The first mechanistic insights into the interaction of Esc(1–21) with CFTR in a cellular context are provided and the NBD1-NBD2 interface is identified as a suitable target for peptide-based potentiators to support the development of Esc(1–21) and its derivatives as novel multifunctional therapeutic candidates for CF treatment.
Abstract
Cystic fibrosis (CF) is a genetic disorder caused by variants in the gene encoding the CFTR ion channel, leading to defective epithelial chloride transport, dehydrated airway mucus, chronic infections, and progressive lung damage. Although CFTR modulators have improved clinical outcomes, a subset of patients remains unresponsive to current therapies, highlighting the need for alternative strategies. The frog skin-derived antimicrobial peptide Esc(1–21) was recently identified as a CFTR potentiator, likely through direct interaction with the interface of the channel’s cytosolic nucleotide-binding domains (NBDs); yet its molecular mechanism is poorly understood. Here, we engineered a cysteine-less CFTR scaffold by introducing cysteine pairs within or outside the predicted peptide-binding region and used molecular dynamics simulation to define the putative interaction site. Disulfide cross-linking assays performed in cells expressing these constructs validated the computational predictions and showed that the peptide selectively prevents cross-link formation only when cysteines are positioned at the NBD interface, thereby supporting a direct and specific interaction with CFTR. Importantly, confocal microscopy provided evidence for peptide intracellular internalization, together with colocalization with the ion channel, indicating that Esc(1–21) can access the cytosolic side of CFTR to enhance its activity. Overall, these findings (i) provide the first mechanistic insights into the interaction of Esc(1–21) with CFTR in a cellular context; (ii) identify the NBD1-NBD2 interface as a suitable target for peptide-based potentiators; and (iii) support the development of Esc(1–21) and its derivatives as novel multifunctional therapeutic candidates for CF treatment.
Acute lung injury and persistent inflammation caused by SARS-CoV-2 may lead to further complications after the virus gets cleared. People with Cystic fibrosis (pwCF) are highly vulnerable to pulmonary diseases due to mutations in the CFTR gene. However, there is limited research about SARS-CoV-2 severity in pwCF and subjects carrying a single variant of this gene.
Middle-aged (4-5 months old) and elderly (>18 months) mice expressing various levels of CFTR (WT, CFTR Knock out (KO), and heterozygous for CFTR (HET)) were infected with the mouse-adapted SARS-CoV-2 virus, MA10. Body weight and body temperature were measured in addition to spike specific antibodies detected by ELISA, flow cytometry on immune cells from blood samples, and lung histology.
Middle-aged KO mice significantly lost less weight than WT and HET mice for both male and female mice. However, HET males lost significantly more weight compared to WT and KO male mice (p = 0.0058 and 0.0004, respectively). HET mice body weight recovery took much longer especially in males for both middle-aged and aged mice (∼28 days for middle-aged mice vs 21 days for KO mice, and 51 days for aged mice vs 25 days for KO mice).
This study highlights the significance of carrying CFTR mutation in susceptibility and lung pathology to COVID-19. Our data show that the severity of SARS-CoV-2 infection in different mice CFTR genotypes is highly dependent on the age and gender.
Internal OSU funding
Viral Immunology (VIR)
M. Wahba, P. Boyaka, E. Cormet-Boyaka et al.· Journal of Immunology· 0 citations
Cystic fibrosis (CF) is a devastating genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. As morbidity and mortality from CF results from a lack of mucus clearance that leads to chronic bacterial infections and progressive loss of lung function, site-specific insertion of a CFTR cDNA into the endogenous CFTR locus in airway basal stem cells (ABSCs) could prove curative for all disease-causing mutations. This study describes the development of genome-editing approach utilizing nonviral reagents, designed to be packaged into nonviral delivery systems. An sgRNA targeting the 5′ untranslated region (UTR) of CFTR was characterized as directing high on-target cutting and displaying a safe off-target profile. Airway cell lines electroporated with chemically modified (1-Aminohexane, AmC6), linear double-stranded DNA (ldsDNA) constructs were utilized as a homology directed repair (HDR) donor, initially optimized with an mCitrine reporter. Expectedly, when the 780 bp mCitrine cDNA was replaced with the 4.4 kb CFTR cDNA, integration efficiency dropped significantly. However, 1%–2% integration of codon-optimized donors was sufficient to restore CFTR expression in the bulk-edited population of a human bronchial epithelial cell line, 16HBE14o- (16HBE), to levels reaching 50% of wild-type expression as measured by western blot. Electrophysiological validation of CFTR ion channel function measured via Ussing chamber assay revealed that these bulk-edited populations exhibit greater than 40% restoration of the chloride ion currents of the measured wild-type controls. These results demonstrate that low levels of CFTR integration can be made therapeutically relevant by optimizing the designs of gene editing reagents. Importantly, this work utilizes nonviral-editing reagents, an essential step toward in vivo gene therapy for CF.
Vrishti Sinha, P. Ayoub, C. Juett et al.· Molecular Therapy: Nucleic A...· 0 citations
Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel in humans and other vertebrates, whose mutation leads to cystic fibrosis. CFTR inhibitory factor — Cif — is a recently discovered bacterial epoxide hydrolase that downregulates CFTR protein upon the bacterial infection. However, its cleaving activity towards fatty acid epoxides — epoxygenase-derived oxylipins — has been recently characterized. We identified a list of host-associated bacteria with putative Cif proteins, identified their most prevalent ecological functions by systematic literature review, and performed similar review for the previously assembled list of host-associated bacteria carrying lipoxygenase (LOX). Both Cif and LOX showed the association with pathogenesis and symbiosis in broad host range, and similar ecological profiles of their carriers suggested that they both might target oxylipin signaling in hosts. We also described the association of Cif with plant hormone biosynthesis and plant growth promotion — which indirectly supports our previous model of bacterial LOX action in plant and vertebrate hosts.
Георгий Фёдорович Куракин· Cytokines and inflammation· 0 citations
Cystic fibrosis (CF) is one of the most common rare genetic diseases. It is caused by pathogenic variants of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. More than 2200 variants have been identified in the CFTR gene that need detailed knowledge and functional characterization in order to develop specific therapeutic strategies. The traditional therapy for CF relied on addressing symptoms through mucolytic and antibiotic treatments, respiratory physiotherapy and aerosol therapy. However, in the last few years, the development of small new molecules targeting and restoring the underlying CFTR channel defect marked an important step in CF treatment. In addition, the implementation of patient-specific cellular models allowed the evaluation of pharmacological responses, leading to therapeutic advances in the direction of personalized treatment. The focus of this review is to describe and discuss the strategies for restoring the CFTR functional defects depending on the specific CFTR pathogenic variants. In particular, the review highlights the possible application of experimental and clinical drugs to CF treatment, which may allow improvements in patients’ quality of life and life expectancy. The in vitro testing of therapeutic drugs (theratyping) is performed nowadays through the use of several cellular models, especially those derived from patient-specific tissues. This topic is a hot point in CF research and the review also aims to provide an overview of the state of the art in theratyping. The novelty of this review is the integrated view of the most recent achievements in precision diagnostics and therapy of CF at the molecular, cellular and clinical level, which are able to change the natural history of this disease.
Sara Allushi, M. Virgulti, Giovanna Blaconà et al.· International Journal of Mol...· 0 citations
A summary of recent literature pertaining to CFTR modulators is provided to enhance the knowledge of the growing body of evidence guiding the next era of CF care, in which disease modification at the molecular level is increasingly achievable.
Dawn Selhorst, E. Stekolchik· Respiratory care· 0 citations
BACKGROUND
Dent disease type 1 is an X-linked proximal tubulopathy caused by mutations in CLCN5, which encodes the chloride/proton exchanger ClC-5. While loss-of-function mutations impair endocytosis and lead to progressive renal disease, the pathological consequences of uncoupling mutations that selectively disrupt Cl-/H+ exchange remain incompletely understood.
METHODS
In this study, we examined the renal phenotype associated with the ClC-5 E211A uncoupling mutation, which abolishes proton transport while preserving chloride conductance, and compared it with that of ClC-5 null mice.
RESULTS
Contrary to the expectation that partial transport activity would mitigate disease severity, E211A mutant mice developed more severe and progressive kidney pathology than null mutants. Both models exhibited early low-molecular-weight proteinuria, including marked urinary excretion of β2-microglobulin and vitamin D-binding protein. However, E211A mutants displayed progressive polyuria, reduced body weight, and pronounced interstitial fibrosis at 5 and 18 months of age, changes not observed in ClC-5 null mice. Renal injury in E211A mutants was characterized by glomerular abnormalities, including enlarged Bowman's spaces, cyst formation, reduced podocyte markers Wilms tumor protein and nephrin, and age-dependent tubular degeneration. ClC-5 E211A protein showed altered localization and reduced expression in vivo, suggesting disrupted trafficking or stability. Phenotypic severity was strongly influenced by genetic background, underscoring its modulatory role in disease expression.
CONCLUSIONS
Together, these findings demonstrate that the ClC-5 E211A uncoupling mutation induces additional adverse effects that exceed those caused by complete loss of ClC-5. Our results reveal that uncoupling mutations can drive progressive kidney damage through mechanisms distinct from gene knockout and highlight the need for mutation-specific therapeutic strategies for Dent disease type 1.
