Correctors Rescue CFTR Mutations in Nucleotide-Binding Domain 1 (NBD1) by Modulating Proteostasis

Area of Science:

• Molecular Biology
• Genetics
• Pharmacology

Background:

• Cystic fibrosis is caused by mutations in the CFTR gene.
• Mutations in the nucleotide-binding domain 1 (NBD1) of CFTR lead to protein misfolding, instability, and degradation.
• Developing therapeutic strategies to correct these mutations is crucial for treating cystic fibrosis.

Purpose of the Study:

• To evaluate the efficacy of small molecule correctors in rescuing specific NBD1 CFTR mutations (A455E, S492F, ΔI507, R560T).
• To investigate the impact of these correctors on CFTR protein expression, stability, and localization.
• To elucidate the molecular mechanisms underlying the rescue effect, including interactions with chaperone proteins and degradation machinery.

Main Methods:

• Transfection of Cos-7 cells and generation of HEK-293 cell lines stably expressing CFTR NBD1 mutants.
• Treatment of mutant cell lines with various small molecule correctors (CFFT-002, CFFT-003, C3, C4, C18).
• Assessment of CFTR protein expression, plasma membrane localization, and binding to histone deacetylases (HDACs) and heat shock proteins (Hsps) using molecular and cellular assays.
• Gene silencing of Hsp27 and Hsp40 to evaluate their role in CFTR mutant rescue.

Main Results:

• The combination of correctors C18 and C4 demonstrated the most significant rescue of CFTR NBD1 mutants.
• Treated mutants exhibited increased CFTR protein expression and enhanced plasma membrane localization.
• A notable decrease in the binding of mutated CFTR to HDAC 6, HDAC 7, Hsp27, and Hsp40 was observed.
• Silencing Hsp27 or Hsp40 individually rescued CFTR mutants, but simultaneous silencing showed no additive effect.

Conclusions:

• Small molecule correctors, particularly the C18+C4 combination, can effectively rescue specific NBD1 mutations in the CFTR gene.
• The rescue mechanism involves stabilizing the CFTR protein and reducing its interaction with endoplasmic reticulum-associated degradation machinery, including HDACs and Hsps.
• Targeting these interactions offers a promising therapeutic avenue for cystic fibrosis patients with NBD1 mutations.