Revertant mutants G550E and 4RK rescue cystic fibrosis mutants in the first nucleotide-binding domain of CFTR by different mechanisms

Roxo-Rosa, Mónica; Xu, Zhe; Schmidt, André; Neto, Mário F.; Cai, Zhiwei; Soares, Cláudio M.; Sheppard, David N.; Amaral, Margarida D.

doi:10.1073/pnas.0608312103

Cited by 104 publications

(144 citation statements)

References 43 publications

(67 reference statements)

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“…Moreover, the CFTR gene harbors isoleucine instead of valine in that position in several mammal species. Our results are concordant with those previously obtained in baby hamster kidney cells overexpressing CFTR 23 (by using Western blot analysis, iodide effluxes, and inside-out patch-clamp experiments in which the V562I mutant behaves like WT-CFTR). However, V562I, which was first reported as a polymorphism, 24 has been reassigned as a non-CF-causing mutation, notably implicated in the CBAVD phenotype.…”

Section: Discussionsupporting

confidence: 92%

Orphan Missense Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator

Fresquet

Clément

Norez

et al. 2011

The Journal of Molecular Diagnostics

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More than 1860 mutations have been found within the human cystic fibrosis transmembrane conductance regulator (CFTR) gene sequence. These mutations can be classified according to their degree of severity in CF disease. Although the most common mutations are well characterized, few data are available for rare mutations. Thus, genetic counseling is particularly difficult when fetuses or patients with CF present these orphan variations. We describe a threestep in vitro assay that can evaluate rare missense CFTR mutation consequences to establish a correlation between genotype and phenotype. By using a green fluorescent protein-tagged CFTR construct, we expressed mutated proteins in COS-7 cells. CFTR trafficking was visualized by confocal microscopy, and the cellular localization of CFTR was determined using intracellular markers. We studied the CFTR maturation process using Western blot analysis and evaluated CFTR channel activity by automated iodide efflux assays. Of six rare mutations that we studied, five have been isolated in our laboratory. The cellular and functional impact that we observed in each case was compared with the clinical data concerning the patients in whom we encountered these mutations. In conclusion, we propose that performing this type of analysis for orphan CFTR missense mutations can improve CF genetic counseling. Cystic fibrosis (CF) is the most common severe autosomal recessive genetic disorder in the white population, with an incidence of approximately 1 in 3500. It results from mutations in the CF transmembrane conductance regulator (CFTR) gene 1,2 that encodes a chloride channel normally expressed at the apical membrane of epithelial cells. 3,4 CFTR is a member of the ATP-binding cassette transporter family. It contains two repeated units composed of a membrane-spanning domain, which includes six transmembrane helices, and a nucleotide-binding domain, which harbors an ATP-binding and hydrolysis site. 5 Both halves are joined by a specific cytoplasmic regulator (R) domain, whose phosphorylation by protein kinase A activates the outward anionic channel activity. 6,7 To date, Ͼ1860 mutations identified within the human CFTR sequence are listed in the CF mutation database (http://www.genet.sickkids.on.ca/Home.html, last accessed March 8, 2011). These mutations can be divided into six classes, according to the mechanism that disrupts CFTR function 8,9 : absence of the protein at the apical plasma membrane because of i) defective protein synthesis or ii) impaired maturation leading to protein degradation, iii) defective regulation of CFTR channel activity, iv) altered ionic selectivity and conductance, v) lowered CFTR mRNA amount, and vi) decreased protein stability.The most common mutation, F508del, is a class II mutation. It is carried by 90% of patients with CF, on at least one allele, and it leads to a severe CF phenotype. In our laboratory, we use the Elucigene CF30 Kit (Gen-Probe, Inc., San Diego, CA), which has been approved in the French national neonatal CF screening program for rou...

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Section: Discussionsupporting

confidence: 92%

Orphan Missense Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator

Fresquet

Clément

Norez

et al. 2011

The Journal of Molecular Diagnostics

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“…Alternatively, they may alter the interaction of CFTR domains while leaving the biochemical and biophysical properties of the isolated NBD unaltered. The suppressors might also have little influence on the properties of the CFTR polypeptide in cis but may alter the interaction of cellular quality control machinery with CFTR, thereby promoting CFTR trafficking in trans (22,23). Finally, suppression of the ⌬F508 defect may be the result of a combination of effects on specific intradomain, interdomain, and cellular components interactions.…”

mentioning

confidence: 99%

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

Thibodeau

Richardson

Wang

et al. 2010

Journal of Biological Chemistry

163

211

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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...

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“…The importance of using recombinant CFTR-expressing cell lines as heterologous model systems is recognized from previous studies (Chang et al, 1999;Mendes et al, 2003;Roxo-Rosa et al, 2006), since endogenous CFTR-expressing models are limited and the expression level is usually too low to study CFTR biogenesis, trafficking and function.…”

Section: Cell Linesmentioning

confidence: 99%

“…Adherent cell lines, such as BHK cells, not expressing (BHK-L) or stably expressing wild type (wt)-, F508del-CFTR (mutant), or F508del/4RK-CFTR ("repaired") are cultivated in DMEM/F12 media containing 5% fetal calf serum (Gibco, Invitrogen) and 500 mM methotrexate (MTXTeva Pharma) in culture flasks (Nunc Prand Products) until 80% of confluence (Roxo-Rosa et al, 2006). Cells are then trypsinized and washed once in PBS (Gibco, Invitrogen) and pellets with 5 Â 10 6 cells should be quickly frozen on dry ice and stored at À80 C until analysis.…”

Section: Cell Linesmentioning

confidence: 99%

See 1 more Smart Citation

Signaling Pathways of Proteostasis Network Unraveled by Proteomic Approaches on the Understanding of Misfolded Protein Rescue

Gomes‐Alves

Neves

Penque

2011

Methods in Enzymology

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Revertant mutants G550E and 4RK rescue cystic fibrosis mutants in the first nucleotide-binding domain of CFTR by different mechanisms

Cited by 104 publications

References 43 publications

Orphan Missense Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator

Orphan Missense Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator

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

Signaling Pathways of Proteostasis Network Unraveled by Proteomic Approaches on the Understanding of Misfolded Protein Rescue

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