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.     

Mutational analysis of the yeast a-factor transporter STE6, a member of the ATP binding cassette (ABC) protein superfamily.

STE6, the yeast a-factor transporter, is a member of the ATP binding cassette protein superfamily, which also includes the mammalian multidrug resistance protein and the cystic fibrosis gene product. These proteins contain two homologous halves, each with six membrane spanning segments and a predicted ATP nucleotide binding domain. To assess the importance of the two halves of STE6, and to examine the functional significance of residues conserved among members of the ATP binding cassette superfamily, we introduced mutations into the nucleotide binding domains of STE6. Our analysis demonstrates that both halves of STE6 are critical for function and that some, but not all, mutations analogous to those known to result in cystic fibrosis impair STE6 activity. To examine further the functional contribution of each half of the STE6 protein, we severed the STE6 coding sequence and expressed the two halves of the transporter as separate polypeptides. Whereas 'half-molecules' are unable to provide transport function individually, co-expression of both half-molecules in the same cell leads to functional reconstitution of STE6-mediated a-factor transport.     

Biosynthesis of mucin type <Emphasis Type="Italic">O</Emphasis>-glycans: Lack of correlation between glycosyltransferase and sulfotransferase activities and CFTR expression

Structural differences have been reported in the glycosylation patterns of cystic fibrosis glycoproteins. Although the gene mutated in cystic fibrosis (CFTR) has been cloned and characterized as a chloride channel, its relationship to the highly viscous mucus and structural glycoprotein and mucin abnormalities in cystic fibrosis still remains to be defined. We have evaluated O-glycan biosynthesis in CHO and BHK cells that express CFTR and F508 CFTR as in vitro models, and utilized the cftr knockout mouse as an in vivo model of CFTR dysfunction. Activities of glycosyltransferases and sulfotransferases synthesizing mucin type O-glycan chains were determined in these models. Differences in transferase activity levels were found between tissues and cell types and during mouse development. No specific patterns of activities were associated with the lack of CFTR or with F508CFTR expression. This suggests that it is not the presence or absence of normal CFTR, or the presence of mutant CFTR alone, but rather cell specific additional factors or pathophysiological consequences that determine the changes in mucin glycosylation in cystic fibrosis.     

Frequency of the F508 deletion in the CFTR gene in Turkish cystic fibrosis patients

Summary The F508 deletion in the cystic fibrosis transmembrane conductance regulator (CFTR) gene was found in 8 out of 30 Turkish cystic fibrosis (CF) chromosomes (27%). Five Turkish ΔF508 CF chromosomes were associated with the risk haplotype B in KM19 (2 allele)/XV2c (1 allele). In the Turkish population, cystic fibrosis is predominantly caused by mutations other than the F508 deletion.     

Altered protein folding may be the molecular basis of most cases of cystic fibrosis.

Experiments have demonstrated that the cystic fibrosis transmembrane conductance regulator protein (CFTR), containing the most common cystic fibrosis (CF)-causing mutation (delta F508), reaches the plasma membrane in reduced amounts. Studies of a peptide model of CFTR indicate that the delta F508 mutated region is more sensitive to denaturating conditions. This paper proposes that altered protein folding accounts for these findings, and, thus, most cases of CF. Significantly, the hypothesis makes specific predictions about the effect of stabilizing conditions on mutant CFTR, and, further, suggests a new class of pharmaceuticals that may prove effective in the treatment of this important genetic disease.     

Rescue of Folding Defects in ABC Transporters Using Pharmacological Chaperones

The ATP-binding cassette (ABC) family of membrane transport proteins is the largest class of transporters in humans (48 members). The majority of ABC transporters function at the cell surface. Therefore, defective folding and trafficking of the protein to the cell surface can lead to serious health problems. The classic example is cystic fibrosis (CF). In most CF patients, there is a deletion of Phe508 in the CFTR protein (ΔF508 CFTR) that results in defective folding and intracellular retention of the protein (processing mutant). A potential treatment for most patients with CF would be to use a ligand(s) of CFTR that acts a pharmacological chaperone to correct the folding defect. The feasibility of such an approach was first demonstrated with the multidrug transporter P-glycoprotein (P-gp), an ABC transporter, and a sister protein of CFTR. It was found that P-gps with mutations at sites equivalent to those found in CFTR processing mutants were rescued when they were expressed in the presence of drug substrates or modulators of P-gp. These compounds acted as pharmacological chaperones and functioned by promoting interactions among the various domains in the protein during the folding process. Several groups have attempted to identify compounds that could rescue the folding defect in ΔF508 CFTR. The best compound identified through high-throughout screening is a quinazoline derivative (CFcor-325). Expression of ΔF508 CFTR as well as other CFTR processing mutants in the presence of 1 μM CFcor-325 promoted folding and trafficking of the mutant proteins to the cell surface in an active conformation. Therefore, CFcor-325 and other quinazoline derivates could be important therapeutic compounds for the treatment of CF.     

Glycosylation and the cystic fibrosis transmembrane conductance regulator

The cystic fibrosis transmembrane conductance regulator (CFTR) has been known for the past 11 years to be a membrane glycoprotein with chloride channel activity. Only recently has the glycosylation of CFTR been examined in detail, by O'Riordan et al in Glycobiology. Using cells that overexpress wild-type (wt)CFTR, the presence of polylactosamine was noted on the fully glycosylated form of CFTR. In the present commentary the results of that work are discussed in relation to the glycosylation phenotype of cystic fibrosis (CF), and the cellular localization and processing of ΔF508 CFTR. The significance of the glycosylation will be known when endogenous CFTR from primary human tissue is examined.     

Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis.

The gene associated with cystic fibrosis (CF) encodes a membrane-associated, N-linked glycoprotein called CFTR. Mutations were introduced into CFTR at residues known to be altered in CF chromosomes and in residues believed to play a role in its function. Examination of the various mutant proteins in COS-7 cells indicated that mature, fully glycosylated CFTR was absent from cells containing delta F508, delta 1507, K464M, F508R, and S5491 cDNA plasmids. Instead, an incompletely glycosylated version of the protein was detected. We propose that the mutant versions of CFTR are recognized as abnormal and remain incompletely processed in the endoplasmic reticulum where they are subsequently degraded. Since mutations with this phenotype represent at least 70% of known CF chromosomes, we argue that the molecular basis of most cystic fibrosis is the absence of mature CFTR at the correct cellular location.     

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

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

Frequency of the ΔF508 mutation and flanking marker haplotypes at the CF locus from 167 Czech families

Summary This study analyses distribution patterns of the ΔF508 mutation of the cystic fibrosis transmembrane conductance regulator gene (CFTR) gene and the cystic fibrosis (CF)-linked marker loci MET, D7S23, D7S399, and D7S8 in a sample of 167 (116 complete) CF families from Bohemia and Moravia (Czechoslovakia). DNA typing was performed by polymerase chain reaction amplification, restriction analysis, and agarose or polyacrylamide gel electrophoresis. The frequency of the ΔF508 mutation in this sample is 67% and the frequency of the B haplotype is 77.6% on CF chromosomes. Linkage disequilibrium was found between ΔF508 and all markers tested.     

Gp78 cooperates with RMA1 in endoplasmic reticulum-associated degradation of CFTRDeltaF508

Misfolded or improperly assembled proteins in the endoplasmic reticulum (ER) are exported into the cytosol and degraded via the ubiquitin–proteasome pathway, a process termed ER-associated degradation (ERAD). Saccharomyces cerevisiae Hrd1p/Der3p is an ER membrane-spanning ubiquitin ligase that participates in ERAD of the cystic fibrosis transmembrane conductance regulator (CFTR) when CFTR is exogenously expressed in yeast cells. Two mammalian orthologues of yeast Hrd1p/Der3p, gp78 and HRD1, have been reported. Here, we demonstrate that gp78, but not HRD1, participates in ERAD of the CFTR mutant CFTRΔF508, by specifically promoting ubiquitylation of CFTRΔF508. Domain swapping experiments and deletion analysis revealed that gp78 binds to CFTRΔF508 through its ubiquitin binding region, the so-called coupling of ubiquitin to ER degradation (CUE) domain. Gp78 polyubiquitylated in vitro an N-terminal ubiquitin-glutathione-S-transferase (GST)-fusion protein, but not GST alone. This suggests that gp78 recognizes the ubiquitin that is already conjugated to CFTRΔF508 and catalyzes further polyubiquitylation of CFTRΔF508 in a manner similar to that of a multiubiquitin chain assembly factor (E4). Furthermore, we revealed by small interfering RNA methods that the ubiquitin ligase RMA1 functioned as an E3 enzyme upstream of gp78. Our data demonstrates that gp78 cooperates with RMA1 with E4-like activity in the ERAD of CFTRΔF508.     

Distribution patterns of the ΔF508 mutation in the CFTR gene on CF-linked marker haplotypes in the German population

Summary We have measured the frequency of the ΔF508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and its association with cystic fibrosis (CF)-linked marker haplotypes in the German population. Based on the analysis of 400 CF chromosomes, the frequency of the ΔF508 mutation is estimated to be 77.3%, the vast majority being associated with marker haplotype KM19-XV2c 2 1. Our data further suggest the presence of another frequent CF mutation associated with this marker haplotype.     

Three point mutations in the CFTR gene in French cystic fibrosis patients: Identification by denaturing gradient gel electrophoresis

Summary The cystic fibrosis (CF) gene was recently identified as a gene spanning 250 kilobases (kbp) and coding for a 1480 amino acid protein, cystic fibrosis transmembrane conductance regulator (CFTR). Approximately 70% of CF mutations involve a three-base-pair deletion in CFTR exon 10, resulting in the loss of a phenylalanine at position 508 in the gene product (ΔF508). In order to screen for other molecular defects, we have used a strategy based on denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified gene segments. This method, which permits rapid detection of any sequence change in a given DNA stretch, was used successfully to analyse 61 non-ΔF508 CF chromosomes from French CF patients. A study of CFTR exons 10, 11, 14a, 15 and 20 detected three mutations located in exons 14a, 15 and 20, along with several nucleotide sequence polymorphisms. These nucleotide changes were identified by direct sequencing of PCR fragments displaying altered electrophoretic behaviour, together with some of the polymorphisms and mutations previously characterized by others. The strategy presented here constitutes a valuable tool for the development of carrier testing for individuals or couples with a family history of cystic fibrosis, and will contribute to deciphering the functionally important regions of the CFTR gene.     

A novel natural product compound enhances cAMP-regulated chloride conductance of cells expressing CFTR[delta]F508

BACKGROUND: Cystic fibrosis (CF) results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel localized at the plasma membrane of diverse epithelia. The most common mutation leading to CF, Delta F508, occurs in the first nucleotide-binding domain (NBD1) of CFTR. The Delta F508 mutation disrupts protein processing, leading to a decreased level of mutant channels at the plasma membrane and reduced transepithelial chloride permeability. Partial correction of the Delta F508 molecular defect in vitro is achieved by incubation of cells with several classes of chemical chaperones, indicating that further investigation of novel small molecules is warranted as a means for producing new therapies for CF. MATERIALS AND METHODS: The yeast two-hybrid assay was used to study the effect of CF-causing mutations on the ability of NBD1 to self-associate and form dimers. A yeast strain demonstrating defective growth as a result of impaired NBD1 dimerization due to Delta F508 was used as a drug discovery bioassay for the identification of plant natural product compounds restoring mutant NBD1 interaction. Active compounds were purified and the chemical structures determined. The purified compounds were tested in epithelial cells expressing CFTR Delta F508 and the resulting effect on transepithelial chloride permeability was assessed using short-circuit chloride current measurements. RESULTS: Wild-type NBD1 of CFTR forms homodimers in a yeast two-hybrid assay. CF-causing mutations within NBD1 that result in defective processing of CFTR (Delta F508, Delta I507, and S549R) disrupted NBD1 interaction in yeast. In contrast, a CF-causing mutation that does not impair CFTR processing (G551D) had no effect on NBD1 dimerization. Using the yeast-based assay, we identified a novel limonoid compound (TS3) that corrected the Delta F508 NBD1 dimerization defect in yeast and also increased the chloride permeability of Fisher Rat Thyroid (FRT) cells stably expressing CFTR Delta F508. CONCLUSION: The establishment of a phenotype for the Delta F508 mutation in the yeast two-hybrid system yielded a simple assay for the identification of small molecules that interact with the mutant NBD1 and restore dimerization. The natural product compound identified using the system (TS3) was found to increase chloride conductance in epithelial cells to an extent comparable to genistein, a known CFTR activator. The yeast system will thus be useful for further identification of compounds with potential for CF drug therapy.     

Cystic Fibrosis: A Disease of Altered Protein Folding

Cystic fibrosis (CF) is caused by mutations in the gene that encodes the cystic fibrosis transmembrane conductance regulator, CFTR. Previously we demonstrated that the common F508 mutation in the first nucleotide binding domain (NBD1) alters the ability of the domain to fold into a functional three-dimensional structure, providing a molecular explanation for the observation that the mutant CFTR is retained in the endoplasmic reticulum and does not traffic to the apical membrane of affected epithelial cells. Notably, when conditions are altered to promote folding of the mutant protein, it can assume a functional conformation. Correcting the folding defect may have therapeutic benefit for the treatment of cystic fibrosis. Here we summarize these results and discuss the implications in vitro folding studies have for understanding the pathobiology of CF.     

Functional analysis of F508del CFTR in native human colon

The major cystic fibrosis mutation F508del has been classified by experiments in animal and cell culture models as a temperature-sensitive mutant defective in protein folding, processing and trafficking, but literature data on F508del CFTR maturation and function in human tissue are inconsistent. In the present study the molecular pathology of F508del CFTR was characterized in freshly excised rectal mucosa by bioelectric measurement of the basic defect and CFTR protein analysis by metabolic labelling or immunoblot. The majority of investigated F508del homozygous subjects expressed low amounts of complex-glycosylated mature F508del CFTR and low residual F508del CFTR-mediated chloride secretory activity in the rectal mucosa. The finding that some F508del CFTR escapes the ER quality control in vivo substantiates the hope that the defective processing and trafficking of F508del CFTR can be corrected by pharmacological agents.     

Delta F508 CFTR pool in the endoplasmic reticulum is increased by calnexin overexpression

The most common cystic fibrosis transmembrane conductance regulator (CFTR) mutant in cystic fibrosis patients, Delta F508 CFTR, is retained in the endoplasmic reticulum (ER) and is consequently degraded by the ubiquitin-proteasome pathway known as ER-associated degradation (ERAD). Because the prolonged interaction of Delta F508 CFTR with calnexin, an ER chaperone, results in the ERAD of Delta F508 CFTR, calnexin seems to lead it to the ERAD pathway. However, the role of calnexin in the ERAD is controversial. In this study, we found that calnexin overexpression partially attenuated the ERAD of Delta F508 CFTR. We observed the formation of concentric membranous bodies in the ER upon calnexin overexpression and that the Delta F508 CFTR but not the wild-type CFTR was retained in the concentric membranous bodies. Furthermore, we observed that calnexin overexpression moderately inhibited the formation of aggresomes accumulating the ubiquitinated Delta F508 CFTR. These findings suggest that the overexpression of calnexin may be able to create a pool of Delta F508 CFTR in the ER.     

Introduction of the most common cystic fibrosis mutation (Delta F508) into human P-glycoprotein disrupts packing of the transmembrane segments

The most common mutation in cystic fibrosis (deletion of phenylalanine 508 (DeltaF508) in the cystic fibrosis conductance transmembrane regulator (CFTR) gene) causes defective synthesis of CFTR protein. To understand how this deletion interferes with protein folding, we made the equivalent deletion (DeltaY490) in P-glycoprotein (P-gp). A Cys-less P-gp with cysteines in transmembrane (TM) 4 or TM5 can be cross-linked with a cysteine in TM12. Deleting Tyr(490) in P-gp resulted in an inactive and defectively processed mutant in which no cross-linking between TM4 or TM5 and TM12 was detected. Expression of the DeltaY490 mutant in the presence of a chemical chaperone corrected the processing defect and yielded active P-gp mutants that could be cross-linked between TM4 or TM5 and TM12. Cross-linking between TM4 or TM5 and TM12 was also detected when residues (483)TIAENIRYG(491) in P-gp were replaced with residues (501)TIKENIIFG(509) from CFTR (P-gp/CFTR). Deleting Phe(508) in the P-gp/CFTR chimera, however, caused defective processing of the mutant protein and no detectable cross-linking between TM4 or TM5 and TM12. The processing defect was corrected with a chemical chaperone and yielded active P-gp/CFTR mutant proteins that could be cross-linked. These results show that deletion at residue 490 disrupts packing of the TM segments possibly by affecting interaction between the first nucleotide-binding domain (Tyr(490)) and the first cytoplasmic loop (Glu(184)).     

Strategies for Correcting the AF508 CFTR Protein-Folding Defect

Many human diseases arise as a result of mutations within genes encoding essential proteins. In many cases, the mutations are not so severe as to render the protein biologically inactive. Rather, the mutations oftentimes result in only subtle protein-folding abnormalities. In the case of the CFTR protein, a mutation leading to the loss of a single amino acid is responsible for the diseased state in the majority of individuals with cystic fibrosis. Here the newly synthesized mutant CFTR protein, missing a phenylalanine residue at position 508 (F508 CFTR), is unable to transit from the endoplasmic reticulum to the plasma membrane, where it functions as a regulator of chloride transport. All of the available evidence indicate that the newly synthesized F508 CFTR protein adopts a slightly altered conformation and therefore is retained at the level of the endoplasmic reticulum, ostensibly by the actions of the cellular quality control system. Because the mutant protein is capable of functioning as a chloride channel, developing ways to elicit its release out of the ER and to the plasma membrane has important clinical implications. Herein, we discuss our recent studies showing that the protein folding defect associated with the F508 CFTR mutation, as well as a number of other temperature-sensitive mutations, can be overcome by strategies designed to influence protein folding inside the cell. Specifically we show that a number of low-molecular-weight compounds, all of which are known to stabilize proteins in their native conformation, are effective in rescuing the folding and/or processing defects associated with different mutations that oftentimes lead to human disease.