 region lies within a surface-exposed loop that has not been assigned any interaction with associated proteins. Here we demonstrate that the pleiotropic protein kinase CK2 that controls protein trafficking, cell proliferation, and development binds wild-type CFTR near F508 and phosphorylates NBD1 at Ser-511 in vivo and that mutation of Ser-511 disrupts CFTR channel gating. Importantly, the interaction of CK2 with NBD1 is selectively abrogated by the DeltaF508 mutation without disrupting four established CFTR-associated kinases and two phosphatases. Loss of CK2 association is functionally corroborated by the insensitivity of DeltaF508-CFTR to CK2 inhibition, the absence of CK2 activity in DeltaF508 CFTR-expressing cell membranes, and inhibition of CFTR channel activity by a peptide that mimics the F508 region of CFTR (but not the equivalent DeltaF508 peptide). Disruption of this CK2-CFTR association is the first described DeltaF508-dependent protein-protein interaction that provides a new molecular paradigm in the most frequent form of cystic fibrosis.     

Diminished Self-Chaperoning Activity of the ΔF508 Mutant of CFTR Results in Protein Misfolding

The absence of a functional ATP Binding Cassette (ABC) protein called the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) from apical membranes of epithelial cells is responsible for cystic fibrosis (CF). Over 90% of CF patients carry at least one mutant allele with deletion of phenylalanine at position 508 located in the N-terminal nucleotide binding domain (NBD1). Biochemical and cell biological studies show that the ΔF508 mutant exhibits inefficient biosynthetic maturation and susceptibility to degradation probably due to misfolding of NBD1 and the resultant misassembly of other domains. However, little is known about the direct effect of the Phe508 deletion on the NBD1 folding, which is essential for rational design strategies of cystic fibrosis treatment. Here we show that the deletion of Phe508 alters the folding dynamics and kinetics of NBD1, thus possibly affecting the assembly of the complete CFTR. Using molecular dynamics simulations, we find that meta-stable intermediate states appearing on wild type and mutant folding pathways are populated differently and that their kinetic accessibilities are distinct. The structural basis of the increased misfolding propensity of the ΔF508 NBD1 mutant is the perturbation of interactions in residue pairs Q493/P574 and F575/F578 found in loop S7-H6. As a proof-of-principle that the S7-H6 loop conformation can modulate the folding kinetics of NBD1, we virtually design rescue mutations in the identified critical interactions to force the S7-H6 loop into the wild type conformation. Two redesigned NBD1-ΔF508 variants exhibited significantly higher folding probabilities than the original NBD1-ΔF508, thereby partially rescuing folding ability of the NBD1-ΔF508 mutant. We propose that these observed defects in folding kinetics of mutant NBD1 may also be modulated by structures separate from the 508 site. The identified structural determinants of increased misfolding propensity of NBD1-ΔF508 are essential information in correcting this pathogenic mutant.