Dataset Information

THE PHARMACOLOGICAL RESCUE OF CFTR BY LUMACAFTOR (VX-809) IN THE CYSTIC FIBROSIS BRONCHIAL EPITHELIUM IS ASSOCIATED WITH AN EXTENSIVE REORGANIZATION OF MITOCHONDRIA.

ABSTRACT: Cystic Fibrosis (CF) is a genetic disorder CF is caused by mutations of the gene encoding for the cystic fibrosis transmembrane conductance regulator protein (CFTR), a transmembrane anion channel expressed at the apical membrane of several organs, including the epithelial cells of the airway. CFTR mutations result in dysfunctional ion transport across the apical membrane at the surface of the epithelia, generating thickened and dehydrated secretions. In the lung, this leads to a decrease in the mucociliary clearance, favoring bacterial colonization and progressive obstruction of the duct. Although over 2000 CFTR variants have been identified so far, the most common mutation is a deletion of the phenylalanine in position 508 (F508del), which shows an allelic frequency of around 90% among CF patients. F508del-CFTR is incorrectly folded, causing its retention at the endoplasmic reticulum (ER) and subsequent proteasomal degradation. Among the several drugs available in CF pharmacological treatment, VX-809 (commercial name Lumacaftor) is the most used drug for patients carrying F508del-CFTR mutation. This drug corrects the aberrant folding of F508del-CFTR by favoring the correct intramolecular interactions, thus enabling a higher number of copies of the defective protein to reach the plasma membrane. We applied SWATH-based proteomics to understand if a pharmacological rescue of F508del-CFTR is associated with changes in global protein expression of the human bronchial epithelium by using the CFBE41o- cell model, a line that stably expresses F508del-CFTR.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Cystic Fibrosis

SUBMITTER: Clarissa Braccia

LAB HEAD: Andrea Armirotti

PROVIDER: PXD028355 | Pride | 2022-08-11

REPOSITORIES: Pride

ACCESS DATA

Publications

CFTR Rescue by Lumacaftor (VX-809) Induces an Extensive Reorganization of Mitochondria in the Cystic Fibrosis Bronchial Epithelium.

Braccia Clarissa C Christopher Josie A JA Crook Oliver M OM Breckels Lisa M LM Queiroz Rayner M L RML Liessi Nara N Tomati Valeria V Capurro Valeria V Bandiera Tiziano T Baldassari Simona S Pedemonte Nicoletta N Lilley Kathryn S KS Armirotti Andrea A

Cells 20220616 12

<h4>Background</h4>Cystic Fibrosis (CF) is a genetic disorder affecting around 1 in every 3000 newborns. In the most common mutation, F508del, the defective anion channel, CFTR, is prevented from reaching the plasma membrane (PM) by the quality check control of the cell. Little is known about how CFTR pharmacological rescue impacts the cell proteome.<h4>Methods</h4>We used high-resolution mass spectrometry, differential ultracentrifugation, machine learning and bioinformatics to investigate both ...[more]

PMID: 35741067

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Project description:Cystic Fibrosis (CF) is a genetic disorder CF is caused by mutations of the gene encoding for the cystic fibrosis transmembrane conductance regulator protein (CFTR), a transmembrane anion channel expressed at the apical membrane of several organs, including the epithelial cells of the airway. CFTR mutations result in dysfunctional ion transport across the apical membrane at the surface of the epithelia, generating thickened and dehydrated secretions. In the lung, this leads to a decrease in the mucociliary clearance, favoring bacterial colonization and progressive obstruction of the duct. Although over 2000 CFTR variants have been identified so far, the most common mutation is a deletion of the phenylalanine in position 508 (F508del), which shows an allelic frequency of around 90% among CF patients. F508del-CFTR is incorrectly folded, causing its retention at the endoplasmic reticulum (ER) and subsequent proteasomal degradation. Among the several drugs available in CF pharmacological treatment, VX-809 (commercial name Lumacaftor) is the most used drug for patients carrying F508del-CFTR mutation. This drug corrects the aberrant folding of F508del-CFTR by favoring the correct intramolecular interactions, thus enabling a higher number of copies of the defective protein to reach the plasma membrane. Localization of Organelle Proteins by Isotope Tagging after Differential ultra-Centrifugation (LOPIT-DC, https://doi.org/10.1038/s41467-018-08191-w) workflow was used to study the spatial localization of proteins in the CFBE41o- cells and how it is impacted by CFTR rescue triggered by VX-809. LOPIT-DC is a powerful and well-established tool for the investigation of the spatial distribution of global proteome within cells, which combines subcellular fractionation based on differential centrifugation, quantitative mass spectrometry, and machine learning. Its application provides a global snapshot of the steady-state subcelluler distribution of proteins. The aim of this project is to identify proteins that change their cellular localization in association with the treatment, to uncover molecules that play a role in the trafficking of the defective CFTR to the plasma membrane.

Project description:Cystic fibrosis (CF) is an inherited, multi-system disease caused by dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, a ubiquitous ion channel important for epithelial hydration. A direct consequence of this dysfunction is impaired mucociliary clearance, chronic airway infection and a persistent neutrophilic inflammatory response that results in progressive loss of lung function, development of respiratory failure and premature death. Partial restoration of CFTR function is now possible for most CF patients through mutation specific CFTR modulators. Ivacaftor monotherapy produces significant clinical improvement in CF patients with the G511D mutation. Dual therapy, combining ivacaftor with lumacaftor or tezacaftor, results in modest clinical improvements in patients homozygous for F508del. More recently, triple therapy with elexacaftor/tezacaftor/ivacaftor (ETI) has led to dramatic improvements in lung function and quality of life in patients homozygous and heterozygous for F508del. Sputum proteomics is a powerful research technique capable of identifying important airway disease mechanisms by interrogating the proteome, an entire set of proteins within biological samples. It has confirmed the central role of neutrophilic immune dysregulation in CF and non-CF bronchiectasis, particularly involving the release of antimicrobial proteins and neutrophil-extracellular traps (NETs), and through impaired anti-inflammatory mechanisms. These processes produce distinct molecular signatures within the sputum proteome that become increasingly abnormal with chronic airway infection and progressive lung disease severity. In CF patients, airway and systemic inflammatory cytokines potentially related to these signatures reduce with the various forms of CFTR modulation. To date, no studies of ETI therapy in CF lung disease have assessed large-scale change in protein expression using untargeted proteomics. We hypothesised that ETI therapy would shift the sputum proteome toward health, potentially normalising airway biology in people with CF. The objectives of this study were to investigate changes in the CF sputum proteome with the introduction of ETI, correlate these with changes in clinical markers of disease severity, and make comparisons with the sputum proteome in healthy controls and in repeat samples from CF patients not suitable for ETI therapy. We also explored which molecular pathways associated with CF lung disease did not change with ETI.

Dataset Information

THE PHARMACOLOGICAL RESCUE OF CFTR BY LUMACAFTOR (VX-809) IN THE CYSTIC FIBROSIS BRONCHIAL EPITHELIUM IS ASSOCIATED WITH AN EXTENSIVE REORGANIZATION OF MITOCHONDRIA.

Publications

CFTR Rescue by Lumacaftor (VX-809) Induces an Extensive Reorganization of Mitochondria in the Cystic Fibrosis Bronchial Epithelium.

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