Production of CFTR-ΔF508 Rabbits.
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ABSTRACT:
SUBMITTER: Yang D
PROVIDER: S-EPMC7862758 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The most common mutation in cystic fibrosis (CF) is a deletion of Phe at position 508 (ΔF508-CFTR). ΔF508-CFTR is a trafficking mutant that is retained in the ER, unable to reach the plasma membrane. To identify compounds and drugs that rescue this trafficking defect, we screened a kinase inhibitor library enriched for small molecules already in the clinic or in clinical trials for the treatment of cancer and inflammation, using our recently developed high-content screen technology (Trzcinska-Daneluti et al. Mol. Cell. Proteomics 8:780, 2009). The top hits of the screen were further validated by (1) biochemical analysis to demonstrate the presence of mature (Band C) ΔF508-CFTR, (2) flow cytometry to reveal the presence of ΔF508-CFTR at the cell surface, (3) short-circuit current (Isc) analysis in Ussing chambers to show restoration of function of the rescued ΔF508-CFTR in epithelial MDCK cells stably expressing this mutant (including EC(50) determinations), and importantly (4) Isc analysis of Human Bronchial Epithelial (HBE) cells harvested from homozygote ΔF508-CFTR transplant patients. Interestingly, several inhibitors of receptor Tyr kinases (RTKs), such as SU5402 and SU6668 (which target FGFRs, VEGFR, and PDGFR) exhibited strong rescue of ΔF508-CFTR, as did several inhibitors of the Ras/Raf/MEK/ERK or p38 pathways (e.g. (5Z)-7-oxozeaenol). Prominent rescue was also observed by inhibitors of GSK-3β (e.g. GSK-3β Inhibitor II and Kenpaullone). These results identify several kinase inhibitors that can rescue ΔF508-CFTR to various degrees, and suggest that use of compounds or drugs already in the clinic or in clinical trials for other diseases can expedite delivery of treatment for CF patients.
| S-EPMC3434788 | biostudies-literature
Project description:(19)F-modified bithiazole correctors and phenylglycine potentiators of the ΔF508-CFTR chloride channel were synthesized and their function assayed in cells expressing human ΔF508-CFTR and a halide-sensitive fluorescent protein. Fluorine was incorporated into each scaffold using prosthetic groups for future biodistribution imaging studies using positron emission tomography (PET). The ΔF508-CFTR corrector and potentiator potencies of the fluorinated analogs were comparable to or better than those of the original compounds.
| S-EPMC3274619 | biostudies-literature
Project description:Production of functional proteins requires multiple steps including gene transcription and post-translational processing. MicroRNAs (miRNA) can regulate individual stages of these processes. Despite the importance of the cystic fibrosis transmembrane conductance regulator (CFTR) channel for epithelial anion transport, how its expression is regulated remains uncertain. We discovered that microRNA-138 regulates CFTR expression through its interactions with the transcriptional regulatory protein SIN3A. Treating airway epithelia with a miR-138 mimic increased CFTR mRNA and also enhanced CFTR abundance and transepithelial Cl- permeability independently of elevated mRNA levels. A miR-138 anti-miR had the opposite effects. Importantly, miR-138 altered the expression of many genes encoding proteins that associate with CFTR and may influence its biosynthesis. The most common CFTR mutation, ΔF508, causes protein misfolding, degradation, and cystic fibrosis. Remarkably, manipulating the miR-138 regulatory network also improved biosynthesis of CFTR-ΔF508 and restored Cl- transport to cystic fibrosis airway epithelia. This novel miRNA-regulated network directs gene expression from the chromosome to the cell membrane, indicating that an individual miRNA can control a cellular process broader than previously recognized. This discovery also provides new therapeutic avenues for restoring CFTR function to cells affected by the most common cystic fibrosis mutation.
2012-09-04 | GSE38956 | GEO
Project description:Cystic fibrosis (CF) is caused by mutations in the CF transmembrane regulator (CFTR) that result in reduced anion conductance at the apical membrane of secretory epithelia. Treatment of CF patients carrying the G551D gating mutation with the potentiator VX-770 (ivacaftor) largely restores channel activity and has shown substantial clinical benefit. However, most CF patients carry the ΔF508 mutation, which impairs CFTR folding, processing, function, and stability. Studies in homozygous ΔF508 CF patients indicated little clinical benefit of monotherapy with the investigational corrector VX-809 (lumacaftor) or VX-770, whereas combination clinical trials show limited but significant improvements in lung function. We show that VX-770, as well as most other potentiators, reduces the correction efficacy of VX-809 and another investigational corrector, VX-661. To mimic the administration of VX-770 alone or in combination with VX-809, we examined its long-term effect in immortalized and primary human respiratory epithelia. VX-770 diminished the folding efficiency and the metabolic stability of ΔF508-CFTR at the endoplasmic reticulum (ER) and post-ER compartments, respectively, causing reduced cell surface ΔF508-CFTR density and function. VX-770-induced destabilization of ΔF508-CFTR was influenced by second-site suppressor mutations of the folding defect and was prevented by stabilization of the nucleotide-binding domain 1 (NBD1)-NBD2 interface. The reduced correction efficiency of ΔF508-CFTR, as well as of two other processing mutations in the presence of VX-770, suggests the need for further optimization of potentiators to maximize the clinical benefit of corrector-potentiator combination therapy in CF.
| S-EPMC4467693 | biostudies-literature
Project description:The G551D cystic fibrosis transmembrane conductance regulator (CFTR) mutation is associated with severe disease in ∼5% of cystic fibrosis patients worldwide. This amino acid substitution in NBD1 results in a CFTR chloride channel characterized by a severe gating defect that can be at least partially overcome in vitro by exposure to a CFTR potentiator. In contrast, the more common ΔF508 mutation is associated with a severe protein trafficking defect, as well as impaired channel function. Recent clinical trials demonstrated a beneficial effect of the CFTR potentiator, Ivacaftor (VX-770), on lung function of patients bearing at least one copy of G551D CFTR, but no comparable effect on ΔF508 homozygotes. This difference in efficacy was not surprising in view of the established difference in the molecular phenotypes of the two mutant channels. Recently, however, it was shown that the structural defect introduced by the deletion of F508 is associated with the thermal instability of ΔF508 CFTR channel function in vitro. This additional mutant phenotype raised the possibility that the differences in the behavior of ΔF508 and G551D CFTR, as well as the disparate efficacy of Ivacaftor, might be a reflection of the differing thermal stabilities of the two channels at 37 °C. We compared the thermal stability of G551D and ΔF508 CFTR in Xenopus oocytes in the presence and absence of CTFR potentiators. G551D CFTR exhibited a thermal instability that was comparable to that of ΔF508 CFTR. G551D CFTR, however, was protected from thermal instability by CFTR potentiators, whereas ΔF508 CFTR was not. These results suggest that the efficacy of VX-770 in patients bearing the G551D mutation is due, at least in part, to the ability of the small molecule to protect the mutant channel from thermal instability at human body temperature.
| S-EPMC4159205 | biostudies-other
Project description:BackgroundWe previously reported that expression of a miR-138 mimic or knockdown of SIN3A in primary cultures of cystic fibrosis (CF) airway epithelia increased ΔF508-CFTR mRNA and protein levels, and partially restored CFTR-dependent chloride transport. Global mRNA transcript profiling in ΔF508-CFBE cells treated with miR-138 mimic or SIN3A siRNA identified two genes, SYVN1 and NEDD8, whose inhibition significantly increased ΔF508-CFTR trafficking, maturation, and function. Little is known regarding the dynamic changes in the CFTR gene network during such rescue events. We hypothesized that analysis of condition-specific gene networks from transcriptomic data characterizing ΔF508-CFTR rescue could help identify dynamic gene modules associated with CFTR biogenesis.MethodsWe applied a computational method, termed M-module, to analyze multiple gene networks, each of which exhibited differential activity compared to a baseline condition. In doing so, we identified both unique and shared gene pathways across multiple differential networks. To construct differential networks, gene expression data from CFBE cells were divided into three groups: (1) siRNA inhibition of NEDD8 and SYVN1; (2) miR-138 mimic and SIN3A siRNA; and (3) temperature (27 °C for 24 h, 40 °C for 24 h, and 27 °C for 24 h followed by 40 °C for 24 h).ResultsInterrogation of individual networks (e.g., NEDD8/SYVN1 network), combinations of two networks (e.g., NEDD8/SYVN1 + temperature networks), and all three networks yielded sets of 1-modules, 2-modules, and 3-modules, respectively. Gene ontology analysis revealed significant enrichment of dynamic modules in pathways including translation, protein metabolic/catabolic processes, protein complex assembly, and endocytosis. Candidate CFTR effectors identified in the analysis included CHURC1, GZF1, and RPL15, and siRNA-mediated knockdown of these genes partially restored CFTR-dependent transepithelial chloride current to ΔF508-CFBE cells.ConclusionsThe ability of the M-module to identify dynamic modules involved in ΔF508 rescue provides a novel approach for studying CFTR biogenesis and identifying candidate suppressors of ΔF508.
| S-EPMC8557508 | biostudies-literature
Project description:The most common cystic fibrosis (CF) causing mutation, deletion of phenylalanine 508 (ΔF508 or Phe508del), results in functional expression defect of the CF transmembrane conductance regulator (CFTR) at the apical plasma membrane (PM) of secretory epithelia, which is attributed to the degradation of the misfolded channel at the endoplasmic reticulum (ER). Deletion of phenylalanine 670 (ΔF670) in the yeast oligomycin resistance 1 gene (YOR1, an ABC transporter) of Saccharomyces cerevisiae phenocopies the ΔF508-CFTR folding and trafficking defects. Genome-wide phenotypic (phenomic) analysis of the Yor1-ΔF670 biogenesis identified several modifier genes of mRNA processing and translation, which conferred oligomycin resistance to yeast. Silencing of orthologues of these candidate genes enhanced the ΔF508-CFTR functional expression at the apical PM in human CF bronchial epithelia. Although knockdown of RPL12, a component of the ribosomal stalk, attenuated the translational elongation rate, it increased the folding efficiency as well as the conformational stability of the ΔF508-CFTR, manifesting in 3-fold augmented PM density and function of the mutant. Combination of RPL12 knockdown with the corrector drug, VX-809 (lumacaftor) restored the mutant function to ~50% of the wild-type channel in primary CFTRΔF508/ΔF508 human bronchial epithelia. These results and the observation that silencing of other ribosomal stalk proteins partially rescue the loss-of-function phenotype of ΔF508-CFTR suggest that the ribosomal stalk modulates the folding efficiency of the mutant and is a potential therapeutic target for correction of the ΔF508-CFTR folding defect.
| S-EPMC4864299 | biostudies-literature
Project description:The most common mutation of CFTR, affecting approximately 90% of CF patients, is a deletion of phenylalanine at position 508 (F508del, ΔF508). Misfolding of ΔF508-CFTR impairs both its trafficking to the plasma membrane and its chloride channel activity. To identify small molecules that can restore channel activity of ΔF508-CFTR, we synthesized and evaluated eighteen novel hydroxypyrazoline analogues as CFTR potentiators. To elucidate potentiation activities of hydroxypyrazolines for ΔF508-CFTR, CFTR activity was measured using a halide-sensitive YFP assay, Ussing chamber assay and patch-clamp technique. Compounds 7p, 7q and 7r exhibited excellent potentiation with EC50 value <10 μM. Among the compounds, 7q (a novel CFTR potentiator, CP7q) showed the highest potentiation activity with EC50 values of 0.88 ± 0.11 and 4.45 ± 0.31 μM for wild-type and ΔF508-CFTR, respectively. In addition, CP7q significantly potentiated chloride conductance of G551D-CFTR, a CFTR gating mutant; its maximal potentiation activity was 1.9 fold higher than the well-known CFTR potentiator genistein. Combination treatment with CP7q and VX-809, a corrector of ΔF508-CFTR, significantly enhanced functional rescue of ΔF508-CFTR compared with VX-809 alone. CP7q did not alter the cytosolic cAMP level and showed no cytotoxicity at the concentration showing maximum efficacy. The hydroxypyrazolines may be potential development candidates for drug therapy of cystic fibrosis.
| S-EPMC4749168 | biostudies-literature
Project description:The molecular mechanism of Endoplasmic Reticulum-associated degradation (ERAD) of Cystic fibrosis transmembrane-conductance regulator (CFTR) is largely unknown. Particularly, it is unknown what ER luminal factor(s) are involved in ERAD. Herein, we used ProtoArray to identify an ER luminal co-chaperone, DNAJB9, which can directly interact with CFTR. For both WT- and ΔF508 (deletion of phenylalanine at position 508, the most common CF-causing mutant)-CFTR, knockdown of DNAJB9 by siRNA increased their expression levels on the cell surface and, consequently, upregulated their function. Furthermore, genetic ablation of DNAJB9 in WT mice increased CFTR expression and enhanced CFTR-dependent fluid secretion in enteroids. Importantly, DNAJB9 deficiency upregulated enteroids' fluid secretion in CF mice (homozygous for ΔF508), and silencing one allele of DNAJB9 is sufficient to rescue ΔF508-CFTR in vitro and in vivo, suggesting that DNAJB9 may be a rate-limiting factor in CFTR ERAD pathway. Our studies identified the first ER luminal co-chaperone involved in CFTR ERAD, and DNAJB9 could be a novel therapeutic target for CF.
| S-EPMC6614449 | biostudies-literature
Project description:Cystic Fibrosis (CF) is an autosomal recessive disorder caused by mutations in the gene encoding the Cystic fibrosis transmembrane conductance regulator (CFTR). ΔF508-CFTR, the most common disease-causing CF mutant, exhibits folding and trafficking defects and is retained in the endoplasmic reticulum, where it is targeted for proteasomal degradation. To identify signaling pathways involved in ΔF508-CFTR rescue, we screened a library of endoribonuclease-prepared short interfering RNAs (esiRNAs) that target ∼750 different kinases and associated signaling proteins. We identified 20 novel suppressors of ΔF508-CFTR maturation, including the FGFR1. These were subsequently validated by measuring channel activity by the YFP halide-sensitive assay following shRNA-mediated knockdown, immunoblotting for the mature (band C) ΔF508-CFTR and measuring the amount of surface ΔF508-CFTR by ELISA. The role of FGFR signaling on ΔF508-CFTR trafficking was further elucidated by knocking down FGFRs and their downstream signaling proteins: Erk1/2, Akt, PLCγ-1, and FRS2. Interestingly, inhibition of FGFR1 with SU5402 administered to intestinal organoids (mini-guts) generated from the ileum of ΔF508-CFTR homozygous mice resulted in a robust ΔF508-CFTR rescue. Moreover, combination of SU5402 and VX-809 treatments in cells led to an additive enhancement of ΔF508-CFTR rescue, suggesting these compounds operate by different mechanisms. Chaperone array analysis on human bronchial epithelial cells harvested from ΔF508/ΔF508-CFTR transplant patients treated with SU5402 identified altered expression of several chaperones, an effect validated by their overexpression or knockdown experiments. We propose that FGFR signaling regulates specific chaperones that control ΔF508-CFTR maturation, and suggest that FGFRs may serve as important targets for therapeutic intervention for the treatment of CF.
| S-EPMC4458721 | biostudies-literature