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Cystic fibrosis (CF), one of the most common fatal hereditary disorders, is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The CFTR gene product is a multidomain adenosine triphosphate-binding cassette (ABC) protein that functions as a chloride (Cl(-)) channel that is regulated by intracellular magnesium [Mg(2+)]i. The most common mutations in CFTR are a deletion of a phenylalanine residue at position 508 (ΔF508-CFTR, 70-80 % of CF phenotypes) and a Gly551Asp substitution (G551D-CFTR, 4-5 % of alleles), which lead to decreased or almost abolished Cl(-) channel function, respectively. Magnesium ions have to be finely regulated within cells for optimal expression and function of CFTR. Therefore, the melastatin-like transient receptor potential cation channel, subfamily M, member 7 (TRPM7), which is responsible for Mg(2+) entry, was studies and [Mg(2+)]i measured in cells stably expressing wildtype CFTR, and two mutant proteins (ΔF508-CFTR and G551D-CFTR). This study shows for the first time that [Mg(2+)]i is decreased in cells expressing ΔF508-CFTR and G551D-CFTR mutated proteins. It was also observed that the expression of the TRPM7 protein is increased; however, membrane localization was altered for both ΔF508del-CFTR and G551D-CFTR. Furthermore, both the function and regulation of the TRPM7 channel regarding Mg(2+) is decreased in the cells expressing the mutated CFTR. Ca(2+) influx via TRPM7 were also modified in cells expressing a mutated CFTR. Therefore, there appears to be a direct involvement of TRPM7 in CF physiopathology. Finally, we propose that the TRPM7 activator Naltriben is a new potentiator for G551D-CFTR as the function of this mutant increases upon activation of TRPM7 by Naltriben.

Cystic fibrosis is the most common lethal autosomal recessive disease in the Caucasian population, and is due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The CFTR protein functions as a chloride channel. Electrophoretic analysis shows that normal CFTR exists in three different molecular weight forms of 127, 131 and 170 kDa (bands A, B and C, respectively) representing different glycosylation forms. Band A is the non-glycosylated form of CFTR, band B is the core-glycosylated CFTR and band C is the mature form of CFTR with complex glycosylation. The glycosylation state of CFTR is representative of its maturation and is an important marker of the protein processing and function. The most common mutation in CF is a missing phenylalanine at position 508 (F508del-CFTR). The misfolded F508del-CFTR protein, which exhibits an altered glycosylation, is observed as bands A and B only and does not traffic correctly to the plasma membrane. Laboratory experiments (SDS-PAGE, immunoblotting, glycosidase digestions, and mobility shift of deglycosylated CFTR) aimed to assess the expression of CFTR and to depict which band is observed have been developed. Nevertheless, these experimental procedures are time consuming and poorly specific. The aim of the present study was to provide an easy, rapid and reproducible methodology to assess whether the CFTR protein within a protein extract is expressed and matured. We show here that surface plasmon resonance (SPR) permits a direct detection of the mature form of CFTR in crude cell lysates, providing a new tool to characterize CFTR in cells without any labelling or pre-treatment before cell lysis. The study of the effects of correctors for F508del-CFTR is the main task of many laboratories. Therefore, the proposed method is likely a useful tool for a rapid detection of mature CFTR in complex samples. We also show here that our method permits the characterization of CFTR in patients' cell extracts in a minimum time.

International audience; Cystic fibrosis (CF), the most common autosomal recessive disease in Caucasians, is due to mutations in the CFTR gene. F508del, the most frequent mutation in patients, impairs CFTR protein folding and biosynthesis. The F508del-CFTR protein is retained in the endoplasmic reticulum (ER) and its traffic to the plasma membrane is altered. Nevertheless, if it reaches the cell surface, it exhibits a Cl(-) channel function despite a short half-life. Pharmacological treatments may target the F508del-CFTR defect directly by binding to the mutant protein or indirectly by altering cellular proteostasis, and promote its plasma membrane targeting and stability. We previously showed that annexine A5 (AnxA5) directly binds to F508del-CFTR and, when overexpressed, promotes its membrane stability, leading to the restoration of some Cl(-) channel function in cells. Because Gonadotropin-Releasing Hormone (GnRH) increases AnxA5 expression in some cells, we tested it in CF cells. We showed that human epithelial cells express GnRH-receptors (GnRH-R) and that GnRH induces an AnxA5 overexpression and an increased Cl(-) channel function in F508del-CFTR cells, due to an increased stability of the protein in the membranes. Beside the numerous physiological implications of the GnRH-R expression in epithelial cells, we propose that a topical use of GnRH is a potential treatment in CF.

The level of circulating anxA5 is correlated to various diseases such as acute myocardial infarction, trauma, thrombosis, inflammation and in some cancers. Our aim was to assess whether a direct approach using surface plasmon resonance (SPR) could be easily used to provide a rapid and cheap alternative to detect anxA5 in blood samples in human. Our results indicate that SPR permits to detect and quantify circulating anxA5 in serum with a minimum time of manipulation. Furthermore, we report here, for the first time, that the level of circulating anxA5 is significantly higher in male than in female (5.43 (± 0.02) and 4.41 (± 0.2)ng/ml, respectively). In conclusion, we found that SPR can be used to rapidly quantify anxA5 in patients and that a gender difference has to be taken into account to explain gender differences in the prevalence of some diseases.

Cystic fibrosis (CF) is the most common lethal autosomal recessive disease in the Caucasian population. It is due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. To date, over 1910 mutations have been identified in the CFTR gene. Among these mutations, the CF-causing missense mutation G551D-CFTR (approx. 5% of cases) encodes for a CFTR chloride channel with normal expression on the cell surface. Nevertheless, it is associated with severe disease due to its altered channel activation. The aim of the present study was to identify specific interacting proteins of G551D-CFTR. Co-immunoprecipitated proteins with G551D-CFTR were resolved by 2D-gel electrophoresis (2-DE). Mass Spectrometry revealed that calumenin was present in the protein complex linked to G551D-CFTR. Despite its basal expression was not modified in G551D-CFTR expressing cells when compared to Wt-CFTR expressing cells, it was more abundant in the G551D-CFTR complex detected by immunoprecipitation. The calumenin-CFTR interaction was also shown by Surface Plasmon Resonance and further confirmed by computational analysis of the predicted calumenin’s partners. Because in our cellular model calumenin was found in the endoplasmic reticulum (ER) by immunofluorescence experiments, we suggest that calumenin is likely involved in the mutated CFTR’s maturation. In conclusion, we showed for the first time that calumenin binds to CFTR and that it is increased in the G551D-CFTR complex. We suggest that it may be involved in the physiopathology of G551D-CFTR and that G551D-CFTR may follow a specific maturation and trafficking pathway. We also hypothesize that UPR may be triggered independently of the retention of G551D-CFTR in the ER because Grp78/Bip expression is increased in the cells. Finally, we propose here that Calumenin is a new CFTR chaperone. Citation: Teng L, Kerbiriou M, Taiya M, Le Hir S, Mignen O, et al. (2012) Proteomic Identification of Calumenin as a G551D CFTR Associated Protein. PLoS ONE 7(6): e40173. doi:10.1371/journal.pone.0040173 Editor: Sue Cotterill, St. Georges University of London, United Kingdom Received October 6, 2011; Accepted June 6, 2012; Published June 29, 2012 Copyright: 2012 Teng et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the association Gaetan Salaun, the ‘‘Etablissement Francais du Sang’’ and the French CF foundation ‘‘Vaincre la Mucoviscidose’’. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: pascal.trouve@univ-brest.fr (PT); claude.ferec@univ-brest.fr (CF)