Disease-relevant proteostasis regulation of cystic fibrosis transmembrane conductance regulator

Abstract

Mismanaged protein trafficking by the proteostasis network contributes to several conformational diseases, including cystic fibrosis, the most frequent lethal inherited disease in Caucasians. Proteostasis regulators, as cystamine, enable the beneficial action of cystic fibrosis transmembrane conductance regulator (CFTR) potentiators in DF508-CFTR airways beyond drug washout. Here we tested the hypothesis that functional CFTR protein can sustain its own plasma membrane (PM) stability. Depletion or inhibition of wild-type CFTR present in bronchial epithelial cells reduced the availability of the small GTPase Rab5 by causing Rab5 sequestration within the detergent-insoluble protein fraction together with its accumulation in aggresomes. CFTR depletion decreased the recruitment of the Rab5 effector early endosome antigen 1 to endosomes, thus reducing the local generation of phosphatidylinositol-3-phosphate. This diverts recycling of surface proteins, including transferrin receptor and CFTR itself. Inhibiting CFTR function also resulted in its ubiquitination and interaction with SQSTM1/p62 at the PM, favoring its disposal. Addition of cystamine prevented the recycling defect of CFTR by enhancing BECN1 expression and reducing SQSTM1 accumulation. Our results unravel an unexpected link between CFTR protein and function, the latter regulating the levels of CFTR surface expression in a positive feed-forward loop, and highlight CFTR as a pivot of proteostasis in bronchial epithelial cells.

Restoration of CFTR function in patients with cystic fibrosis carrying the F508del-CFTR mutation

“…This indicates that prior re-establishment of autophagy prolongs the persistence at the epithelial surface of a sufficient amount of functional F508del-CFTR to interrupt, for a while, the negative loop that compromises its PM residence and function. 31 We have reported that a CFTR-sufficient environment is required to allow F508del-CFTR to traffic to and reside at the PM of bronchial epithelial cells. 31 Accordingly, when F508del-CFTR is transfected into cells, the resulting protein is not expressed in the PM of CFTR-deficient HeLa cells, yet is capable of trafficking to and residing at the PM of CFTRsufficient normal bronchial epithelial cells, unless these latter cells are treated with .…”

“…31 We have reported that a CFTR-sufficient environment is required to allow F508del-CFTR to traffic to and reside at the PM of bronchial epithelial cells. 31 Accordingly, when F508del-CFTR is transfected into cells, the resulting protein is not expressed in the PM of CFTR-deficient HeLa cells, yet is capable of trafficking to and residing at the PM of CFTRsufficient normal bronchial epithelial cells, unless these latter cells are treated with . 31 Indeed, the functional inhibition of CFTR in normal bronchial epithelial cells ignites the removal of endogenous wild-type CFTR from the PM by diverting CFTR recycling to lysosomal degradation.…”

“…31 Accordingly, when F508del-CFTR is transfected into cells, the resulting protein is not expressed in the PM of CFTR-deficient HeLa cells, yet is capable of trafficking to and residing at the PM of CFTRsufficient normal bronchial epithelial cells, unless these latter cells are treated with . 31 Indeed, the functional inhibition of CFTR in normal bronchial epithelial cells ignites the removal of endogenous wild-type CFTR from the PM by diverting CFTR recycling to lysosomal degradation. 31 Proteostasis regulators such as cystamine interrupt this negative feed forward loop (in which a functional CFTR defect entails the destruction of PM-sessile CFTR protein) and create a permissive environment for the restoration of CFTR function.…”

Erratum: A novel treatment of cystic fibrosis acting on-target: cysteamine plus epigallocatechin gallate for the autophagy-dependent rescue of class II-mutated CFTR

“…Sulfosuccinimidyl-6-(biotinamido) hexanoate (sulfo-NHS-LC-Biotin, Pierce, Rockford, IL, USA, 21335), was dissolved at 1 mg/ml in PBS (Gibco/Thermo Scientific Milan, Italy, 18912-014, pH 8.2), as described. 22 After homogenization with a Potter-Elvehjem pestle, the cells were centrifuged at 2300 × g for 15 min at 4°C. Supernatant fractions that contain the cytoplasmic and PM fractions were centrifuged 1 h at 16 000 × g at 4°C; the pellet that contains the intact membrane, was solubilized in Buffer A (20 mM Tris-HCl, pH 7.4, 2 mM EDTA, 20 mM 2-mercaptoethanol, 1 × PMSF, 1 μg/ml inhibitor protease cocktail (Sigma Aldrich, P8340)+1% Triton X-100 (Sigma Aldrich, X-100-RS) and centrifuged 1 h at 60 000 × g in the ultracentrifuge.…”

“…Nasal brushing. Freshly isolated brushed nasal epithelial cells were obtained by nasal brushing, as previously described, 20,22 from enrolled CF patients and from five non-CF consenting healthy volunteers. Brushes with cells were rapidly transferred in 15-ml sterilized tubes containing RPMI1640 medium (Invitrogen, Carlsbad, CA, USA) with 1% penicillin-streptomycin (Lonza Group LTD, Basel, Switzerland, 17-602E).…”