Full-length Cystic Fibrosis Transmembrane Conductance Regulator Research Articles

Lentiviral Gene Therapy for Cystic Fibrosis: A Promising Approach and First-in-Human Trial.

Cystic fibrosis (CF) is a genetic disease caused by mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene. Although CF is a multiorgan disease, the leading causes of morbidity and mortality are related to progressive lung disease. Current understanding of the effects of the broad spectrum of CFTR mutations on CFTR function has allowed for the development of CFTR modulator therapies. Despite the remarkable impact that these therapies have had, there remains a significant proportion of people with CF (estimated at 10-15% of the global CF population) who are genetically ineligible for, or intolerant of, current CFTR-targeting therapies and whose therapeutic needs remain unmet. Inhaled genetic therapies offer the prospect of addressing the unmet pulmonary treatment need in people with CF, with several approaches, including gene addition therapy (the focus of this review), RNA-based therapies, antisense oligonucleotides, and gene editing, being explored. Various nonviral and viral vectors have been investigated for CF gene addition therapy for mutation-agnostic restoration of CFTR function in the lungs. Lentiviral vectors offer the prospect of highly efficient and long-lasting gene expression, and the potential to be safely and, in contrast to other commonly used viral vectors, effectively redosed. A third-generation lentiviral vector pseudotyped with Sendai virus F and HN envelope proteins (rSIV.F/HN) has been developed for the treatment of CF. Promising preclinical results support the progression of this vector carrying a full-length CFTR transgene (BI 3720931) into a first-in-human clinical trial expected to begin in 2024.

DNA-PKcs inhibition improves sequential gene insertion of the full-length CFTR cDNA in airway stem cells

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Although many people with CF (pwCF) are treated using CFTR modulators, some are non-responsive due to their genotype or other uncharacterized reasons. Autologous airway stem cell therapies, in which the CFTR cDNA has been replaced, may enable a durable therapy for all pwCF. Previously, CRISPR-Cas9 with two AAVs was used to sequentially insert two halves of the CFTR cDNA and an enrichment cassette into the CFTR locus. However, the editing efficiency was <10% and required enrichment to restore CFTR function. Further improvement in gene insertion may enhance cell therapy production. To improve CFTR cDNA insertion in human airway basal stem cells (ABCs), we evaluated the use of the small molecules AZD7648 and ART558 which inhibit non-homologous end joining (NHEJ) and micro-homology mediated end joining (MMEJ). Adding AZD7648 alone improved gene insertion by 2-3-fold. Adding both ART558 and AZD7648 improved gene insertion but induced toxicity. ABCs edited in the presence of AZD7648 produced differentiated airway epithelial sheets with restored CFTR function after enrichment. Adding AZD7648 did not increase off-target editing. Further studies are necessary to validate if AZD7648 treatment enriches cells with oncogenic mutations.

DNA-PKcs Inhibition Improves Sequential Gene Insertion of the Full-Length CFTR cDNA in Airway Stem Cells.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Although many people with CF (pwCF) are treated using CFTR modulators, some are non-responsive due to their genotype or other uncharacterized reasons. Autologous airway stem cell therapies, in which the CFTR cDNA has been replaced, may enable a durable therapy for all pwCF. Previously, CRISPR-Cas9 with two AAVs was used to sequentially insert two halves of the CFTR cDNA and an enrichment cassette into the CFTR locus. However, the editing efficiency was <10% and required enrichment to restore CFTR function. Further improvement in gene insertion may enhance cell therapy production. To improve CFTR cDNA insertion in human airway basal stem cells (ABCs), we evaluated the use of the small molecules AZD7648 and ART558 which inhibit non-homologous end joining (NHEJ) and micro-homology mediated end joining (MMEJ). Adding AZD7648 alone improved gene insertion by 2-3-fold. Adding both ART558 and AZD7648 improved gene insertion but induced toxicity. ABCs edited in the presence of AZD7648 produced differentiated airway epithelial sheets with restored CFTR function after enrichment. Adding AZD7648 did not increase off-target editing. Further studies are necessary to validate if AZD7648 treatment enriches cells with oncogenic mutations.

Readthrough-induced misincorporated amino acid ratios guide mutant-specific therapeutic approaches for two CFTR nonsense mutations.

Cystic fibrosis (CF) is a monogenic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Premature termination codons (PTCs) represent ∼9% of CF mutations that typically cause severe expression defects of the CFTR anion channel. Despite the prevalence of PTCs as the underlying cause of genetic diseases, understanding the therapeutic susceptibilities of their molecular defects, both at the transcript and protein levels remains partially elucidated. Given that the molecular pathologies depend on the PTC positions in CF, multiple pharmacological interventions are required to suppress the accelerated nonsense-mediated mRNA decay (NMD), to correct the CFTR conformational defect caused by misincorporated amino acids, and to enhance the inefficient stop codon readthrough. The G418-induced readthrough outcome was previously investigated only in reporter models that mimic the impact of the local sequence context on PTC mutations in CFTR. To identify the misincorporated amino acids and their ratios for PTCs in the context of full-length CFTR readthrough, we developed an affinity purification (AP)-tandem mass spectrometry (AP-MS/MS) pipeline. We confirmed the incorporation of Cys, Arg, and Trp residues at the UGA stop codons of G542X, R1162X, and S1196X in CFTR. Notably, we observed that the Cys and Arg incorporation was favored over that of Trp into these CFTR PTCs, suggesting that the transcript sequence beyond the proximity of PTCs and/or other factors can impact the amino acid incorporation and full-length CFTR functional expression. Additionally, establishing the misincorporated amino acid ratios in the readthrough CFTR PTCs aided in maximizing the functional rescue efficiency of PTCs by optimizing CFTR modulator combinations. Collectively, our findings contribute to the understanding of molecular defects underlying various CFTR nonsense mutations and provide a foundation to refine mutation-dependent therapeutic strategies for various CF-causing nonsense mutations.

Exon-skipping antisense oligonucleotides for cystic fibrosis therapy.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF), and the CFTR-W1282X nonsense mutation causes a severe form of CF. Although Trikafta and other CFTR-modulation therapies benefit most CF patients, targeted therapy for patients with the W1282X mutation is lacking. The CFTR-W1282X protein has residual activity but is expressed at a very low level due to nonsense-mediated messenger RNA (mRNA) decay (NMD). NMD-suppression therapy and read-through therapy are actively being researched for CFTR nonsense mutants. NMD suppression could increase the mutant CFTR mRNA, and read-through therapies may increase the levels of full-length CFTR protein. However, these approaches have limitations and potential side effects: because the NMD machinery also regulates the expression of many normal mRNAs, broad inhibition of the pathway is not desirable, and read-through drugs are inefficient partly because the mutant mRNA template is subject to NMD. To bypass these issues, we pursued an exon-skipping antisense oligonucleotide (ASO) strategy to achieve gene-specific NMD evasion. A cocktailof two splice-site-targeting ASOs induced the expression of CFTR mRNA without the premature-termination-codon-containing exon 23 (CFTR-Δex23), which is an in-frame exon. Treatment of human bronchial epithelial cells with this cocktail of ASOs that target the splice sites flanking exon 23 results in efficient skipping of exon 23 and an increase in CFTR-Δex23 protein. The splice-switching ASO cocktail increases the CFTR-mediated chloride current in human bronchial epithelial cells. Our results set the stage for developing an allele-specific therapy for CF caused by the W1282X mutation.

Targeted replacement of full-length CFTR in human airway stem cells by CRISPR-Cas9 for pan-mutation correction in the endogenous locus

Cystic fibrosis (CF) is a monogenic disease caused by impaired production and/or function of the CF transmembrane conductance regulator (CFTR) protein. Although we have previously shown correction of the most common pathogenic mutation, there are many other pathogenic mutations throughout the CF gene. An autologous airway stem cell therapy in which the CFTR cDNA is precisely inserted into the CFTR locus may enable the development of a durable cure for almost all CF patients, irrespective of the causal mutation. Here, we use CRISPR-Cas9 and two adeno-associated viruses (AAVs) carrying the two halves of the CFTR cDNA to sequentially insert the full CFTR cDNA along with a truncated CD19 (tCD19) enrichment tag in upper airway basal stem cells (UABCs) and human bronchial epithelial cells (HBECs). The modified cells were enriched to obtain 60%-80% tCD19+ UABCs and HBECs from 11 different CF donors with a variety of mutations. Differentiated epithelial monolayers cultured at air-liquid interface showed restored CFTR function that was >70% of the CFTR function in non-CF controls. Thus, our study enables the development of a therapy for almost all CF patients, including patients who cannot be treated using recently approved modulator therapies.

The CFTR P67L variant reveals a key role for N-terminal lasso helices in channel folding, maturation, and pharmacologic rescue

Patients with cystic fibrosis (CF) harboring the P67L variant in the cystic fibrosis transmembrane conductance regulator (CFTR) often exhibit a typical CF phenotype, including severe respiratory compromise. This rare mutation (reported in <300 patients worldwide) responds robustly to CFTR correctors, such as lumacaftor and tezacaftor, with rescue in model systems that far exceed what can be achieved for the archetypical CFTR mutant F508del. However, the specific molecular consequences of the P67L mutation are poorly characterized. In this study, we conducted biochemical measurements following low-temperature growth and/or intragenic suppression, which suggest a mechanism underlying P67L that (1) shares key pathogenic features with F508del, including off-pathway (non-native) folding intermediates, (2) is linked to folding stability of nucleotide-binding domains 1 and 2, and (3) demonstrates pharmacologic rescue that requires domains in the carboxyl half of the protein. We also investigated the “lasso” helices 1 and 2, which occur immediately upstream of P67. Based on limited proteolysis, pulse chase, and molecular dynamics analysis of full-length CFTR and a series of deletion constructs, we argue that P67L and other maturational processing (class 2) defects impair the integrity of the lasso motif and confer misfolding of downstream domains. Thus, amino-terminal missense variants elicit a conformational change throughout CFTR that abrogates maturation while providing a robust substrate for pharmacologic repair.

Identification of Compounds That Promote Readthrough of Premature Termination Codons in the CFTR.

Cystic fibrosis (CF) is caused by a mutation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, which disrupts an ion channel involved in hydration maintenance via anion homeostasis. Nearly 5% of CF patients possess one or more copies of the G542X allele, which results in a stop codon at residue 542, preventing full-length CFTR protein synthesis. Identifying small-molecule modulators of mutant CFTR biosynthesis that affect the readthrough of this and other premature termination codons to synthesize a fully functional CFTR protein represents a novel target area of drug discovery. We describe the implementation and integration for large-scale screening of a homogeneous, 1536-well functional G542X-CFTR readthrough assay. The assay uses HEK 293 cells engineered to overexpress the G542X-CFTR mutant, whose functional activity is monitored with a membrane potential dye. Cells are co-incubated with a CFTR amplifier and CFTR corrector to maximize mRNA levels and trafficking of CFTR to the cell surface. Compounds that allow translational readthrough and synthesis of functional CFTR chloride channels are reflected by changes in membrane potential in response to cAMP stimulation with forskolin and CFTR channel potentiation with genistein. Assay statistics yielded Z′ values of 0.69 ± 0.06. As further evidence of its suitability for high-throughput screening, we completed automated screening of approximately 666,000 compounds, identifying 7761 initial hits. Following secondary and tertiary assays, we identified 188 confirmed hit compounds with low and submicromolar potencies. Thus, this approach takes advantage of a phenotypic screen with high-throughput scalability to identify new small-molecule G542X-CFTR readthrough modulators.

CFTR trafficking mutations disrupt cotranslational protein folding by targeting biosynthetic intermediates

Protein misfolding causes a wide spectrum of human disease, and therapies that target misfolding are transforming the clinical care of cystic fibrosis. Despite this success, however, very little is known about how disease-causing mutations affect the de novo folding landscape. Here we show that inherited, disease-causing mutations located within the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) have distinct effects on nascent polypeptides. Two of these mutations (A455E and L558S) delay compaction of the nascent NBD1 during a critical window of synthesis. The observed folding defect is highly dependent on nascent chain length as well as its attachment to the ribosome. Moreover, restoration of the NBD1 cotranslational folding defect by second site suppressor mutations also partially restores folding of full-length CFTR. These findings demonstrate that nascent folding intermediates can play an important role in disease pathogenesis and thus provide potential targets for pharmacological correction.

A helper-dependent adenoviral vector rescues CFTR to wild-type functional levels in cystic fibrosis epithelial cells harbouring class I mutations.

Cystic fibrosis (CF) is a genetic disorder affecting multiple organs, including the pancreas, hepatobiliary system and reproductive organs; however, lung disease is responsible for the majority of morbidity and mortality. Management of CF involves CF transmembrane conductance regulator (CFTR) modulator agents including corrector drugs to augment cellular trafficking of mutant CFTR as well as potentiators that open defective CFTR channels. These therapies are poised to help most individuals with CF, with the notable exception of individuals with class I mutations where full-length CFTR protein is not produced. For these mutations, gene replacement has been suggested as a potential solution.In this work, we used a helper-dependent adenoviral vector (HD-CFTR) to express CFTR in nasal epithelial cell cultures derived from CF subjects with class I CFTR mutations.CFTR function was significantly restored in CF cells by HD-CFTR and reached healthy control functional levels as detected by Ussing chamber and membrane potential (FLIPR) assay. A dose-response relationship was observed between the amount of vector used and subsequent functional outcomes; small amounts of HD-CFTR were sufficient to correct CFTR function. At higher doses, HD-CFTR did not increase CFTR function in healthy control cells above baseline values. This latter observation allowed us to use this vector to benchmark in vitro efficacy testing of CFTR-modulator drugs.In summary, we demonstrate the potential for HD-CFTR to inform in vitro testing and to restore CFTR function to healthy control levels in airway cells with class I or CFTR nonsense mutations.

Potentially lethal cystic fibrosis gene variant in the orangutan.

A syndrome of chronic upper and lower airway disease leading to increased morbidity and mortality occurs primarily in captive orangutans. Similarities in symptoms to the inherited human respiratory disease, cystic fibrosis, led us to hypothesize that orangutan respiratory disease is a result of variants in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. We identified the nonsense variant, c.484A>T (p.Lys162X), in heterozygosity in an unaffected orangutan. Analysis of the pedigree of this orangutan confirmed that both his sire and deceased fetus also harbored the c.484A>T allele. An expression minigene harboring c.484A>T produced no full-length CFTR protein in HEK293 cells. Finally, the c.484A>T CFTR messenger RNA abundance was severely reduced in primary nasal epithelial cells of the orangutan indicating that c.484A>T (p.Lys162X) is potentially lethal. Genetic screening of the captive orangutan population could be used to prevent transmission of this potentially lethal variant, and thus aid in the conservation of this critically endangered species.

Cystic Fibrosis Gene Mutation Frequency Among a Group of Suspected Children in King Hussein Medical Center.

Introduction:Cystic fibrosis (CF) is a genetic multisystem disorder that affects mostly the lungs, but other organs such as liver, pancreas and intestine also affected. CF is inherited in an autosomal recessive manner and occurs in males and females equally. Cystic fibrosis Transmembrane Conductance Regulator (CFTR) mutations are classified into five classes. Class 1 (non-functional protein), class 2 (near-absence of mature CFTR protein at the apical cell membrane), class 3 (full-length CFTR protein incorporated into the cell membrane), class 4 (reduced conductance CFTR mutation), and class 5 (reduced amount of CFTR protein with normal function). Globally F508 mutation is the most common.Aim:The aim of this study was to determine the frequency of CFTR gene mutation in Jordanian populations attending a major hospital (KHMC).Material and Methods:This is a retrospective study was conducted on 777 sera samples for patients clinically suspected to have cystic fibrosis over a six year period 1/1/2013-1/10/2018. The patient’s age range between 1year and 33 years, of which 59.2% (460) were male and 40.8% (317) female. Blood samples were analyzed at Princess Iman Centre for Research and Laboratory Sciences at King Hussein Medical Centre. The samples were tested for 34 mutations of CFTR gene using CF Strip Assay VIENNA LAB Diagnostics GmbH, Austria by polymerase chain reaction (PCR).Results:A total of 777 patients samples were analyzed for cystic gene mutations. Twelve (12) mutations were identified. In 49 patients (6.3%) were heterozygous genotype mutant and 28 (3.6%) were homozygous. The most frequent mutation F508del was found in 32/77 (41.5%). 20 (25.9%) of them were heterozygous genotype mutant and 12 (15.6%) were homozygous genotype mutant. The second frequent mutation was N1303K with frequency rate 15.6% (12/77), 9 (11.7%) of them were heterozygous and 3 (3.9%) were homozygous. Regarding frequency of cystic fibrosis gene mutation depending on sex, 55.8% (43/77) of mutations were found in male, whereas 44.2% (34/77) in female.Conclusion:Our findings suggest that cystic fibrosis in Jordan is not a rare disease, and found that the most frequent CFTR gene mutation was F508del, which is in keeping with results from other Mediterranean countries.

R-Domain Phosphorylation by Protein Kinase A Stimulates Dissociation of Unhydrolyzed ATP from the First Nucleotide-Binding Site of the Cystic Fibrosis Transmembrane Conductance Regulator.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an asymmetric ATP-binding cassette transporter in which ATP hydrolysis occurs only at the second of the two composite nucleotide-binding sites whereas there are noncanonical substitutions of key catalytic residues in the first site. Therefore, in widely accepted models of CFTR function, ATP is depicted as remaining bound at the first site while it is hydrolyzed at the second site. However, the long lifetime of ATP at nucleotide-binding domain 1 (NBD1) had been measured under conditions where the channel had not been activated by phosphorylation. Here we show that phosphorylation by protein kinase A (PKA), obligatory for channel activation, strongly accelerates dissociation of the unhydrolyzed ATP from NBD1 of both full-length and NBD2-deleted CFTR. This stimulation of nucleotide release results from phosphorylation of the CFTR regulatory domain (residues 634-835) (R-domain). Mimicking phosphorylation by mutating the eight phosphorylation sites in the R-domain (8SE) has the same robust effect on accelerating the dissociation of ATP from NBD1. These findings provide new insight into relationships between R-domain phosphorylation and ATP binding and hydrolysis, the two main CFTR regulatory pathways.

Structural stability of purified human CFTR is systematically improved by mutations in nucleotide binding domain 1

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is an ABC transporter containing two transmembrane domains forming a chloride ion channel, and two nucleotide binding domains (NBD1 and NBD2). CFTR has presented a formidable challenge to obtain monodisperse, biophysically stable protein. Here we report a comprehensive study comparing effects of single and multiple NBD1 mutations on stability of both the NBD1 domain alone and on purified full length human CFTR. Single mutations S492P, A534P, I539T acted additively, and when combined with M470V, S495P, and R555K cumulatively yielded an NBD1 with highly improved structural stability. Strategic combinations of these mutations strongly stabilized the domain to attain a calorimetric Tm > 70 °C. Replica exchange molecular dynamics simulations on the most stable 6SS-NBD1 variant implicated fluctuations, electrostatic interactions and side chain packing as potential contributors to improved stability. Progressive stabilization of NBD1 directly correlated with enhanced structural stability of full-length CFTR protein. Thermal unfolding of the stabilized CFTR mutants, monitored by changes in intrinsic fluorescence, demonstrated that Tm could be shifted as high as 67.4 °C in 6SS-CFTR, more than 20 °C higher than wild-type. H1402S, an NBD2 mutation, conferred CFTR with additional thermal stability, possibly by stabilizing an NBD-dimerized conformation. CFTR variants with NBD1-stabilizing mutations were expressed at the cell surface in mammalian cells, exhibited ATPase and channel activity, and retained these functions to higher temperatures. The capability to produce enzymatically active CFTR with improved structural stability amenable to biophysical and structural studies will advance mechanistic investigations and future cystic fibrosis drug development.

Structural effects of extracellular loop mutations in CFTR helical hairpins

Missense mutations constitute 40% of 2000 cystic fibrosis-phenotypic mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) database, yet the precise mechanism as to how a point mutation can render the entire 1480-residue CFTR protein dysfunctional is not well-understood. Here we investigate the structural effects of two CF-phenotypic mutations – glutamic acid to glycine at position 217 (E217G) and glutamine to arginine at position 220 (Q220R) - in the extracellular (ECL2) loop region of human CFTR using helical hairpin constructs derived from transmembrane (TM) helices 3 and 4 of the first membrane domain. We systematically replaced the wild type (WT) residues E217 and Q220 with the subset of missense mutations that could arise through a single nucleotide change in their respective codons. Circular dichroism spectra of E217G revealed that a significant increase in helicity vs. WT arises in the membrane-mimetic environment of sodium dodecylsulfate (SDS) micelles, while this mutant showed a similar gel shift to WT on SDS-PAGE gels. In contrast, the CF-mutant Q220R showed similar helicity but an increased gel shift vs. WT. These structural variations are compared with the maturation levels of the corresponding mutant full-length CFTRs, which we found are reduced to approx. 50% for E217G and 30% for Q220R vs. WT. The overall results with CFTR hairpins illustrate the range of impacts that single mutations can evoke in intramolecular protein-protein and/or protein-lipid interactions - and the levels to which corresponding mutations in full-length CFTR may be flagged by quality control mechanisms during biosynthesis.

Toward inclusive therapy with CFTR modulators: Progress and challenges.

Cystic fibrosis is caused by gene mutations that result in an abnormal Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein on the surface of cells. CFTR modulators are a novel class of drugs that directly target the molecular defect. CFTR modulators include potentiators that result in improved activity of the channel; correctors that help the protein traffic to the cell surface properly; and readthrough agents that restore full-length CFTR by suppression of premature termination codons, among other novel classes more recently established. While some of these drugs, CFTR potentiators in particular, have provided remarkable improvements for CF patients, others have yet to achieve profoundly improved outcomes, and many CF patients are not yet impacted by CFTR modulators due to lack of knowledge regarding susceptibility of their mutations to treatment. One limitation to expanding these types of therapies to the maximum number of patients with CF is the lack of rigorously validated clinical biomarkers that can determine efficacy on an individual basis, as well as few pre-clinical tools that can predict whether an individual with a rare combination of mutant alleles will respond to a particular CFTR modulator regimen. In this review, we discuss the various groups of CFTR modulators and their status in clinical development, as well as address the current literature on biomarkers, pre-clinical cell-based tools, and the role of pharmacometrics in creating therapeutic strategies to improve the lives of all patients with cystic fibrosis, regardless of their specific mutation.

Molecular dynamics of the cryo-EM CFTR structure

Cystic fibrosis (CF), a lethal monogenic disease, is caused by mutant variants of the CF transmembrane conductance regulator (CFTR). Recent advances in single molecule cryo-EM methods enabled structural determination of full-length human and zebrafish CFTR, achieving an important milestone for CF drug development. To relate these structures to the gating cycle, we examined its dynamic features using molecular dynamics simulations. Our results show that the nucleotide binding domains (NBDs) in this bottom-open apo conformation exhibit motions related to dimerization and the bottom-closed apo CFTR model indicates opening of NBDs in contrast to transporters. These observations help in understanding the properties of CFTR chloride channel distinct from transporters and in proper interpretation of available structural information on this ABC protein.

Attenuation of Phosphorylation-dependent Activation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by Disease-causing Mutations at the Transmission Interface

Cystic fibrosis transmembrane conductance regulator (CFTR) is a multidomain membrane protein that functions as a phosphorylation-regulated anion channel. The interface between its two cytosolic nucleotide binding domains and coupling helices conferred by intracellular loops extending from the channel pore domains has been referred to as a transmission interface and is thought to be critical for the regulated channel activity of CFTR. Phosphorylation of the regulatory domain of CFTR by protein kinase A (PKA) is required for its channel activity. However, it was unclear if phosphorylation modifies the transmission interface. Here, we studied purified full-length CFTR protein using spectroscopic techniques to determine the consequences of PKA-mediated phosphorylation. Synchrotron radiation circular dichroism spectroscopy confirmed that purified full-length wild-type CFTR is folded and structurally responsive to phosphorylation. Intrinsic tryptophan fluorescence studies of CFTR showed that phosphorylation reduced iodide-mediated quenching, consistent with an effect of phosphorylation in burying tryptophans at the transmission interface. Importantly, the rate of phosphorylation-dependent channel activation was compromised by the introduction of disease-causing mutations in either of the two coupling helices predicted to interact with nucleotide binding domain 1 at the interface. Together, these results suggest that phosphorylation modifies the interface between the catalytic and pore domains of CFTR and that this modification facilitates CFTR channel activation.

A sequence upstream of canonical PDZ-binding motif within CFTR COOH-terminus enhances NHERF1 interaction.

The development of cystic fibrosis transmembrane conductance regulator (CFTR) targeted therapy for cystic fibrosis has generated interest in maximizing membrane residence of mutant forms of CFTR by manipulating interactions with scaffold proteins, such as sodium/hydrogen exchange regulatory factor-1 (NHERF1). In this study, we explored whether COOH-terminal sequences in CFTR beyond the PDZ-binding motif influence its interaction with NHERF1. NHERF1 displayed minimal self-association in blot overlays (NHERF1, Kd = 1,382 ± 61.1 nM) at concentrations well above physiological levels, estimated at 240 nM from RNA-sequencing and 260 nM by liquid chromatography tandem mass spectrometry in sweat gland, a key site of CFTR function in vivo. However, NHERF1 oligomerized at considerably lower concentrations (10 nM) in the presence of the last 111 amino acids of CFTR (20 nM) in blot overlays and cross-linking assays and in coimmunoprecipitations using differently tagged versions of NHERF1. Deletion and alanine mutagenesis revealed that a six-amino acid sequence 1417EENKVR1422 and the terminal 1478TRL1480 (PDZ-binding motif) in the COOH-terminus were essential for the enhanced oligomerization of NHERF1. Full-length CFTR stably expressed in Madin-Darby canine kidney epithelial cells fostered NHERF1 oligomerization that was substantially reduced (∼5-fold) on alanine substitution of EEN, KVR, or EENKVR residues or deletion of the TRL motif. Confocal fluorescent microscopy revealed that the EENKVR and TRL sequences contribute to preferential localization of CFTR to the apical membrane. Together, these results indicate that COOH-terminal sequences mediate enhanced NHERF1 interaction and facilitate the localization of CFTR, a property that could be manipulated to stabilize mutant forms of CFTR at the apical surface to maximize the effect of CFTR-targeted therapeutics.

Phosphorylation Modifies Coupling of the Membrane Domains and NBD1 of Full Length CFTR

Cystic fibrosis is a common genetic recessive disease caused by mutations of the Cystic fibrosis transmembrane conductance regulator (CFTR), an ATP-Binding Cassette (ABC) anion channel. CFTR consists of two membrane spanning domains (MSDs), two nucleotide binding domains (NBDs), intracellular loops (ICLs) that “couple” MSDs and NBDs, and a regulatory (R) domain. CFTR activity is predominantly regulated by protein kinase A phosphorylation at the R domain. However, little is known of the effect of phosphorylation on intramolecular interactions of full length CFTR. Intrinsic tryptophan fluorescence studies of a similar ABC transporter, BtuCD, have shown that urea dissociates the ICL:NBD interface which we were interested to apply on purified full length CFTR. Our results were similar to BtuCD studies, suggesting the dissociation of ICL:NBD interface of full length CFTR. Phosphorylation modified urea denaturation like VX-661, a suggested modulator of ICL:NBD1 interface. Phosphorylation and VX-661 similarly modified quenching of tryptophan residues suggesting that monitored tryptophan residues were located within an electrostatic environment. Our biophysical studies led us to hypothesize that phosphorylation may modify the intramolecular interactions at the ICL:NBD1 interface of full length CFTR. A phosphorylation site on the regulatory insertion, which has been suggested to open up upon phosphorylation to allow interaction of ICLs to NBD1 is located near many tryptophan residues from the CFTR homology model. To specifically study the interactions, cysteine crosslinking studies were conducted on a Wt CFTR lacking native cysteines with V510C (NBD1) and A1067C (ICL4) engineered and transfected in HEK cells. Phosphorylation significantly enhanced the crosslinking and interaction at the ICL4:NBD1 more than the ICL:NBD1 modulator VX-661. This study provides insight into the effect of phosphorylation on the intramolecular interactions of full length CFTR.