The Cystic Fibrosis-causing Mutation ΔF508 Affects Multiple Steps in Cystic Fibrosis Transmembrane Conductance Regulator Biogenesis

Abstract

The deletion of phenylalanine 508 in the first nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator is directly associated with >90% of cystic fibrosis cases. This mutant protein fails to traffic out of the endoplasmic reticulum and is subsequently degraded by the proteasome. The effects of this mutation may be partially reversed by the application of exogenous osmolytes, expression at low temperature, and the introduction of second site suppressor mutations. However, the specific steps of folding and assembly of full-length cystic fibrosis transmembrane conductance regulator (CFTR) directly altered by the disease-causing mutation are unclear. To elucidate the effects of the ⌬F508 mutation, on various steps in CFTR folding, a series of misfolding and suppressor mutations in the nucleotide binding and transmembrane domains were evaluated for effects on the folding and maturation of the protein. The results indicate that the isolated NBD1 responds to both the ⌬F508 mutation and intradomain suppressors of this mutation. In addition, identification of a novel second site suppressor of the defect within the second transmembrane domain suggests that ⌬F508 also effects interdomain interactions critical for later steps in the biosynthesis of CFTR.The maturation of polytopic multidomain membrane proteins is a complex process that requires the proper folding and assembly of individual domains to form a functional complex (1). These processes may be tightly coupled and occur simultaneously or may proceed in a hierarchical fashion. In addition, these processes may proceed in either a co-or post-translational manner (2, 3). The unique nature of these proteins often requires chaperone systems to promote the proper interactions both within and across multiple protein domains. Perturbations that alter the structures of the individual domains or that alter the interactions of these multi-domain complexes are recognized by the cellular quality control (QC) machines, which ultimately target the newly synthesized protein for maturation or degradation.Studies of the cystic fibrosis transmembrane conductance regulator (CFTR), 5 the protein whose loss results in cystic fibrosis (CF) have provided insight into the folding of polytopic membrane proteins (4). CFTR is a member of the ABC-transporter family of proteins and is composed of five distinct domains; two transmembrane domains, TMD1 and TMD2; two nucleotide binding domains, NBD1 and NBD2; and a regulatory domain, R (4). The most common CF-causing mutation, the deletion of phenylalanine 508 (⌬F508), is located in the N-terminal cytoplasmic NBD1 (5-9). This single amino acid deletion results in a dramatic reduction of mature, plasma membrane resident CFTR. The immature protein is arrested in an intermediate conformational state that is recognized by the cellular quality control machinery and targeted for degradation by the ubiquitin-proteasome system (10 -13). Previous work has shown that the ⌬F508 CFTR can be "rescued" by a variety of treatments; tha...

Cystic Fibrosis Transmembrane Conductance Regulator (ABCC7) Structure

“…Although the inability to date to purify the TMDs of CFTR in a native conformational state has prevented direct biophysical evaluation of this hypothesis, its validity is supported by the results from mutagenesis experiments conducted by several different laboratories Loo et al 2010;Thibodeau et al 2010;Mendoza et al 2012;Rabeh et al 2012). These experiments provide evidence that the defective trafficking of F508del-CFTR in tissue culture cells can be suppressed by strengthening the interaction between the perturbed surface on NBD1 and the cognate region of the TMD.…”

“…However, the local structural perturbations caused by the F508del mutation in the folded conformation of NBD1 are likely to contribute at least indirectly to pathogenesis by disrupting structural interactions between NBD1 and the TMDs of CFTR (Lewis et al 2005Serohijos et al 2008;He et al 2010;Loo et al 2010;Thibodeau et al 2010). Recently published work suggests that developing effective drugs to correct the molecular defects caused by the F508del mutation in CFTR may require correction of these disrupted interdomain interactions (Mendoza et al 2012;Rabeh et al 2012).…”

Increased folding and channel activity of a rare cystic fibrosis mutant with CFTR modulators

“…The answer to this question is not clear, but it appears that the ⌬F508 mutation causes global defects in CFTR that involve misfolding of nucleotide binding domain 1 and failure of nucleotide binding domain 1 to make proper intramolecular contacts required to stabilize the native CFTR structure (7,13,20,22,25). Studies on the mechanism of Corr-4a action suggest that it acts to stabilize the TM regions of CFTR (9,28).…”

“…Since CFTRV232D channels could reach the plasma membrane, we asked if channel activity could be stimulated by small-molecule potentiators of CFTR gating (25). As with the experimental protocol in Fig.…”

Genetic, cell biological, and clinical interrogation of theCFTR mutation c.3700 A>G (p.Ile1234Val) informs strategies for future medical intervention

“…7,8 The p.Gly551Asp mutation causes defective channel gating, [9][10][11] and VX-770 is thought to be effective in partially restoring lung function in patients with p.Gly551Asp because it enhances the channel activity of this mutant. [12][13][14][15] The most common mutation in Europe and North America, p.Phe508del, has been studied extensively and has been found to impair CFTR protein folding during synthesis. [16][17][18][19] Knowledge regarding the molecular defects caused by p.Phe508del has driven the development of targeted, interventional compounds, such as VX-809 (or Lumacaftor).…”