scholarly journals Targeting Nonsense: Optimization of 1,2,4-Oxadiazole TRIDs to Rescue CFTR Expression and Functionality in Cystic Fibrosis Cell Model Systems

2020 ◽  
Vol 21 (17) ◽  
pp. 6420
Author(s):  
Ivana Pibiri ◽  
Raffaella Melfi ◽  
Marco Tutone ◽  
Aldo Di Leonardo ◽  
Andrea Pace ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Fluorescent Protein ◽  
Cell Model ◽  
Yellow Fluorescent Protein ◽  
Premature Termination Codon ◽  
Severe Form ◽  
Model Systems ◽  
Functional Protein ◽  
Nonsense Mutations ◽  
Translational Readthrough

Cystic fibrosis (CF) patients develop a severe form of the disease when the cystic fibrosis transmembrane conductance regulator (CFTR) gene is affected by nonsense mutations. Nonsense mutations are responsible for the presence of a premature termination codon (PTC) in the mRNA, creating a lack of functional protein. In this context, translational readthrough-inducing drugs (TRIDs) represent a promising approach to correct the basic defect caused by PTCs. By using computational optimization and biological screening, we identified three new small molecules showing high readthrough activity. The activity of these compounds has been verified by evaluating CFTR expression and functionality after treatment with the selected molecules in cells expressing nonsense–CFTR–mRNA. Additionally, the channel functionality was measured by the halide sensitive yellow fluorescent protein (YFP) quenching assay. All three of the new TRIDs displayed high readthrough activity and low toxicity and can be considered for further evaluation as a therapeutic approach toward the second major cause of CF.

scholarly journals Genetic and transgenic reagents for Drosophila simulans, D. mauritiana, D. yakuba, D. santomea and D. virilis

2016 ◽  
Author(s):  
David L. Stern ◽  
Justin Crocker ◽  
Yun Ding ◽  
Nicolas Frankel ◽  
Gretchen Kappes ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Integration Site ◽  
Yellow Fluorescent Protein ◽  
Model Systems ◽  
Enhanced Yellow Fluorescent Protein ◽  
Functional Studies ◽  
Site Specific Integration ◽  
Genetic Strains ◽  
Fluorescent Molecules ◽  
Integration Efficiency

AbstractSpecies of the Drosophila melanogaster species subgroup, including the species D. simulans, D. mauritiana, D. yakuba, and D. santomea, have long served as model systems for studying evolution. Studies in these species have been limited, however, by a paucity of genetic and transgenic reagents. Here we describe a collection of transgenic and genetic strains generated to facilitate genetic studies within and between these species. We have generated many strains of each species containing mapped piggyBac transposons including an enhanced yellow fluorescent protein gene expressed in the eyes and a phiC31 attP site-specific integration site. We have tested a subset of these lines for integration efficiency and reporter gene expression levels. We have also generated a smaller collection of other lines expressing other genetically encoded fluorescent molecules in the eyes and a number of other transgenic reagents that will be useful for functional studies in these species. In addition, we have mapped the insertion locations of 58 transposable elements in D. virilis that will be useful for genetic mapping studies.

scholarly journals Confirmation of Connexin45 Underlying Weak Gap Junctional Intercellular Coupling in HeLa Cells

Biomolecules ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 1389 ◽  
Author(s):  
Eun Ju Choi ◽  
Nicolás Palacios-Prado ◽  
Juan C. Sáez ◽  
Jinu Lee
Keyword(s):  
Hela Cells ◽  
Fluorescent Protein ◽  
Cell Model ◽  
Lucifer Yellow ◽  
Yellow Fluorescent Protein ◽  
Pcr Analysis ◽  
Gap Junctional Intercellular Communication ◽  
Null Cell ◽  
Cx43 Expression ◽  
Gap Junctional

Gap junctions (GJs) are intercellular channels that connect adjacent cells electrically and metabolically. The iodide-yellow fluorescent protein (I-YFP) gap junctional intercellular communication (GJIC) assay is a recently developed method with high sensitivity. HeLa cells have been widely used as GJ-deficient cells for GJ-related research. Herein, we present evidence showing that HeLa cells have functional GJs comprising connexin (Cx) 45 using the I-YFP GJ assay and CRISPR/Cas9 system. We conducted the I-YFP GJIC assay in HeLa cells, which revealed a weak level of GJIC that could not be detected by the Lucifer yellow scrape-loading assay. The mRNA expression of GJB5 (Cx31.1), GJA1 (Cx43), and GJC1 (Cx45) was detected in HeLa cells by RT-PCR analysis. Knocking out GJC1 (Cx45) abolished GJIC, as analyzed by the I-YFP assay and dual whole-cell patch-clamp assay. These results suggest that HeLa cells express Cx45-based GJs and that the I-YFP GJIC assay can be used for cells with weak GJIC, such as Cx45-expressing HeLa cells. Further, GJC1 (Cx45)-knockout HeLa cells are more suitable as a GJ-null cell model for transfection experiments than wild-type HeLa cells. This experimental design was successfully applied to knock out Cx43 expression and GJIC in A549 lung cancer cells and can thus be used to identify major Cxs in other cell types and to establish GJ assay systems for different Cxs.

Degradation of channelopsin-2 in the absence of retinal and degradation resistance in certain mutants

2013 ◽  
Vol 394 (2) ◽  
pp. 271-280 ◽  
Author(s):  
Sybille Ullrich ◽  
Ronnie Gueta ◽  
Georg Nagel
Keyword(s):  
Amino Acids ◽  
Western Blot ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Aromatic Amino Acids ◽  
Animal Cells ◽  
Functional Protein ◽  
Enhanced Resistance ◽  
Chloride Pump ◽  
Natronomonas Pharaonis

Abstract Channelrhodopsin-2 is a light-gated cation channel from the green alga Chlamydomonas reinhardtii. It is functional in animal cells and therefore widely used for light-activated depolarization, especially in neurons. To achieve a fully functional protein, the chromophore all-trans-retinal is needed. It has not been investigated whether or not the apoprotein is stable without its cofactor until now. Here we show that channelopsin-2 (Chop2, protein without bound retinal) is much more prone to degradation than channelrhodopsin-2 (protein with retinal). Constructs of Chop2 fused to yellow fluorescent protein (Chop2::YFP) in the absence and presence of retinal confirm this observation by exhibiting strongly differing fluorescence. We present mutants of Chop2 with highly increased stability in the absence of retinal. Substitution of threonine 159 with aromatic amino acids causes enhanced resistance to degradation in the absence of retinal, which is confirmed by fluorescence intensity, the increase in photocurrents on the addition of retinal to previously expressed protein, and Western blot analysis. Exchanging threonine 159 with cysteine, however, increases photocurrents due to better binding of retinal, without obvious stabilization against degradation of the retinal-free opsin. We also show that the light-activated hyperpolarizing chloride pump halorhodopsin from Natronomonas pharaonis (NpHR) is not prone to retinal-dependent degradation.

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.

scholarly journals Ataluren—Promising Therapeutic Premature Termination Codon Readthrough Frontrunner

2021 ◽  
Vol 14 (8) ◽  
pp. 785
Author(s):  
Sylwia Michorowska
Keyword(s):  
Cystic Fibrosis ◽  
Clinical Trials ◽  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Premature Termination Codon ◽  
Hereditary Disease ◽  
In Vitro Activity ◽  
Nonsense Mutations ◽  
Expression Of Genes

Around 12% of hereditary disease-causing mutations are in-frame nonsense mutations. The expression of genes containing nonsense mutations potentially leads to the production of truncated proteins with residual or virtually no function. However, the translation of transcripts containing premature stop codons resulting in full-length protein expression can be achieved using readthrough agents. Among them, only ataluren was approved in several countries to treat nonsense mutation Duchenne muscular dystrophy (DMD) patients. This review summarizes ataluren’s journey from its identification, via first in vitro activity experiments, to clinical trials in DMD, cystic fibrosis, and aniridia. Additionally, data on its pharmacokinetics and mechanism of action are presented. The range of diseases with underlying nonsense mutations is described for which ataluren therapy seems to be promising. What is more, experiments in which ataluren did not show its readthrough activity are also included, and reasons for their failures are discussed.

scholarly journals Exon-skipping antisense oligonucleotides for cystic fibrosis therapy

2022 ◽  
Vol 119 (3) ◽  
pp. e2114858118
Author(s):  
Young Jin Kim ◽  
Nicole Sivetz ◽  
Jessica Layne ◽  
Dillon M. Voss ◽  
Lucia Yang ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Messenger Rna ◽  
Residual Activity ◽  
Exon Skipping ◽  
Premature Termination Codon ◽  
Severe Form ◽  
Bronchial Epithelial Cells ◽  
Human Bronchial Epithelial Cells ◽  
Bronchial Epithelial

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 cocktail of 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.

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