Network Biology Identifies Novel Regulators of CFTR Trafficking and Membrane Stability

Abstract

In cystic fibrosis, the most common disease-causing mutation is F508del, which causes not only intracellular retention and degradation of CFTR, but also defective channel gating and decreased membrane stability of the small amount that reaches the plasma membrane (PM). Thus, pharmacological correction of mutant CFTR requires targeting of multiple cellular defects in order to achieve clinical benefit. Although small-molecule compounds have been identified and commercialized that can correct its folding or gating, an efficient retention of F508del CFTR at the PM has not yet been explored pharmacologically despite being recognized as a crucial factor for improving functional rescue of chloride transport. In ongoing efforts to determine the CFTR interactome at the PM, we used three complementary approaches: targeting proteins binding to tyrosine-phosphorylated CFTR, protein complexes involved in cAMP-mediated CFTR stabilization at the PM, and proteins selectively interacting at the PM with rescued F508del-CFTR but not wt-CFTR. Using co-immunoprecipitation or peptide–pull down strategies, we identified around 400 candidate proteins through sequencing of complex protein mixtures using the nano-LC Triple TOF MS technique. Key candidate proteins were validated for their robust interaction with CFTR-containing protein complexes and for their ability to modulate the amount of CFTR expressed at the cell surface of bronchial epithelial cells. Here, we describe how we explored the abovementioned experimental datasets to build a protein interaction network with the aim of identifying novel pharmacological targets to rescue CFTR function in cystic fibrosis (CF) patients. We identified and validated novel candidate proteins that were essential components of the network but not detected in previous proteomic analyses.

Treatment of Polarized Cystic Fibrosis Airway Cells With HGF Prevents VX-661-Rescued F508del-CFTR Destabilization Caused by Prolonged Co-exposure to VX-770

“…By immunolabeling polarized CFBE cells treated with either vehicle (DMSO), or 3 µM of either VX-809 or VX-661 for 15 days, we could observe that exposure to VX-661 resulted in a clearly better structured epithelial-like monolayer, when compared to VX-809, and also elicited apparent stronger CFTR staining at the apical membrane of the polarized cell monolayer ( Figure 2A ). Using a previously described methodology ( Loureiro et al, 2019 ) to quantify apical (AP), basolateral (BL) and total (TL = AP + BL) immunofluorescent CFTR signals, we confirmed that, despite producing equivalent levels of total rF508del protein, prolonged treatment with VX-661 resulted in a small (∼1.5-fold) but significant ( p < 0.05) increase in apical rF508del abundance over that produced by equivalent treatment with VX-809 ( Figure 2B ). Repeating these experiments using the previously characterized model of polarized CFBE cell co-expressing F508del-CFTR and the YFP-F46L/H148Q/I152L halide sensor ( Matos et al, 2018 ; Loureiro et al, 2019 ) allowed us to confirm that forskolin-stimulated activity of CFTR was indeed higher in VX-661-treated cells, although not sufficient to reach statistical significance over cells similarly treated with VX-809 ( Figures 2C,D ).…”

“…Using a previously described methodology ( Loureiro et al, 2019 ) to quantify apical (AP), basolateral (BL) and total (TL = AP + BL) immunofluorescent CFTR signals, we confirmed that, despite producing equivalent levels of total rF508del protein, prolonged treatment with VX-661 resulted in a small (∼1.5-fold) but significant ( p < 0.05) increase in apical rF508del abundance over that produced by equivalent treatment with VX-809 ( Figure 2B ). Repeating these experiments using the previously characterized model of polarized CFBE cell co-expressing F508del-CFTR and the YFP-F46L/H148Q/I152L halide sensor ( Matos et al, 2018 ; Loureiro et al, 2019 ) allowed us to confirm that forskolin-stimulated activity of CFTR was indeed higher in VX-661-treated cells, although not sufficient to reach statistical significance over cells similarly treated with VX-809 ( Figures 2C,D ). In both cases, CFTR activity was similarly inhibited by the presence of CFTR inhibitor 172 (inh172; Figures 2C,D ).…”

“…Samples were analyzed by immunoblotting as previously described ( Matos et al, 2018 ; Loureiro et al, 2019 ). Antibodies used for WB were: mouse anti-CFTR clone 596 (obtained through the UNC CFTR antibody distribution program sponsored by CFFT), mouse anti-α-Tubulin clone B-5-1-2 (Sigma-Aldrich), mouse-anti-E-cadherin (Transduction Laboratories), mouse-anti-CK18 (Millipore), rabbit-anti-ZO-1, mouse-anti-CK8 and rabbit-anti-Ki-67 (all from Santa Cruz Biotechnology).…”

“…The most frequent mutation, F508del, is present in at least one allele of 80–85% of CF individuals worldwide and causes the protein to misfold and be prematurely degraded by the ER quality control mechanism (ERQC) ( Farinha and Matos, 2016 ). The very few F508del-CFTR molecules that manage to escape ERQC in CF cells bare a deficiency in channel gating, and a highly decreased half-life at the PM of epithelial cells ( Farinha et al, 2013 ; Farinha and Matos, 2016 ; Loureiro et al, 2019 ).…”

Exploring the basic mechanisms in Cystic Fibrosis: Promoting data presentation and discussion at the 16th ECFS Basic Science Conference

“…Focus on this CFTR "regulome" will generate novel important insights which will facilitate identification of novel therapeutic targets. This will be of particular relevance to patients carrying CFTR genotypes that are not responsive to CFTR-directed approaches [20], and deepen our understanding of how these interactions shape CFTR fate in the cell [21,22].…”