CFTR: folding, misfolding and correcting the ΔF508 conformational defect

Abstract

Cystic fibrosis (CF), the most common lethal genetic disease in the Caucasian population, is caused by loss-of-function mutations of the CF transmembrane conductance regulator (CFTR), a cyclic AMP-regulated plasma membrane chloride channel. The most common mutation, deletion of phenylalanine 508 (ΔF508), impairs CFTR folding and, consequently, its biosynthetic and endocytic processing as well as chloride channel function. Pharmacological treatments may target the ΔF508 CFTR structural defect directly by binding to the mutant protein and/or indirectly by altering cellular protein homeostasis (proteostasis) to promote ΔF508 CFTR plasma membrane targeting and stability. This review discusses recent basic research aimed at elucidating the structural and trafficking defects of ΔF508 CFTR, a prerequisite for the rational design of CF therapy to correct the loss-of-function phenotype.

Figure I

(a) YFP potentiator assay. Fisher rat thyroid (FRT) cells co-expressing human ΔF508 CFTR and a halide-sensing YFP are incubated at a reduced temperature (27 °C) for 18–24 h before assay to target ΔF508 CFTR to the plasma membrane. Test compounds are added for 10 min in the presence of forskolin before iodide (I⁻) addition. ΔF508 CFTR function is assayed in a plate reader by quantifying YFP fluorescence quenching in response to iodide addition, (b) Corrector assays. YFP-based assay: cells are incubated with test compounds at 37 °C for 24 h ΔF508 CFTR function is assayed by iodide addition in the presence of forskolin and the potentiator genistein. Membrane potential based assay (V_m): ΔF508 CFTR activation is monitored by potential-sensitive fluorescent dyes, using a FRET-based assay in low chloride medium. Immunodetection assay: the plasma membrane density of ΔF508 CFTR exposing a 3xHA epitope tag at its exofacial surface is detected by cell surface ELISA.

Figure 1

CFTR predicted structure and folding model, (a) Homology model of human CFTR in the outward-facing configuration. CFTR structure was visualized with MacPyMOL based on the model of Mornon et al. [99]. The nucleotide binding domains (NBDs) 1 and 2, the regulatory domain (R) and the membrane spanning domains (MSDs) 1 and 2 of CFTR are color coded, and the F508 amino acid residue is indicated. The interface between the NBDs and the MSDs formed by the cytoplasmic loops (CLs) 1–4 are shown in the insert, (b) Cooperative folding of WT CFTR (upper panel) and misassembly of ΔF508 CFTR (lower panel). Whereas individual domains can achieve loosely folded conformations and domain assembly cotranslationally, the compactly folded, native tertiary structure with native NBDs–MSDs interfaces are formed post translationally and minimally requires assembly of MSD1–NBD1–R–MSD2 [30]. Thermodynamic and kinetic destabilization of ΔF508 NBD1 and disruption of NBD1–CL4 and CL1 interface compromises the cooperative domain assembly with conformational destabilization of the four domains to variable extents [30, 37, 39]. The protease susceptibility of NBD1 and NBD2 increases nearly 2- to 5-fold and 60-fold, respectively, in ΔF508 CFTR [27, 30, 45]. The estimated folding free energy of individual domains is color-coded based on inference from in vitro folding energetic studies and in vivo processing of domain combinations [–40].

Figure 2

Possible impact of the ΔF508 mutation and corrector molecules on the energetics of folding of the isolated NBD1 domain. Kinetic and thermodynamic destabilization of the isolated NBD1 by the ΔF508 mutation, as compared with WT (a) may lead to increased folding activation energy ΔG_t² (b), decreased folding free energy ΔG⁰ (c) or a combination of these (d). The NBD1 folding scheme assumes a simplified two state folding mechanism with a single transition state (T) between the native (N) and unfolded (U) states, (e) Whereas pharmacological chaperones (PCs) could preferentially normalize the native state (N) folding energetics (right), proteostasis regulators (PRs) may preferentially accelerate domain folding by suppressing the transition state (T) free energy (center). The altered N and T states of the mutant are indicated by a star.

Figure 3

Selected constituents of the ER and peripheral quality control machinery that limit ΔF508 CFTR accumulation at the plasma membrane, (a) Endoplasmic reticulum (ER) quality control (QC). E3 (CHIP, Rma1, Neddr4-2 and Fbs1) and E4 (gp78) ubiquitin (Ub) ligases that contribute to the ubiquitination of non-native nascent CFTR chains are numbered. COMMD1 inhibits peripheral ubiquitination with an unknown mechanism [100]. Co-chaperones that participate in the regulation of Hsc70/Hsp70 and Hsp90 activity are not included, except for DNAJB12, a J-domain protein that facilitates Rma1- and Hsc70-dependent ubiquitination of ΔF508 CFTR. Deubiquitination by the ER-anchored USP19 or soluble UCH-L1 can metabolically stabilize ΔF508 CFTR. (b) Peripheral QC. Chaperone-dependent or chaperone-independent ubiquitination contribute to the rapid internalization and recycling defect of non-native CFTR from the PM. Following endocytosis, ubiquitinated channels are recognized by the ESCRT0-I components (e.g. Hrs, STAM-1 and Tsg101) at early endosomes and rerouted from the constitutive recycling pathway towards lysosomal degradation. Inhibition of E3 ligase and chaperone-dependent recruitment or overexpression of Usp10 can metabolically stabilize CFTR at the PM by facilitating recycling and delaying lysosomal delivery.

Figure 4

Small molecule potentiators and correctors of ΔF508 CFTR. Chemical structures of indicated ΔF508 CFTR potentiators and correctors.