Small-molecule correctors divert CFTR-F508del from ERAD by stabilizing sequential folding states

Over 80% of people with cystic fibrosis (CF) carry the F508del mutation in the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride ion channel at the apical plasma membrane (PM) of epithelial cells. F508del impairs CFTR folding causing it to be destroyed by endoplasmic reticulum a...

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Published in:Molecular biology of the cell 2024-02, Vol.35 (2), p.ar15
Main Authors: Riepe, Celeste, Wąchalska, Magda, Deol, Kirandeep K, Amaya, Anais K, Porteus, Matthew H, Olzmann, James A, Kopito, Ron R
Format: Article
Language:English
Subjects:
Benzodioxoles - pharmacology
Benzodioxoles - therapeutic use
Chloride channels
Cystic fibrosis
Cystic Fibrosis - drug therapy
Cystic Fibrosis Transmembrane Conductance Regulator - metabolism
Endoplasmic reticulum
Endoplasmic Reticulum-Associated Degradation
Humans
Identification and classification
Ligases - genetics
Ligases - metabolism
Mitochondrial Proteins - metabolism
Mutation
Protein Folding
Special Issue on Protein Quality Control
Ubiquitin-Protein Ligases - metabolism
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Summary:Over 80% of people with cystic fibrosis (CF) carry the F508del mutation in the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride ion channel at the apical plasma membrane (PM) of epithelial cells. F508del impairs CFTR folding causing it to be destroyed by endoplasmic reticulum associated degradation (ERAD). Small-molecule correctors, which act as pharmacological chaperones to divert CFTR-F508del from ERAD, are the primary strategy for treating CF, yet corrector development continues with only a rudimentary understanding of how ERAD targets CFTR-F508del. We conducted genome-wide CRISPR/Cas9 knockout screens to systematically identify the molecular machinery that underlies CFTR-F508del ERAD. Although the ER-resident ubiquitin ligase, RNF5 was the top E3 hit, knocking out only modestly reduced CFTR-F508del degradation. Sublibrary screens in an knockout background identified RNF185 as a redundant ligase and demonstrated that CFTR-F508del ERAD is robust. Gene-drug interaction experiments illustrated that correctors tezacaftor (VX-661) and elexacaftor (VX-445) stabilize sequential, RNF5-resistant folding states. We propose that binding of correctors to nascent CFTR-F508del alters its folding landscape by stabilizing folding states that are not substrates for RNF5-mediated ubiquitylation.
ISSN:1059-1524
1939-4586
1939-4586
DOI:10.1091/mbc.E23-08-0336