Alteration of CFTR transmembrane span integration by disease-causing mutations

Many missense mutations in the cystic fibrosis transmembrane conductance regulator protein (CFTR) result in its misfolding, endoplasmic reticulum (ER) accumulation, and, thus, cystic fibrosis. A number of these mutations are located in the predicted CFTR transmembrane (TM) spans and have been projected to alter span integration. However, the boundaries of the spans have not been precisely defined experimentally. In this study, the ER luminal integration profiles of TM1 and TM2 were determined using the ER glycosylation machinery, and the effects of the CF-causing mutations G85E and G91R thereon were assessed. The mutations either destabilize the integrated conformation or alter the TM1 ER integration profile. G85E misfolding is based in TM1 destabilization by glutamic acid and loss of glycine and correlates with the temperature-insensitive ER accumulation of immature full-length CFTR harboring the mutation. By contrast, temperature-dependent misfolding owing to the G91R mutation depends on the introduction of the basic side chain rather than the loss of the glycine. This work demonstrates that CF-causing mutations predicted to have similar effects on CFTR structure actually result in disparate molecular perturbations that underlie ER accumulation and the pathology of CF.     

Targets for cystic fibrosis therapy: proteomic analysis and correction of mutant cystic fibrosis transmembrane conductance regulator

Proteomic analysis has proved to be an important tool for understanding the complex nature of genetic disorders, such as cystic fibrosis (CF), by defining the cellular protein environment (proteome) associated with wild-type and mutant proteins. Proteomic screens identified the proteome of CF transmembrane conductance regulator (CFTR), and provided fundamental information to studies designed for understanding the crucial components of physiological CFTR function. Simultaneously, high-throughput screens for small-molecular correctors of CFTR mutants provided promising candidates for therapy. The majority of CF cases are caused by nucleotide deletions (ΔF508 CFTR; >75%), resulting in CFTR misfolding, or insertion of premature termination codons (～10%), leading to unstable mRNA and reduced levels of truncated dysfunctional CFTR. In this article, we review recent results of proteomic screens, developments in identifying correctors for the most frequent CFTR mutants, and comment on how integration of the knowledge gained from these studies may aid in finding a cure for CF and a number of other genetic disorders.     

CFTR: folding, misfolding and correcting the ΔF508 conformational defect

Cystic fibrosis (CF), the most common lethal genetic disease in the Caucasian population, is caused by loss-of-function mutations of the CF transmembrane conductance regulator (CFTR), a cyclic AMP-regulated plasma membrane chloride channel. The most common mutation, deletion of phenylalanine 508 (ΔF508), impairs CFTR folding and, consequently, its biosynthetic and endocytic processing as well as chloride channel function. Pharmacological treatments may target the ΔF508 CFTR structural defect directly by binding to the mutant protein and/or indirectly by altering cellular protein homeostasis (proteostasis) to promote ΔF508 CFTR plasma membrane targeting and stability. This review discusses recent basic research aimed at elucidating the structural and trafficking defects of ΔF508 CFTR, a prerequisite for the rational design of CF therapy to correct the loss-of-function phenotype.     

VX-809 corrects folding defects in cystic fibrosis transmembrane conductance regulator protein through action on membrane-spanning domain 1

Cystic fibrosis (CF) is a fatal genetic disorder associated with defective hydration of lung airways due to the loss of chloride transport through the CF transmembrane conductance regulator protein (CFTR). CFTR contains two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a regulatory domain, and its channel assembly requires multiple interdomain contacts. The most common CF-causing mutation, F508del, occurs in NBD1 and results in misfolding and premature degradation of F508del-CFTR. VX-809 is an investigational CFTR corrector that partially restores CFTR function in people who are homozygous for F508del-CFTR. To identify the folding defect(s) in F508del-CFTR that must be repaired to treat CF, we explored the mechanism of VX-809 action. VX-809 stabilized an N-terminal domain in CFTR that contains only MSD1 and efficaciously restored function to CFTR forms that have missense mutations in MSD1. The action of VX-809 on MSD1 appears to suppress folding defects in F508del-CFTR by enhancing interactions among the NBD1, MSD1, and MSD2 domains. The ability of VX-809 to correct F508del-CFTR is enhanced when combined with mutations that improve F508del-NBD1 interaction with MSD2. These data suggest that the use of VX-809 in combination with an additional CFTR corrector that suppresses folding defects downstream of MSD1 may further enhance CFTR function in people with F508del-CFTR.     

Correction of the CF defect by curcumin: hypes and disappointments

Cystic fibrosis (CF), the most-common lethal hereditary disease in the white population, is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The mutation that is most frequently responsible for the disease, DeltaF508, causes misfolding and retention of the CFTR protein in the endoplasmic reticulum. This leads to a series of cellular dysfunctions and results in a multi-organ disease. In a recent report, Egan et al.(1) demonstrated that curcumin, a non-toxic natural product and major constituent of turmeric spice, corrected the CF defects in DeltaF508 CF mice. This paper aroused a lot of attention and hopes were raised that curcumin might produce similar effects in human, giving an efficient treatment for most CF patients. However, skepticism is growing since subsequent studies fail to reproduce these initial exciting results. Thus, although herbal medicines and dietary supplements can be desirable alternatives to classical pharmacological compounds, their efficacy needs careful evaluation both in vivo and ex vivo.     

C-terminal truncations destabilize the cystic fibrosis transmembrane conductance regulator without impairing its biogenesis. A novel class of mutation.

Defective cAMP-stimulated chloride conductance of the plasma membrane of epithelial cell is the hallmark of cystic fibrosis (CF) and results from mutations in the cystic fibrosis transmembrane conductance regulator, CFTR. In the majority of CF patients, mutations in the CFTR lead to its misfolding and premature degradation at the endoplasmic reticulum (ER). Other mutations impair the cAMP-dependent activation or the ion conductance of CFTR chloride channel. In the present work we identify a novel mechanism leading to reduced expression of CFTR at the cell surface, caused by C-terminal truncations. The phenotype of C-terminally truncated CFTR, representing naturally occurring premature termination and frameshift mutations, were examined in transient and stable heterologous expression systems. Whereas the biosynthesis, processing, and macroscopic chloride channel function of truncated CFTRs are essentially normal, the degradation rate of the mature, complex-glycosylated form is 5- to 6-fold faster than the wild type CFTR. These experiments suggest that the C terminus has a central role in maintaining the metabolic stability of the complex-glycosylated CFTR following its exit from the ER and provide a plausible explanation for the severe phenotype of CF patients harboring C-terminal truncations.     

Increased folding and channel activity of a rare cystic fibrosis mutant with CFTR modulators

Cystic fibrosis (CF) is a lethal recessive genetic disease caused by mutations in the CFTR gene. The gene product is a PKA-regulated anion channel that is important for fluid and electrolyte transport in the epithelia of lung, gut, and ducts of the pancreas and sweat glands. The most common CFTR mutation, ΔF508, causes a severe, but correctable, folding defect and gating abnormality, resulting in negligible CFTR function and disease. There are also a large number of rare CF-related mutations where disease is caused by CFTR misfolding. Yet the extent to which defective biogenesis of these CFTR mutants can be corrected is not clear. CFTRV232D is one such mutant that exhibits defective folding and trafficking. CFTRΔF508 misfolding is difficult to correct, but defective biogenesis of CFTRV232D is corrected to near wild-type levels by small-molecule folding correctors in development as CF therapeutics. To determine if CFTRV232D protein is competent as a Cl(-) channel, we utilized single-channel recordings from transfected human embryonic kidney (HEK-293) cells. After PKA stimulation, CFTRV232D channels were detected in patches with a unitary Cl(-) conductance indistinguishable from that of CFTR. Yet the frequency of detecting CFTRV232D channels was reduced to ～20% of patches compared with 60% for CFTR. The folding corrector Corr-4a increased the CFTRV232D channel detection rate and activity to levels similar to CFTR. CFTRV232D-corrected channels were inhibited with CFTR(inh-172) and stimulated fourfold by the CFTR channel potentiator VRT-532. These data suggest that CF patients with rare mutations that cause CFTR misfolding, such as CFTRV232D, may benefit from treatment with folding correctors and channel potentiators in development to restore CFTRΔF508 function.     

Reduced number of CFTR molecules in erythrocyte plasma membrane of cystic fibrosis patients

Cystic fibrosis (CF), the most common genetic disease among Caucasians, is caused by mutations in the gene encoding CFTR (cystic fibrosis transmembrane conductance regulator). The most frequent mutation, ΔF508, results in protein misfolding and, as a consequence, prevents CFTR from reaching its final location at the cell surface. CFTR is expressed in various cell types including red blood cells. The functional role of CFTR in erythrocytes is still unclear. Since the number of CFTR copies in a single erythrocyte of healthy donors and CF patients with a homozygous ΔF508 mutation is unknown, we counted CFTR, localized in erythrocyte plasma membrane, at the single molecule level. A novel experimental approach combining atomic force microscopy with quantum-dot-labeled anti-CFTR antibodies, used as topographic surface markers, was employed to detect individual CFTR molecules. Analysis of erythrocyte plasma membranes taken from healthy donors and CF patients with a homozygous ΔF508 mutation reveals mean (SEM) values of 698 (12.8) (n=542) and 172 (3.8) (n=538) CFTR molecules per red blood cell, respectively. We conclude that erythrocytes reflect the CFTR status of the organism and that quantification of CFTR in a blood sample could be useful in the diagnosis of CFTR related diseases.     

Reduced number of CFTR molecules in erythrocyte plasma membrane of cystic fibrosis patients

Cystic fibrosis (CF), the most common genetic disease among Caucasians, is caused by mutations in the gene encoding CFTR (cystic fibrosis transmembrane conductance regulator). The most frequent mutation, DeltaF508, results in protein misfolding and, as a consequence, prevents CFTR from reaching its final location at the cell surface. CFTR is expressed in various cell types including red blood cells. The functional role of CFTR in erythrocytes is still unclear. Since the number of CFTR copies in a single erythrocyte of healthy donors and CF patients with a homozygous DeltaF508 mutation is unknown, we counted CFTR, localized in erythrocyte plasma membrane, at the single molecule level. A novel experimental approach combining atomic force microscopy with quantum-dot-labeled anti-CFTR antibodies, used as topographic surface markers, was employed to detect individual CFTR molecules. Analysis of erythrocyte plasma membranes taken from healthy donors and CF patients with a homozygous DeltaF508 mutation reveals mean (SEM) values of 698 (12.8) (n=542) and 172 (3.8) (n=538) CFTR molecules per red blood cell, respectively. We conclude that erythrocytes reflect the CFTR status of the organism and that quantification of CFTR in a blood sample could be useful in the diagnosis of CFTR related diseases.     

The Cystic Fibrosis-causing Mutation ΔF508 Affects Multiple Steps in Cystic Fibrosis Transmembrane Conductance Regulator Biogenesis

The deletion of phenylalanine 508 in the first nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator is directly associated with >90% of cystic fibrosis cases. This mutant protein fails to traffic out of the endoplasmic reticulum and is subsequently degraded by the proteasome. The effects of this mutation may be partially reversed by the application of exogenous osmolytes, expression at low temperature, and the introduction of second site suppressor mutations. However, the specific steps of folding and assembly of full-length cystic fibrosis transmembrane conductance regulator (CFTR) directly altered by the disease-causing mutation are unclear. To elucidate the effects of the ΔF508 mutation, on various steps in CFTR folding, a series of misfolding and suppressor mutations in the nucleotide binding and transmembrane domains were evaluated for effects on the folding and maturation of the protein. The results indicate that the isolated NBD1 responds to both the ΔF508 mutation and intradomain suppressors of this mutation. In addition, identification of a novel second site suppressor of the defect within the second transmembrane domain suggests that ΔF508 also effects interdomain interactions critical for later steps in the biosynthesis of CFTR.     

Hsp90 cochaperone Aha1 downregulation rescues misfolding of CFTR in cystic fibrosis

The pathways that distinguish transport of folded and misfolded cargo through the exocytic (secretory) pathway of eukaryotic cells remain unknown. Using proteomics to assess global cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein interactions (the CFTR interactome), we show that Hsp90 cochaperones modulate Hsp90-dependent stability of CFTR protein folding in the endoplasmic reticulum (ER). Cell-surface rescue of the most common disease variant that is restricted to the ER, DeltaF508, can be initiated by partial siRNA silencing of the Hsp90 cochaperone ATPase regulator Aha1. We propose that failure of DeltaF508 to achieve an energetically favorable fold in response to the steady-state dynamics of the chaperone folding environment (the "chaperome") is responsible for the pathophysiology of CF. The activity of cargo-associated chaperome components may be a common mechanism regulating folding for ER exit, providing a general framework for correction of misfolding disease.     

Structural models of CFTR–AMPK and CFTR–PKA interactions: R-domain flexibility is a key factor in CFTR regulation

Cystic fibrosis (CF), the most common lethal genetic disease among Caucasians, is caused by mutations in cystic fibrosis transmembrane conductance regulator (CFTR). CFTR’s main role is to transport chloride ions across epithelial cell membranes. It also regulates many cell functions. However, the exact role of CFTR in cellular processes is not yet fully understood. It is recognized that a key factor in CFTR-related regulation is its phosphorylation state. The important kinases regulating CFTR are cAMP-dependent protein kinase A (PKA) and 5′-AMP-activated protein kinase (AMPK). PKA and AMPK have opposite effects on CFTR activity despite their highly similar structures and recognition motifs. Utilizing homology modeling, in silico mutagenesis and literature mining, we supplement available information regarding the atomic-resolution structures of PKA, AMPK and CFTR, and the complexes CFTR–PKA and CFTR–AMPK. The atomic-resolution structural predictions reveal an unexpected availability of CFTR Ser813 for phosphorylation by both PKA and AMPK. These results indicate the key role of the structural flexibility of the serine-rich R-domain in CFTR regulation by phosphorylation.     

Distinct proteostasis states drive pharmacologic chaperone susceptibility for cystic fibrosis transmembrane conductance regulator misfolding mutants

Pharmacological chaperones represent a class of therapeutic compounds for treating protein misfolding diseases. One of the most prominent examples is the FDA-approved pharmacological chaperone lumacaftor (VX-809), which has transformed cystic fibrosis (CF) therapy. CF is a fatal disease caused by mutations in the CF transmembrane conductance regulator (CFTR). VX-809 corrects folding of F508del CFTR, the most common patient mutation, yet F508del exhibits only mild VX-809 response. In contrast, rarer mutations P67L and L206W are hyperresponsive to VX-809, while G85E is nonresponsive. Despite the clinical success of VX-809, the mechanistic origin for the distinct susceptibility of mutants remains unclear. Here we use interactomics to characterize the impact of VX-809 on proteostasis interactions of P67L and L206W and compare these with F508del and G85E. We determine that hyperresponsive mutations P67L and L206W exhibit decreased interactions with proteasomal and autophagy degradation machinery compared with F508del and G85E. We then show inhibiting the proteasome attenuates P67L and L206W VX-809 response. Our data suggest a previously unidentified but required role for protein degradation in VX-809 correction. Furthermore, we present an approach for identifying proteostasis characteristics of mutant-specific therapeutic response to pharmacological chaperones.     

Spectrum of CFTR mutations on Réunion Island: impact on neonatal screening

The large heterogeneity in the cystic fibrosis (CF) gene is the main difficulty for genotype characterization. Numerous studies have reported considerable variations in frequencies of CF transmembrane conductance regulator (CFTR) mutations in different populations, such as African, Asian, or European populations. To completely characterize the spectrum of mutations in the CFTR gene in the Réunion Island population, we screened 228 CF chromosomes using denaturing high-pressure liquid chromatography and denaturing gradient gel electrophoresis following by direct sequencing. We identified 27 mutations, accounting for 93% of CF chromosomes. They included three novel mutations (M1T, 3121-3C-->G, and L1324P), which are described in this paper. The detection of such a high proportion of Réunion Island CFTR mutations is important for improving neonatal screening of CF on Réunion Island.     

Processing mutations located throughout the human multidrug resistance P-glycoprotein disrupt interactions between the nucleotide binding domains

The most common cause of cystic fibrosis is misfolding of the cystic fibrosis transmembrane conductance regulator (CFTR) protein because of deletion of residue Phe-508 (DeltaF508). P-glycoprotein (P-gp) is an ideal model protein for studying how mutations disrupt folding of ATP-binding cassette proteins such as CFTR because specific chemical chaperones can be used to correct folding defects. Interactions between the nucleotide binding domains (NBDs) are critical because ATP binds at the interface between the NBDs. Here, we used disulfide cross-linking between cysteines in the Walker A sites and the LSGGQ signature sequences to test whether processing mutations located throughout P-gp disrupted interactions between the NBDs. We found that mutations present in the cytoplasmic loops, transmembrane segments, and linker regions or deletion of Tyr-490 (equivalent to Phe-508 in CFTR) inhibited cross-linking between the NBDs. Deletion of Phe-508 in the P-gp/CFTR chimera also inhibited cross-linking between the NBDs. Cross-linking was restored, however, when the mutants were expressed in the presence of the chemical chaperone cyclosporin A. The "rescued" mutants exhibited drug-stimulated ATPase activity, and cross-linking between the NBDs was inhibited by vanadate trapping of nucleotide. These results together with our previous findings (Loo, T. W., Bartlett, M. C., and Clarke, D. M. (2002) J. Biol. Chem. 277, 27585-27588) indicate that processing mutations disrupt interactions among all four domains. It appears that cross-talk between the cytoplasmic and the transmembrane domains is required for establishment of proper domain-domain interactions that occur during folding of ATP-binding cassette protein transporters.     

Two frameshift mutations in the cystic fibrosis gene

Cystic fibrosis (CF) is a recessive disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. We have identified in exon 7 two frameshift mutations, one caused by a two-nucleotide insertion and the other caused by a one-nucleotide deletion; these mutations--CF1154insTC and CF1213delT, respectively, are predicted to shift the reading frame of the protein and to introduce UAA(ochre) termination codons at residues 369 and 368.     

Mechanisms of protein misfolding in conformational lung diseases

Genetic or environmentally-induced alterations in protein structure interfere with the correct folding, assembly and trafficking of proteins. In the lung the expression of misfolded proteins can induce a variety of pathogenetic effects. Cystic fibrosis (CF) and alpha-1 antitrypsin (AAT) deficiency are two major clinically relevant pulmonary disorders associated with protein misfolding. Both are genetic diseases the primary causes of which are expression of mutant alleles of the cystic fibrosis transmembrane conductance regulator (CFTR) and SERPINA1, respectively. The most common and best studied mutant forms of CFTR and AAT are ΔF508 CFTR and the Glu342Lys mutant of AAT called ZAAT, respectively. Non-genetic mechanisms can also damage protein structure and induce protein misfolding in the lung. Cigarette-smoke contains oxidants and other factors that can modify a protein's structure, and is one of the most significant environmental causes of protein damage within the lung. Herein we describe the mechanisms controlling the folding of wild type and mutant versions of CFTR and AAT proteins, and explore the consequences of cigarette-smoke-induced effects on the protein folding machinery in the lung.     

Targeted therapy for cystic fibrosis: cystic fibrosis transmembrane conductance regulator mutation-specific pharmacologic strategies

Cystic fibrosis (CF) results from the absence or dysfunction of a single protein, the CF transmembrane conductance regulator (CFTR). CFTR plays a critical role in the regulation of ion transport in a number of exocrine epithelia. Improvement or restoration of CFTR function, where it is deficient, should improve the CF phenotype. There are >1000 reported disease-causing mutations of the CFTR gene. Recent investigations have afforded a better understanding of the mechanism of dysfunction of many of these mutant CFTRs, and have allowed them to be classified according to their mechanism of dysfunction. These data, as well as an enhanced understanding of the role of CFTR in regulating epithelial ion transport, have led to the development of therapeutic strategies based on pharmacologic enhancement or repair of mutant CFTR dysfunction. The strategy, termed 'protein repair therapy', is aimed at improving the regulation of epithelial ion transport by mutant CFTRs in a mutation-specific fashion. The grouping of CFTR gene mutations, according to mechanism of dysfunction, yields some guidance as to which pharmacologic repair agents may be useful for specific CFTR mutations. Recent data has suggested that combinations of pharmacologic repair agents may be necessary to obtain clinically meaningful CFTR repair. Nevertheless, such strategies to improve mutant CFTR function hold great promise for the development of novel therapies aimed at correcting the underlying pathophysiology of CF.