Human Cystic Fibrosis Transmembrane Conductance Research Articles

ATP-dependent thermoring basis for the heat unfolding of the first nucleotide-binding domain isolated from human CFTR.

Traditionally, the thermostability of a protein is defined by a melting temperature, at which half of the protein is unfolded. However, this definition cannot indicate the structural origin of a heat-induced unfolding pathway. Here, the thermoring structures were studied on the ATP-dependent heat-induced unfolding of the first nucleotide-binding domain from the human cystic fibrosis transmembrane conductance regulator. The results showed that initial theoretical and experimental melting thresholds aligned well after three structural perturbations including the F508del mutation, the most common cause of cystic fibrosis. This alignment further demonstrated that the heat-induced unfolding process began with the disruption of the least-stable noncovalent interaction within the biggest thermoring along the single peptide chain. The C-terminal region, which was related to the least-stable noncovalent interaction and the ATP-dependent dimerization of two nucleotide-binding domains, emerged as a crucial determinant of the thermal stability of the isolated protein and a potential interfacial drug target to alleviate the thermal defect caused by the F508del mutation. This groundbreaking discovery significantly advances our understanding of protein activity, thermal stability, and molecular pathology.

Systematic optimization of prime editing for the efficient functional correction of CFTR F508del in human airway epithelial cells.

Prime editing (PE) enables precise and versatile genome editing without requiring double-stranded DNA breaks. Here we describe the systematic optimization of PE systems to efficiently correct human cystic fibrosis (CF) transmembrane conductance regulator (CFTR) F508del, a three-nucleotide deletion that is the predominant cause of CF. By combining six efficiency optimizations for PE-engineered PE guide RNAs, the PEmax architecture, the transient expression of a dominant-negative mismatch repair protein, strategic silent edits, PE6 variants and proximal 'dead' single-guide RNAs-we increased correction efficiencies for CFTR F508del from less than 0.5% in HEK293T cells to 58% in immortalized bronchial epithelial cells (a 140-fold improvement) and to 25% in patient-derived airway epithelial cells. The optimizations also resulted in minimal off-target editing, in edit-to-indel ratios 3.5-fold greater than those achieved by nuclease-mediated homology-directed repair, and in the functional restoration of CFTR ion channels to over 50% of wild-type levels (similar to those achieved via combination treatment with elexacaftor, tezacaftor and ivacaftor) in primary airway cells. Our findings support the feasibility of a durable one-time treatment for CF.

621 Administration of SP-101 and doxorubicin results in robust and durable human cystic fibrosis transmembrane conductance regulator minigene transgene expression in the airways of ferrets and corrects human cystic fibrosis airway epithelia in vitro

621 Administration of SP-101 and doxorubicin results in robust and durable human cystic fibrosis transmembrane conductance regulator minigene transgene expression in the airways of ferrets and corrects human cystic fibrosis airway epithelia in vitro

666 Ivacaftor boosts cystic fibrosis transmembrane conductance regulator–mediated nasal potential difference in cystic fibrosis transmembrane conductance regulator knockout mice treated with human cystic fibrosis transmembrane conductance regulator messenger ribonucleic acid lipid nanoparticle

666 Ivacaftor boosts cystic fibrosis transmembrane conductance regulator–mediated nasal potential difference in cystic fibrosis transmembrane conductance regulator knockout mice treated with human cystic fibrosis transmembrane conductance regulator messenger ribonucleic acid lipid nanoparticle

685 Cryo-electron microscopy reconstruction of the conformational ensemble adopted by human cystic fibrosis transmembrane conductance regulator–G551D provides insight into the folding pathway and the mechanism of action of the dual corrector/potentiator elexacaftor

685 Cryo-electron microscopy reconstruction of the conformational ensemble adopted by human cystic fibrosis transmembrane conductance regulator–G551D provides insight into the folding pathway and the mechanism of action of the dual corrector/potentiator elexacaftor

Seamless Gene Correction in the Human Cystic Fibrosis Transmembrane Conductance Regulator Locus by Vector Replacement and Vector Insertion Events.

Background: Gene correction via homology directed repair (HDR) in patient-derived induced pluripotent stem (iPS) cells for regenerative medicine are becoming a more realistic approach to develop personalized and mutation-specific therapeutic strategies due to current developments in gene editing and iPSC technology. Cystic fibrosis (CF) is the most common inherited disease in the Caucasian population, caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Since CF causes significant multi-organ damage and with over 2,000 reported CFTR mutations, CF patients could be one prominent population benefiting from gene and cell therapies. When considering gene-editing techniques for clinical applications, seamless gene corrections of the responsible mutations, restoring native “wildtype” DNA sequence without remnants of drug selectable markers or unwanted DNA sequence changes, would be the most desirable approach. Result: The studies reported here describe the seamless correction of the W1282X CFTR mutation using CRISPR/Cas9 nickases (Cas9n) in iPS cells derived from a CF patient homozygous for the W1282X Class I CFTR mutation. In addition to the expected HDR vector replacement product, we discovered another class of HDR products resulting from vector insertion events that created partial duplications of the CFTR exon 23 region. These vector insertion events were removed via intrachromosomal homologous recombination (IHR) enhanced by double nicking with CRISPR/Cas9n which resulted in the seamless correction of CFTR exon 23 in CF-iPS cells. Conclusion: We show here the removal of the drug resistance cassette and generation of seamless gene corrected cell lines by two independent processes: by treatment with the PiggyBac (PB) transposase in vector replacements or by IHR between the tandemly duplicated CFTR gene sequences.

Heat treatment of thioredoxin fusions increases the purity of α-helical transmembrane protein constructs.

Membrane proteins play key roles in cellular signaling and transport, represent the majority of drug targets, and are implicated in many diseases. Their relevance renders them important subjects for structural, biophysical, and functional investigations. However, obtaining membrane proteins in high purities is often challenging with conventional purification steps alone. To address this issue, we present here an approach to increase the purity of α‐helical transmembrane proteins. Our approach exploits the Thioredoxin (Trx) tag system, which is able to confer some of its favorable properties, such as high solubility and thermostability, to its fusion partners. Using Trx fusions of transmembrane helical hairpin constructs derived from the human cystic fibrosis transmembrane conductance regulator (CFTR) and a bacterial ATP synthase, we establish conditions for the successful implementation of the selective heat treatment procedure to increase sample purity. We further examine systematically its efficacy with respect to different incubation times and temperatures using quantitative gel electrophoresis. We find that minute‐timescale heat treatment of Trx‐tagged fusion constructs with temperatures ranging from 50 to 90°C increases the purity of the membrane protein samples from ~60 to 98% even after affinity purification. We show that this single‐step approach is even applicable in cases where regular selective heat purification from crude extracts, as reported for Trx fusions to soluble proteins, fails. Overall, our approach is easy to integrate into existing purification strategies and provides a facile route for increasing the purity of membrane protein constructs after purification by standard chromatography approaches.

Cholesterol Interaction Directly Enhances Intrinsic Activity of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR).

The recent cryo-electron microscopy structures of zebrafish and the human cystic fibrosis transmembrane conductance regulator (CFTR) provided unprecedented insights into putative mechanisms underlying gating of its anion channel activity. Interestingly, despite predictions based on channel activity measurements in biological membranes, the structure of the detergent purified, phosphorylated, and ATP-bound human CFTR protein did not reveal a stably open conduction pathway. This study tested the hypothesis that the functional properties of the detergent solubilized CFTR protein used for structural determinations are different from those exhibited by CFTR purified under conditions that retain associated lipids native to the membrane. It was found that CFTR purified together with phospholipids and cholesterol using amphipol: A8-35, exhibited higher rates of catalytic activity, phosphorylation dependent channel activation and potentiation by the therapeutic compound, ivacaftor, than did CFTR purified in detergent. The catalytic activity of phosphorylated CFTR detergent micelles was rescued by the addition of phospholipids plus cholesterol, but not by phospholipids alone, arguing for a specific role for cholesterol in modulating this function. In summary, these studies highlight the importance of lipid interactions in the intrinsic activities and pharmacological potentiation of CFTR.

Orphan designation: Codon-optimised human cystic fibrosis transmembrane conductance regulator messenger ribonucleic acid complexed with lipid-based nanoparticles, Treatment of cystic fibrosis

Orphan designation: Codon-optimised human cystic fibrosis transmembrane conductance regulator messenger ribonucleic acid complexed with lipid-based nanoparticles, Treatment of cystic fibrosis

Differential thermostability and response to cystic fibrosis transmembrane conductance regulator potentiators of human and mouse F508del-CFTR.

Cross-species comparative studies have highlighted differences between human and mouse cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial Cl− channel defective in cystic fibrosis (CF). Here, we compare the impact of the most common CF mutation F508del on the function of human and mouse CFTR heterologously expressed in mammalian cells and their response to CFTR modulators using the iodide efflux and patch-clamp techniques. Once delivered to the plasma membrane, human F508del-CFTR exhibited a severe gating defect characterized by infrequent channel openings and was thermally unstable, deactivating within minutes at 37°C. By contrast, the F508del mutation was without effect on the gating pattern of mouse CFTR, and channel activity demonstrated thermostability at 37°C. Strikingly, at all concentrations tested, the clinically approved CFTR potentiator ivacaftor was without effect on the mouse F508del-CFTR Cl− channel. Moreover, eight CFTR potentiators, including ivacaftor, failed to generate CFTR-mediated iodide efflux from CHO cells expressing mouse F508del-CFTR. However, they all produced CFTR-mediated iodide efflux with human F508del-CFTR-expressing CHO cells, while fifteen CFTR correctors rescued the plasma membrane expression of both human and mouse F508del-CFTR. Interestingly, the CFTR potentiator genistein enhanced CFTR-mediated iodide efflux from CHO cells expressing either human or mouse F508del-CFTR, whereas it only potentiated human F508del-CFTR Cl− channels in cell-free membrane patches, suggesting that its action on mouse F508del-CFTR is indirect. Thus, the F508del mutation has distinct effects on human and mouse CFTR Cl− channels.

TALEN-Mediated Gene Targeting for Cystic Fibrosis-Gene Therapy.

Cystic fibrosis (CF) is an inherited monogenic disorder, amenable to gene-based therapies. Because CF lung disease is currently the major cause of mortality and morbidity, and the lung airway is readily accessible to gene delivery, the major CF gene therapy effort at present is directed to the lung. Although airway epithelial cells are renewed slowly, permanent gene correction through gene editing or targeting in airway stem cells is needed to perpetuate the therapeutic effect. Transcription activator-like effector nuclease (TALEN) has been utilized widely for a variety of gene editing applications. The stringent requirement for nuclease binding target sites allows for gene editing with precision. In this study, we engineered helper-dependent adenoviral (HD-Ad) vectors to deliver a pair of TALENs together with donor DNA targeting the human AAVS1 locus. With homology arms of 4 kb in length, we demonstrated precise insertion of either a LacZ reporter gene or a human cystic fibrosis transmembrane conductance regulator (CFTR) minigene (cDNA) into the target site. Using the LacZ reporter, we determined the efficiency of gene integration to be about 5%. In the CFTR vector transduced cells, we were able to detect CFTR mRNA expression using qPCR and function correction using fluorometric image plate reader (FLIPR) and iodide efflux assays. Taken together, these findings suggest a new direction for future in vitro and in vivo studies in CF gene editing.

Ligand binding to a remote site thermodynamically corrects the F508del mutation in the human cystic fibrosis transmembrane conductance regulator

Many disease-causing mutations impair protein stability. Here, we explore a thermodynamic strategy to correct the disease-causing F508del mutation in the human cystic fibrosis transmembrane conductance regulator (hCFTR). F508del destabilizes nucleotide-binding domain 1 (hNBD1) in hCFTR relative to an aggregation-prone intermediate. We developed a fluorescence self-quenching assay for compounds that prevent aggregation of hNBD1 by stabilizing its native conformation. Unexpectedly, we found that dTTP and nucleotide analogs with exocyclic methyl groups bind to hNBD1 more strongly than ATP and preserve electrophysiological function of full-length F508del-hCFTR channels at temperatures up to 37 °C. Furthermore, nucleotides that increase open-channel probability, which reflects stabilization of an interdomain interface to hNBD1, thermally protect full-length F508del-hCFTR even when they do not stabilize isolated hNBD1. Therefore, stabilization of hNBD1 itself or of one of its interdomain interfaces by a small molecule indirectly offsets the destabilizing effect of the F508del mutation on full-length hCFTR. These results indicate that high-affinity binding of a small molecule to a remote site can correct a disease-causing mutation. We propose that the strategies described here should be applicable to identifying small molecules to help manage other human diseases caused by mutations that destabilize native protein conformation.

Cellular mechanism for herbal medicine Junchoto to facilitate intestinal Cl\u2212/water secretion that involves cAMP-dependent activation of CFTR

Constipation is a common symptom frequently compromising the quality of daily life. Several mechanistically different drugs have been used to mitigate constipation, including Japanese herbal (Kampo) medicines. However, the mechanisms of their actions are often not well understood. Here we aimed to investigate the molecular mechanisms underlying the effects of Junchoto (JCT), a Kampo medicine empirically prescribed for chronic constipation. Cl− channel activity was measured by the patch-clamp method in human cystic fibrosis transmembrane conductance regulator (CFTR)-expressing HEK293T cells and human intestinal Caco-2 cells. cAMP was measured by a luciferase-based assay. Cell volume change was measured by a particle-sizing and particle-counting analyzer and video-microscopic measurement. In both CFTR-expressing HEK293T and Caco-2 cells, JCT dose-dependently induced whole-cell currents showing typical biophysical and pharmacological features of CFTR. Robust expression of CFTR was confirmed by RT-PCR and Western blotting in Caco-2 cells. Luciferase-based measurement revealed that JCT increases intracellular cAMP levels. Administration of the adenylate cyclase inhibitor SQ22536 or CFTR inhibitor-172, or treatment with small interfering RNAs (siRNA) targeting CFTR, abolished JCT-induced whole-cell currents, suggesting that elevated intracellular cAMP by JCT causes activation of CFTR in Caco-2 cells. Finally, blockade of CFTR activity by CFTR inhibitor-172 or siRNA-knockdown of CFTR or application of SQ22536 markedly reduced the degree of cell volume decrease induced by JCT. JCT can induce a Cl− efflux through the CFTR channel to promote water secretion, and this effect is likely mediated by increased cAMP production.

Sites associated with Kalydeco binding on human Cystic Fibrosis Transmembrane Conductance Regulator revealed by Hydrogen/Deuterium Exchange

Cystic Fibrosis (CF) is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). Mutations associated with CF cause loss-of-function in CFTR leading to salt imbalance in epithelial tissues. Kalydeco (also called VX-770 or ivacaftor) was approved for CF treatment in 2012 but little is known regarding the compound’s interactions with CFTR including the site of binding or mechanisms of action. In this study we use hydrogen/deuterium exchange (HDX) coupled with mass spectrometry to assess the conformational dynamics of a thermostabilized form of CFTR in apo and ligand-bound states. We observe HDX protection at a known binding site for AMPPNP and significant protection for several regions of CFTR in the presence of Kalydeco. The ligand-induced changes of CFTR in the presence of Kalydeco suggest a potential binding site.

Removal of the Fe(iii) site promotes activation of the human cystic fibrosis transmembrane conductance regulator by high-affinity Zn(ii) binding.

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is activated by ATP binding at the interface of two cytoplasmic nucleotide binding domains (NBDs) and phosphorylation of the regulatory (R) domain by protein kinase A (PKA). The human CFTR has two functionally active thiol groups for gating regulation by chemical modification. Although modification of C832 in the R domain with N-ethylmaleimide promotes channel opening, glutathionylation of C1344 in NBD2 inhibits channel opening. Our recent studies demonstrated that the N-ethylmaleimide-induced potentiation involves a high-affinity inhibitory Fe3+ site at the interface between the R domain and intracellular loop 3 (ICL3). However, it is unknown whether the glutathionylation-evoked inhibition implies another stimulatory metal site. Here, Fe3+-insensitive mutations at the R-ICL3 interface were employed to further examine whether Zn2+ potentiated the activity of the human CFTR channel by targeting C1344 once the interfacial Fe3+ bridge was disrupted. The results showed that internal nanomolar Zn2+ increased its activity by about two- to threefold at a low level of protein kinase A, and the increase was reversed by EDTA or DTT or reduced glutathione but suppressed by a high level of protein kinase A, N-ethylmaleimide modification or a C1344A mutation. It is interesting that this Zn2+-triggered potentiation is not found in the wild type human CFTR to which endogenous Fe3+ is bound. Thus, the high-affinity binding of Zn2+ to C1344 in NBD2 may stimulate human CFTR activity in a phosphorylation-dependent manner, but the primary binding of Fe3+ to the ICL3-R interface may prohibit this stimulation.

Learning from each other: ABC transporter regulation by protein phosphorylation in plant and mammalian systems.

The ABC (ATP-binding cassette) transporter family in higher plants is highly expanded compared with those of mammalians. Moreover, some members of the plant ABC subfamily B (ABCB) display very high substrate specificity compared with their mammalian counterparts that are often associated with multi-drug resistance phenomena. In this review, we highlight prominent functions of plant and mammalian ABC transporters and summarize our knowledge on their post-transcriptional regulation with a focus on protein phosphorylation. A deeper comparison of regulatory events of human cystic fibrosis transmembrane conductance regulator (CFTR) and ABCB1 from the model plant Arabidopsis reveals a surprisingly high degree of similarity. Both physically interact with orthologues of the FK506-binding proteins that chaperon both transporters to the plasma membrane in an action that seems to involve heat shock protein (Hsp)90. Further, both transporters are phosphorylated at regulatory domains that connect both nt-binding folds. Taken together, it appears that ABC transporters exhibit an evolutionary conserved but complex regulation by protein phosphorylation, which apparently is, at least in some cases, tightly connected with protein-protein interactions (PPI).

Molecular Basis for Fe(III)-Independent Curcumin Potentiation of Cystic Fibrosis Transmembrane Conductance Regulator Activity.

Curcumin potentiates the phosphorylation-dependent activity of human cystic fibrosis transmembrane conductance regulator (CFTR) in Fe(3+)-dependent and Fe(3+)-independent manners. Although the Fe(3+)-dependent curcumin potentiation results from removal of endogenous inhibitory Fe(3+) at the interface of the regulatory (R) domain and intracellular loop (ICL) 3, the molecular mechanism of Fe(3+)-independent curcumin potentiation is still unknown. Here, HEK-293T cells cultured in an Fe(3+)-containing medium were transiently transfected with CFTR constructs, and the role of a highly conserved ICL1/ICL4 interface and a stimulatory phosphorylation site S795 or S813 at the C-terminus of the R domain in this potentiation pathway was investigated. The results showed that highly conserved aromatic and positively charged residues at the ICL1/ICL4 interface and phosphorylation site S813 were sensitive to curcumin regardless of whether Fe(3+) and nucleotide-binding domain 2 were removed. More importantly, spontaneous disulfide cross-linking between curcumin-sensitive ICL1 and S795 was observed to be enough to promote channel opening as curcumin did. Therefore, the phosphorylated R domain, once released from ICL3, may function as a length- and gating-regulatory cross-linker between two transmembrane domains to promote the stimulatory interactions between the R domain and the ICL1/ICL4 interface. Curcumin may potentiate CFTR activity not only by removing inhibitory Fe(3+) to release the R domain from ICL3 but also by stabilizing the stimulatory R-ICL1/ICL4 interactions. Therefore, the future potentiators with two linked aromatic rings could utilize this potentiation pathway to bypass the need for ATP to rescue the activity of cystic fibrosis mutants with an ATP-dependent gating defect.

Design and characterization of a membrane protein unfolding platform in lipid bilayers.

Accurate measurement of membrane protein stability—and particularly how it may vary as a result of disease-phenotypic mutations—ideally requires a denaturant that can unfold a membrane-embedded structure while leaving the solubilizing environment unaffected. The steric trap method fulfills this requirement by using monovalent streptavidin (mSA) molecules to unfold membrane proteins engineered with two spatially close biotin tags. Here we adapted this method to an 87-residue helix-loop-helix (hairpin) construct derived from helices 3 and 4 in the transmembrane domain of the human cystic fibrosis transmembrane conductance regulator (CFTR), wherein helix-helix tertiary interactions are anticipated to confer a portion of construct stability. The wild type CFTR TM3/4 hairpin construct was modified with two accessible biotin tags for mSA-induced unfolding, along with two helix-terminal pyrene labels to monitor loss of inter-helical contacts by pyrene excimer fluorescence. A series of eight constructs with biotin tags at varying distances from the helix-terminal pyrene labels were expressed, purified and labeled appropriately; all constructs exhibited largely helical circular dichroism spectra. We found that addition of mSA to an optimized construct in lipid vesicles led to a complete and reversible loss in pyrene excimer fluorescence and mSA binding, and hence hairpin unfolding—results further supported by SDS-PAGE visualization of mSA bound and unbound species. While some dimeric/oligomeric populations persist that may affect quantitation of the unfolding step, our characterization of the design yields a promising prototype of a future platform for the systematic study of membrane protein folding in a lipid bilayer environment.

Interplay between inhibitory ferric and stimulatory curcumin regulates phosphorylation-dependent human cystic fibrosis transmembrane conductance regulator and ΔF508 activity.

Curcumin potentiates cystic fibrosis transmembrane conductance regulator (CFTR) activation in an ATP-independent but phosphorylation-dependent manner. The underlying molecular mechanisms are unclear. Here, HEK-293T cells cultured in an Fe(3+)-containing medium were transiently transfected with CFTR constructs, and the role of the inhibitory Fe(3+) bridge between intracellular loop 3 and the regulatory domain of CFTR in this pathway was investigated. The results showed that ethylenediaminetetraacetic acid (EDTA) stimulated phosphorylation-dependent CFTR activation and the stimulation was suppressed by the deletion of the regulatory domain or the insertion of a C832A mutation that removes the Fe(3+)-binding interface. Furthermore, curcumin potentiation of CFTR was significantly weakened not only by Fe(3+)-insensitive mutations at the interface between the regulatory domain and intracellular loop 3 but also by N-ethylmaleimide or EDTA pretreatment that removes Fe(3+). More importantly, potentiation of CFTR was completely suppressed by sufficient Fe(3+). Finally, the insertion of Fe(3+)-insensitive H950R/S768R increased the curcumin-independent activity of ΔF508 but weakened its curcumin potentiation. Thus, Fe(3+) homeostasis in epithelia may play a critical role in regulating CFTR activity, and targeting Fe(3+)-chelating potentiators may direct new therapies for cystic fibrosis.

A survey of detergents for the purification of stable, active human cystic fibrosis transmembrane conductance regulator (CFTR)

Structural knowledge of the cystic fibrosis transmembrane conductance regulator (CFTR) requires developing methods to purify and stabilize this aggregation-prone membrane protein above 1mg/ml. Starting with green fluorescent protein- and epitope-tagged human CFTR produced in mammalian cells known to properly fold and process CFTR, we devised a rapid tandem affinity purification scheme to minimize CFTR exposure to detergent in order to preserve its ATPase function. We compared a panel of detergents, including widely used detergents (maltosides, neopentyl glycols (MNG), C12E8, lysolipids, Chaps) and innovative detergents (branched alkylmaltosides, facial amphiphiles) for CFTR purification, function, monodispersity and stability. ATPase activity after reconstitution into proteoliposomes was 2–3 times higher when CFTR was purified using facial amphiphiles. ATPase activity was also demonstrated in purified CFTR samples without detergent removal using a novel lipid supplementation assay. By electron microscopy, negatively stained CFTR samples were monodisperse at low concentration, and size exclusion chromatography showed a predominance of monomer even after CFTR concentration above 1mg/ml. Rates of CFTR aggregation quantified in an electrophoretic mobility shift assay showed that detergents which best preserved reconstituted ATPase activity also supported the greatest stability, with CFTR monomer half-lives of 6–9days in MNG or Chaps, and 12–17days in facial amphiphile. Cryoelectron microscopy of concentrated CFTR in MNG or facial amphiphile confirmed mostly monomeric protein, producing low resolution reconstructions in conformity with similar proteins. These protocols can be used to generate samples of pure, functional, stable CFTR at concentrations amenable to biophysical characterization.