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

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• Over 80% of cystic fibrosis (CF) patients have the F508del mutation, leading to misfolded cystic fibrosis transmembrane conductance regulator (CFTR) protein.
• Misfolded CFTR is degraded via endoplasmic reticulum associated degradation (ERAD), a process targeted by current CF therapies.
• A deeper understanding of CFTR-F508del ERAD machinery is needed for improved corrector development.

Purpose of the Study:

• To systematically identify the molecular machinery involved in CFTR-F508del ERAD using genome-wide CRISPR/Cas9 screens.
• To investigate the role of identified components, particularly E3 ligases, in CFTR-F508del degradation.
• To explore how CFTR corrector drugs interact with the ERAD pathway.

Main Methods:

• Genome-wide CRISPR/Cas9 knockout screens to identify genes regulating CFTR-F508del ERAD.
• Sublibrary screens in RNF5 knockout cells to identify redundant ERAD machinery.
• Gene-drug interaction experiments to assess the impact of correctors on CFTR folding and degradation.

Main Results:

• RNF5, an ER-resident ubiquitin ligase, was identified as a key player in CFTR-F508del ERAD, though its knockout only partially reduced degradation.
• RNF185 was identified as a redundant ligase, highlighting the robustness of the CFTR-F508del ERAD pathway.
• CFTR corrector drugs, tezacaftor and elexacaftor, were shown to stabilize RNF5-resistant folding states of CFTR-F508del.

Conclusions:

• CFTR-F508del ERAD is a complex and robust process involving multiple E3 ligases, including RNF5 and RNF185.
• CFTR corrector drugs function by stabilizing specific folding intermediates, diverting CFTR-F508del from ERAD-mediated degradation.
• This research provides crucial insights into CFTR protein quality control, potentially guiding the development of more effective CF therapies.