The CFTR Folding Pathway: Implications for the Identification and Development of CF Therapeutics
Mendoza, Juan Luis
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The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein is a member of the ABC transporter superfamily, important for Cl- conductance at the apical cell membrane. Loss-of-function of CFTR leads to Cystic Fibrosis (CF), a fatal genetic disease affecting 70,000 people worldwide. There are hundreds of CF causing mutations with the most common being ΔF508, present in at least one allele in 90% of CF patients. CFTR, comprising of 1480 amino acids, folds into five domains important for forming the channel through the membrane, and the regulation of channel function. F508 is located in Nucleotide Binding Domain 1 (NBD1) and is predicted to be at the interface with Intracellular Loop 4 (ICL4) of Transmembrane Domain 2 (TMD2). Studies of the isolated NBD1 demonstrate that the ΔF508 mutation impacts the folding pathway and stability of the domain. Misfolding of NBD1 contributes to the trafficking defect of the intact protein and subsequent loss-of-function. Conversely, second-site suppressor mutations, which more than compensate for defects of the mutant NBD1 domain, only partially rescue CFTR trafficking, suggesting that the deletion also affects other steps along the folding pathway. The aim of this work was to identify positions in CFTR critical for defining the folding pathway. We used a computational approach and two in vitro folding assays to monitor folding of the isolated NBD1 domain and trafficking of full-length CFTR. These data establish a correlation between the folding of the isolated NBD1 domain and maturation of full-length CFTR. Further, NBD1 second-site suppressor mutations in the ΔF508, F508K (NBD1/ICL4 interface disrupting mutation), and R1070W (ΔF508 NBD1/ICL4 interface stabilizing mutation) backgrounds suggest that ΔF508 CFTR is defective in two steps of CFTR biogenesis: 1) stability and efficiency of folding of the NBD1 domain, and 2) NBD1/ICL4 docking. We demonstrate that efficient rescue of ΔF508 CFTR requires correction the two distinct defects. This work has implications for the discovery and development of CF therapeutics by providing a framework for understanding the observed ceiling in the efficacy of either suppressor mutations or corrector compounds, which likely correct a single defect.