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  • Small molecule correctors divert CFTR-F508del from ERAD by stabilizing sequential folding states. bioRxiv : the preprint server for biology Riepe, C., Wachalska, M., Deol, K. K., Amaya, A. K., Porteus, M. H., Olzmann, J. A., Kopito, R. R. 2023


    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 RNF5 only modestly reduced CFTR-F508del degradation. Sublibrary screens in an RNF5 knockout background identified RNF185 as a redundant ligase, demonstrating that CFTR-F508del ERAD is highly buffered. Gene-drug interaction experiments demonstrated 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.SIGNIFICANCE STATEMENT: Clinically effective small molecule cystic fibrosis (CF) correctors divert mutant CFTR molecules from ER-associated degradation (ERAD). However, the mechanisms underlying CFTR ERAD are not well-understood.The authors used CRISPR knockout screens to identify ERAD machinery targeting CFTR-F508del and found that the pathway is highly buffered, with RNF185 serving as a redundant ubiquitin ligase for RNF5. Gene-drug interaction experiments demonstrated that correctors act synergistically by stabilizing sequential RNF5-resistant folding states.Inhibiting proteostasis machinery is a complementary approach for enhancing current CF corrector therapies.

    View details for DOI 10.1101/2023.09.15.556420

    View details for PubMedID 37745470