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 ΔF508-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.
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Abbreviations
- CF:
-
cystic fibrosis
- CFTR:
-
cystic fibrosis transmembrane conductance regulator
- PRs:
-
proteostasis regulators
- TGM2:
-
transglutaminase-2
- ROS:
-
reactive oxygen species
- PtdIns3K:
-
type III phosphatidylinositol 3-kinase
- PtdIns3P:
-
phosphatidylinositol 3-phosphate
- EEA-1:
-
early endosome antigen 1
- TfR:
-
transferrin receptor
- PM:
-
plasma membrane
- EGFR:
-
epidermal growth factor receptor
- Vps:
-
vacuolar protein sorting
- 3-MA:
-
3-methyl-adenine
- LE:
-
late endosome
- PNS:
-
post-nuclear supernatant
- MTT:
-
3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide
- UVRAG:
-
UV-irradiation-resistance-associated gene
- CHX:
-
cycloheximide
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Acknowledgements
This work was supported by the European Institute for Research in Cystic Fibrosis and Italian Cystic Fibrosis Association (LM), the Programma di Ricerca Scientifica di Rilevante Interesse Nazionale (2008RMJB3A_004, 2008) of the ministero dell’Istruzione, dell’Università e della Ricerca (LM, VR), RO1 HL093004 (EMB), Telethon Grant No. GGP12128 (LM, EMB, VR, MCM), Ligue Nationale contre le Cancer (Equipe labellisée) (GK), AXA Chair for Longevity Research, Agence Nationale pour la Recherche (ANR) (GK), European Commission (ArtForce) (GK), Fondation pour la Recherche Médicale (FRM), Institut National du Cancer (INCa), Cancéropôle Ile-de-France (GK), Fondation Bettencourt-Schueller and the LabEx Onco-Immunology (GK). We thank Noboru Mizushima (The Tokyo Metropolitan Institute of Medical Sciences, Tokyo, Japan) for the gift of the pcDNA3-HA-Beclin1 expression vectors, Dieter C. Gruenert (University of California, San Francisco, CA, USA) for the gift of CFBE41o- cell lines, S. Corvera (University of Massachusetts Medical School, Worcester, MA, USA) for the gift of the GFP-FYVESARA. We thank Alessandro Luciani (European Institute for Research in Cystic Fibrosis, Milan) for technical assistance in confocal microscopy.
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VRV co-designed the research concept, performed surface biotinylation and membrane fractionation, immunoblot and immunoprecipitation experiments, cell cultures and transfections and analyzed data. SE performed immunoblot and immunoprecipitation experiments, confocal microscopy, cell cultures and transfections and analyzed data. EMB provided the scientific knowledge, contributed to the discussion, interpretation and analysis of the data. MV provided the scientific knowledge, supervised confocal microscopic studies and contributed to the discussion, interpretation and analysis of the data. SC provided the SQSTM1 plasmids, provided the scientific knowledge on SQSTM1 mutants and contributed to the analysis of the data. ADM and AL provided the scientific knowledge and contributed to the discussion, interpretation and analysis of the data. SG, MPM and RC contributed to the discussion of data. VR, MCM and GK co-designed the research concept, co-supervised the project, provided the scientific knowledge and contributed to the discussion, interpretation and analysis of the data. LM designed the research concept, planned the overall experimental design and supervised the study. LM, VR and GK wrote the paper.
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Villella, V., Esposito, S., Bruscia, E. et al. Disease-relevant proteostasis regulation of cystic fibrosis transmembrane conductance regulator. Cell Death Differ 20, 1101–1115 (2013). https://doi.org/10.1038/cdd.2013.46
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DOI: https://doi.org/10.1038/cdd.2013.46
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