Emerging knowledge indicates the difficulty in categorizing unusual cystic fibrosis (CF) mutations, with regard to both pathogenic mechanism and theratype. As case in point, we present data concerning P67L mutation of the cystic fibrosis transmembrane conductance regulator (CFTR), a defect carried by a small number of individuals with CF and sometimes attributed to a channel conductance abnormality. Findings from our laboratory and others establish that P67L causes protein misfolding, disrupts maturation, confers gating defects, is thermally stable, and exhibits near normal conductance. These results provide one framework by which rare CF alleles such as P67L can be more comprehensively profiled vis-à-vis molecular pathogenesis. We also demonstrate that emerging CF treatments — ivacaftor and lumacaftor — can mediate pronounced pharmacologic activation of P67L CFTR. Infrequent CF alleles are often improperly characterized, in part, due to the small numbers of patients involved. Moreover, access to new personalized treatments among patients with ultra-orphan genotypes has been limited by difficulty arranging phase III clinical trials, and off-label prescribing has been impaired by high drug cost and difficulty arranging third party reimbursement. Rare CFTR mutations such as P67L are emblematic of the challenges to “precision” medicine, including use of the best available mechanistic knowledge to treat patients with unusual forms of disease.

Synonymous or silent mutations are often overlooked in genetic analyses for disease-causing mutations unless they are directly associated with potential splicing defects. More recent studies, however, indicate that some synonymous single polynucleotide polymorphisms (sSNPs) are associated with changes in protein expression, and in some cases, protein folding and function. The impact of codon usage and mRNA structural changes on protein translation rates and how they can affect protein structure and function is just beginning to be appreciated. Examples are given here that demonstrate how synonymous mutations alter the translational kinetics and protein folding and/or function. The mechanism for how this occurs is based on a model in which codon usage modulates the translational rate by introducing pauses caused by nonoptimal or rare codons or by introducing changes in the mRNA structure, and this in turn influences co-translational folding. Two examples of this include the multidrug resistance protein (p-glycoprotein) and the cystic fibrosis transmembrane conductance regulator gene (CFTR). CFTR is also used here as a model to illustrate how synonymous mutations can be examined using in silico predictive methods to identify which sSNPs have the potential to change protein structure. The methodology described here can be used to help identify “non-silent” synonymous mutations in other genes.

CFTR (cystic fibrosis transmembrane conductance regulator) is expressed in the apical membrane of epithelial cells. Cell-surface CFTR levels are regulated by endocytosis and recycling. A number of adaptor proteins including AP-2 (μ2 subunit) and Dab2 (Disabled-2) have been proposed to modulate CFTR internalization. In the present study we have used siRNA (small interfering RNA)-mediated silencing of these adaptors to test their roles in the regulation of CFTR cell-surface trafficking and stability in human airway epithelial cells. The results indicate that μ2 and Dab2 performed partially overlapping, but divergent, functions. While μ2 depletion dramatically decreased CFTR endocytosis with little effect on the half-life of the CFTR protein, Dab2 depletion increased the CFTR half-life ~3-fold, in addition to inhibiting CFTR endocytosis. Furthermore, Dab2 depletion inhibited CFTR trafficking from the sorting endosome to the recycling compartment, as well as delivery of CFTR to the late endosome, thus providing a mechanistic explanation for increased CFTR expression and half-life. To test whether two E3 ligases were required for the endocytosis and/or down-regulation of surface CFTR, we siRNA-depleted CHIP [C-terminus of the Hsc (heat-shock cognate) 70-interacting protein] and c-Cbl (casitas B-lineage lymphoma). We demonstrate that CHIP and c-Cbl depletion have no effect on CFTR endocytosis, but c-Cbl depletion modestly enhanced the half-life of CFTR. The results of the present study define a significant role for Dab2 both in the endocytosis and post-endocytic fate of CFTR.

The ΔF508 mutant form of the cystic fibrosis transmembrane conductance regulator (ΔF508 CFTR) that is normally degraded by the ER-associated degradative pathway can be rescued to the cell surface through low-temperature (27°C) culture or small molecular corrector treatment. However, it is unstable on the cell surface, and rapidly internalized and targeted to the lysosomal compartment for degradation. To understand the mechanism of this rapid turnover, we examined the role of two adaptor complexes (AP-2 and Dab2) and three E3 ubiquitin ligases (c-Cbl, CHIP, and Nedd4-2) on low-temperature rescued ΔF508 CFTR endocytosis and degradation in human airway epithelial cells. Our results demonstrate that siRNA depletion of either AP-2 or Dab2 inhibits ΔF508 CFTR endocytosis by 69% and 83%, respectively. AP-2 or Dab2 depletion also increases the rescued protein half-life of ΔF508 CFTR by ~18% and ~91%, respectively. In contrast, the depletion of each of the E3 ligases had no effect on ΔF508 CFTR endocytosis, whereas CHIP depletion significantly increased the surface half-life of ΔF508 CFTR. To determine where and when the ubiquitination occurs during ΔF508 CFTR turnover, we monitored the ubiquitination of rescued ΔF508 CFTR during the time course of CFTR endocytosis. Our results indicate that ubiquitination of the surface pool of ΔF508 CFTR begins to increase 15 min after internalization, suggesting that CFTR is ubiquitinated in a post-endocytic compartment. This post-endocytic ubiquination of ΔF508 CFTR could be blocked by either inhibiting endocytosis, by siRNA knockdown of CHIP, or by treating cells with the CFTR corrector, VX-809. Our results indicate that the post-endocytic ubiquitination of CFTR by CHIP is a critical step in the peripheral quality control of cell surface ΔF508 CFTR.