scholarly journals CFTR mRNAs with nonsense codons are degraded by the SMG6-mediated endonucleolytic pathway

2021 ◽  
Author(s):  
Edward Sanderlin ◽  
Melissa Keenan ◽  
Martin Mense ◽  
Alexey Revenko ◽  
Brett Monia ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Exon Junction Complex ◽  
Transmembrane Conductance Regulator ◽  
Loss Of Function ◽  
Transmembrane Conductance ◽  
Nonsense Mutations ◽  
Nonsense Mediated Decay ◽  
Nonsense Codon ◽  
Translational Readthrough ◽  
Nonsense Codons

Abstract Cystic fibrosis is caused by loss of function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene resulting in severe lung disease. Nearly 10% of cystic fibrosis patients have at least one CFTR allele with a nonsense mutation that generates a nonsense codon in the mRNA. Nonsense mutations can result in significant reduction of gene expression partially due to rapid mRNA degradation through the nonsense-mediated decay (NMD) pathway. It has not been thoroughly investigated which branch of the NMD pathway governs the decay of CFTR mRNAs containing nonsense codons. Here we utilized antisense oligonucleotides targeting NMD factors to evaluate the regulation of nonsense codon-containing CFTR mRNAs by the NMD pathway. Interestingly, we found that CFTR mRNAs with G542X, R1162X, and W1282X nonsense codons require UPF2, UPF3, and exon junction complex proteins for NMD, whereas CFTR mRNAs with the Y122X nonsense codon do not. Furthermore, we demonstrated that all evaluated CFTR mRNAs harboring nonsense codons were degraded by the SMG6-mediated endonucleolytic pathway rather than the SMG5/SMG7-mediated exonucleolytic pathway. Finally, we found that stabilization of CFTR mRNAs by NMD inhibition alone improved functional W1282X protein production, and improved the efficiency of aminoglycoside translational readthrough of CFTR-Y122X, -G542X, and -R1162X mRNAs.

Characterizing diverse orthologues of the cystic fibrosis transmembrane conductance regulator protein for structural studies

2015 ◽  
Vol 43 (5) ◽  
pp. 894-900 ◽  
Author(s):  
Naomi L. Pollock ◽  
Tracy L. Rimington ◽  
Robert C. Ford
Keyword(s):  
Cystic Fibrosis ◽  
Structural Data ◽  
Transmembrane Conductance Regulator ◽  
Loss Of Function ◽  
Transmembrane Conductance ◽  
Functional Studies ◽  
Regulator Protein ◽  
Organ Systems ◽  
Cftr Protein ◽  
Cystic Fibrosis Transmembrane

As an ion channel, the cystic fibrosis transmembrane conductance regulator (CFTR) protein occupies a unique niche within the ABC family. Orthologues of CFTR are extant throughout the animal kingdom from sharks to platypods to sheep, where the osmoregulatory function of the protein has been applied to differing lifestyles and diverse organ systems. In humans, loss-of-function mutations to CFTR cause the disease cystic fibrosis, which is a significant health burden in populations of white European descent. Orthologue screening has proved fruitful in the pursuit of high-resolution structural data for several membrane proteins, and we have applied some of the princples developed in previous studies to the expression and purification of CFTR. We have overexpressed this protein, along with evolutionarily diverse orthologues, in Saccharomyces cerevisiae and developed a purification to isolate it in quantities sufficient for structural and functional studies.

scholarly journals Differential Effects of Oleuropein and Hydroxytyrosol on Aggregation and Stability of CFTR NBD1-ΔF508 Domain

2021 ◽  
Vol 1 (3) ◽  
pp. 204-215
Author(s):  
Christopher S. Robinson ◽  
Jennifer A. Wyderko ◽  
Yeng Vang ◽  
Galen Martin ◽  
Robert T. Youker
Keyword(s):  
Cystic Fibrosis ◽  
Phenolic Compounds ◽  
Aggregate Size ◽  
Docking Studies ◽  
Antagonistic Effect ◽  
Transmembrane Conductance Regulator ◽  
Loss Of Function ◽  
Transmembrane Conductance ◽  
Differential Scanning Fluorimetry ◽  
And Function

Cystic Fibrosis (CF) is caused by loss of function mutations in the Cystic Fibrosis transmembrane conductance regulator (CFTR). The folding and assembly of CFTR is inefficient. Deletion of F508 in the first nucleotide binding domain (NBD1-ΔF508) further disrupts protein stability leading to endoplasmic reticulum retention and proteasomal degradation. Stabilization and prevention of NBD1-ΔF508 aggregation is critical to rescuing the folding and function of the entire CFTR channel. We report that the phenolic compounds Oleuropein and Hydroxytryosol reduce aggregation of NBD1-ΔF508. The NBD1-ΔF508 aggregate size was smaller in the presence of Hydroxytryosol as determined by dynamic light scattering. Neither phenolic compound increased the thermal stability of NBD1-ΔF508 as measured by differential scanning fluorimetry. Interestingly, Hydroxytyrosol inhibited the stabilizing effect of the indole compound BIA, a known stabilizer, on NBD1-ΔF508. Molecular docking studies predicted that Oleuropein preferred to bind in the F1-type core ATP-binding subdomain in NBD1. In contrast, Hydroxytyrosol preferred to bind in the α4/α5/α6 helical bundle of the ABCα subdomain of NBD1 next to the putative binding site for BIA. This result suggests that Hydroxytyrosol interferes with BIA binding, thus providing an explanation for the antagonistic effect on NBD1 stability upon incubation with both compounds. To our knowledge, these studies are the first to explore the effects of these two phenolic compounds on the aggregation and stability of NBD1-ΔF508 domain of CFTR.

scholarly journals Modulation of Ion Transport to Restore Airway Hydration in Cystic Fibrosis

Genes ◽  
2021 ◽  
Vol 12 (3) ◽  
pp. 453
Author(s):  
James A. Reihill ◽  
Lisa E. J. Douglas ◽  
S. Lorraine Martin
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Genetic Disorder ◽  
Airway Epithelial Cells ◽  
Transmembrane Conductance Regulator ◽  
Airway Epithelial ◽  
Loss Of Function ◽  
Transmembrane Conductance ◽  
Infection And Inflammation ◽  
Cf Transmembrane Conductance Regulator

Cystic fibrosis (CF) is a life-limiting genetic disorder caused by loss-of-function mutations in the gene which codes for the CF transmembrane conductance regulator (CFTR) Cl− channel. Loss of Cl− secretion across the apical membrane of airway lining epithelial cells results in dehydration of the airway surface liquid (ASL) layer which impairs mucociliary clearance (MCC), and as a consequence promotes bacterial infection and inflammation of the airways. Interventions that restore airway hydration are known to improve MCC. Here we review the ion channels present at the luminal surface of airway epithelial cells that may be targeted to improve airway hydration and MCC in CF airways.

scholarly journals Integrity and Stability of PTC Bearing CFTR mRNA and Relevance to Future Modulator Therapies in Cystic Fibrosis

Genes ◽  
2021 ◽  
Vol 12 (11) ◽  
pp. 1810
Author(s):  
Luka A. Clarke ◽  
Vanessa C. C. Luz ◽  
Szymon Targowski ◽  
Sofia S. Ramalho ◽  
Carlos M. Farinha ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Premature Termination Codon ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Nonsense Mediated Decay ◽  
Steady State Levels ◽  
Quality Control Mechanism ◽  
Cystic Fibrosis Transmembrane ◽  
Mediated Reduction ◽  
Made In

Major advances have recently been made in the development and application of CFTR (cystic fibrosis transmembrane conductance regulator) mutation class-specific modulator therapies, but to date, there are no approved modulators for Class I mutations, i.e., those introducing a premature termination codon (PTC) into the CFTR mRNA. Such mutations induce nonsense-mediated decay (NMD), a cellular quality control mechanism that reduces the quantity of PTC bearing mRNAs, presumably to avoid translation of potentially deleterious truncated CFTR proteins. The NMD-mediated reduction of PTC-CFTR mRNA molecules reduces the efficacy of one of the most promising approaches to treatment of such mutations, namely, PTC readthrough therapy, using molecules that induce the incorporation of near-cognate amino acids at the PTC codon, thereby enabling translation of a full-length protein. In this study, we measure the effect of three different PTC mutations on the abundance, integrity, and stability of respective CFTR mRNAs, using CFTR specific RT-qPCR-based assays. Altogether, our data suggest that optimized rescue of PTC mutations has to take into account (1) the different steady-state levels of the CFTR mRNA associated with each specific PTC mutation; (2) differences in abundance between the 3′ and 5′ regions of CFTR mRNA, even following PTC readthrough or NMD inhibition; and (3) variable effects on CFTR mRNA stability for each specific PTC mutation.

scholarly journals Nanomolar-potency ‘co-potentiator’ therapy for cystic fibrosis caused by a defined subset of minimal function CFTR mutants

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Puay-Wah Phuan ◽  
Joseph-Anthony Tan ◽  
Amber A. Rivera ◽  
Lorna Zlock ◽  
Dennis W. Nielson ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Class Ii ◽  
Bronchial Epithelial Cell ◽  
Therapeutic Benefit ◽  
Initial Structure ◽  
Transmembrane Conductance Regulator ◽  
Loss Of Function ◽  
Nonsense Mutations ◽  
Minimal Function ◽  
Cftr Mutations

AbstractAvailable CFTR modulators provide no therapeutic benefit for cystic fibrosis (CF) caused by many loss-of-function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, including N1303K. We previously introduced the concept of ‘co-potentiators’ (combination-potentiators) to rescue CFTR function in some minimal function CFTR mutants. Herein, a screen of ~120,000 drug-like synthetic small molecules identified active co-potentiators of pyrazoloquinoline, piperidine-pyridoindole, tetrahydroquinoline and phenylazepine classes, with EC50 down to ~300 nM following initial structure-activity studies. Increased CFTR chloride conductance by up to 8-fold was observed when a co-potentiator (termed ‘Class II potentiator’) was used with a classical potentiator (‘Class I potentiator’) such as VX-770 or GLPG1837. To investigate the range of CFTR mutations benefitted by co-potentiators, 14 CF-associated CFTR mutations were studied in transfected cell models. Co-potentiator efficacy was found for CFTR missense, deletion and nonsense mutations in nucleotide binding domain-2 (NBD2), including W1282X, N1303K, c.3700A > G and Q1313X (with corrector for some mutations). In contrast, CFTR mutations G85E, R334W, R347P, V520F, R560T, A561E, M1101K and R1162X showed no co-potentiator activity, even with corrector. Co-potentiator efficacy was confirmed in primary human bronchial epithelial cell cultures generated from a N1303K homozygous CF subject. The Class II potentiators identified here may have clinical benefit for CF caused by mutations in the NBD2 domain of CFTR.

CFTR variants are associated with chronic bronchitis in smokers

2022 ◽  
pp. 2101994
Author(s):  
Aabida Saferali ◽  
Dandi Qiao ◽  
Wonji Kim ◽  
Karen Raraigh ◽  
Hara Levy ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Chronic Bronchitis ◽  
Meta Analysis ◽  
P Value ◽  
Transmembrane Conductance Regulator ◽  
Loss Of Function ◽  
Transmembrane Conductance ◽  
Clinical Consequences ◽  
History Of ◽  
Coding Variants

IntroductionLoss of function variants in both copies of the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF); however, there is evidence that reduction in CFTR function due to the presence of one deleterious variant can have clinical consequences. Here, we hypothesize that CFTR variants in individuals with a history of smoking are associated with COPD and related phenotypes.MethodsWhole genome sequencing was performed through the NHLBI TOPMed program in 8597 subjects from the COPDGene study, an observational study of current and former smokers. We extracted clinically annotated CFTR variants and performed single variant and variant-set testing for COPD and related phenotypes. Replication was performed in 2,118 subjects from the Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints (ECLIPSE) study.ResultsWe identified 301 coding variants within the CFTR gene boundary: 147 of these have been reported in individuals with CF, including 36 CF-causing variants. We found that CF causing variants were associated with chronic bronchitis in variant-set testing in COPDGene (one sided p-value=0.0025, OR=1.53) and in meta-analysis of COPDGene and ECLIPSE (one sided p-value=0.0060, OR=1.52). Single variant testing revealed that the F508del variant was associated with chronic bronchitis in COPDGene (one sided p-value=0.015, OR=1.47). In addition, we identified 32 subjects with two or more CFTR variants on separate alleles, and these subjects were enriched for COPD cases (p=0.010).ConclusionsCigarette smokers who carry one deleterious CFTR variant have higher rates of chronic bronchitis, while presence of two CFTR variants may be associated with COPD. These results indicate that genetically-mediated reduction in CFTR function contributes to COPD related phenotypes, in particular chronic bronchitis.

scholarly journals Enamel Pathology Resulting from Loss of Function in the Cystic Fibrosis Transmembrane Conductance Regulator in a Porcine Animal Model

2011 ◽  
Vol 194 (2-4) ◽  
pp. 249-254 ◽  
Author(s):  
Eugene H. Chang ◽  
Rodrigo S. Lacruz ◽  
Timothy G. Bromage ◽  
Pablo Bringas, Jr. ◽  
Michael J. Welsh ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Animal Model ◽  
Transmembrane Conductance Regulator ◽  
Loss Of Function ◽  
Transmembrane Conductance ◽  
Cystic Fibrosis Transmembrane
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