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General introduction to section C: Biochemistry and Biophysics of CFTR
Journal of Cystic Fibrosis 3 (2004) 67 www.elsevier.com/locate/jcf
General introduction to section C: Biochemistry and Biophysics of CFTR Aleksander Edelman a,*, Margarida D. Amaral b,c,1 a
INSERM U467, Faculte´ de Me´decine Necker-Enfants Malades, 156, rue de Vaugirard, 75015 Paris cedex 15, France b Centre of Human Genetics, National Institute of Health, Campo Grande-C8, 1749-016 Lisboa, Portugal c Department of Chemistry and Biochemistry, University of Lisboa, Campo Grande-C8, 1749-016 Lisboa, Portugal
The cystic fibrosis gene encodes the cystic fibrosis transmembrane conductance regulator (CFTR) protein whicha is a 1480 amino acid, glycosylated membrane protein that functions as a cAMP-dependent chloride (Cl ) channel. It is mainly expressed in secretory epithelia. The F508delCFTR protein, resultant from the mutant gene found in 70% of CF chromosomes, is aberrantly folded, mostly blocked for maturation and degraded in the ER by the ubiquitin proteosome pathway. This mutant still functions as a cAMPdependent Cl channel to some degree if the protein is directed to the plasma membrane by some strategy that corrects its cellular location. The properties of wild-type and mutant CFTR proteins are studied extensively using different biochemical and biophysical approaches. Although these studies are being developed for more than 15 years, many dark areas in the research field of CFTR protein still need to be unmasked. For example, the production of specific antiCFTR antibodies was one of the main problems for Western immunoprecipitation and immunocytochemistry experiments. Today, the evaluation of existing antibodies shows that only few anti-CFTR antibodies are suitable for native tissue analysis, but several can be used for cell line experiments. The applicability of existing anti-CFTR antibodies is discussed here [1], as well as the basic biochemical techniques that are currently used for the study of CFTR protein produced in vivo [2] and the techniques for its in vitro study [3]. New studies based on proteomic approaches are reviewed here [4]. These are being developed to get insights on proteins differentially expressed in CF versus non-CF cells/tissues or on proteins interacting with CFTR/F508delCFTR that may be involved in the traffic/degradation and or activity of CFTR/F508del-CFTR. The recent elucidation of murine CFTR nucleotide binding domain 1 (NBD1) called for a discussion and review of the CFTR structural aspects [5]. The role of normal and mutant CFTR on the glycosylation pattern of other proteins
and its consequences for host – bacterial interactions in the light of recently published data are also discussed in this section [6]. Novel insights on lipidic alterations in CF and possible consequences for CF deserve our attention and are also reported from a methodological point of view [7]. We believe that we were able to gather in this section (often as authors in one joint manuscript) key groups in each subarea dealt with. All articles are outlines/reviews of various protocols available at the European Working Group on CFTR Expression website [8] that should be used extensively as supplementary material. Our aim was to provide a series of methodologies that hopefully will be useful to the novice and experienced researcher alike by reducing effort and research time of all with an interest in this field.
References [1] Mendes F, Farinha CM, Roxo-Rosa M, Fanen P, Edelman A, Dormer RL, et al. Antibodies for CFTR studies. J Cyst Fibros 2004;3:69 – 72. [2] Farinha CM, Penque D, Roxo-Rosa M, Lukacs G, Dormer RL, McPherson M, et al. Biochemical methods to assess CFTR expression and membrane localization. J Cyst Fibros 2004;3:73 – 7. [3] Benos DJ, Berdiev BK, Ismailov II, Ostedgaard L, Kogan I, Li C, et al. Methods to study CFTR protein in vitro. J Cyst Fibros 2004;3:79 – 83. [4] Roxo-Rosa M, Davezac N, Bensalem N, Majumder M, Heda GD, Simas A, et al. Proteomics techniques for cystic fibrosis research. J Cyst Fibros 2004;3:85 – 9. [5] Dorwart M, Thibodeau P, Thomas P. Cystic fibrosis: recent structural insights. J Cyst Fibros 2004;3:91 – 4. [6] Rhim AD, Stoykova LI, Trindade AJ, Glick MC, Scanlin TF. Altered terminal glycosylation and the pathophysiology of CF lung disease. J Cyst Fibros 2004;3:95 – 6. [7] Ollero M. Methods for the study of lipid metabolites in cystic fibrosis. J Cyst Fibros 2004;3:97 – 8. [8] European Working Group on CFTR Expression. The Online Virtual Repository of Methods and Reagents for CFTR Expression and Functional Studies (section C). http://central.igc.gulbenkian.pt/cftr/ vr/biochemistry.html.
* Corresponding author. Tel.: +33-1-40-61-56-21; fax: +33-1-40-6155-91. E-mail addresses: [email protected] (A. Edelman), [email protected] (M.D. Amaral). 1 Tel.: +351-21-751-92-33; fax: +351-21-750-00-88. 1569-1993/$ - see front matter D 2004 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jcf.2004.05.015
General introduction to section C: Biochemistry and Biophysics of CFTR Aleksander Edelman a,*, Margarida D. Amaral b,c,1 a
INSERM U467, Faculte´ de Me´decine Necker-Enfants Malades, 156, rue de Vaugirard, 75015 Paris cedex 15, France b Centre of Human Genetics, National Institute of Health, Campo Grande-C8, 1749-016 Lisboa, Portugal c Department of Chemistry and Biochemistry, University of Lisboa, Campo Grande-C8, 1749-016 Lisboa, Portugal
The cystic fibrosis gene encodes the cystic fibrosis transmembrane conductance regulator (CFTR) protein whicha is a 1480 amino acid, glycosylated membrane protein that functions as a cAMP-dependent chloride (Cl ) channel. It is mainly expressed in secretory epithelia. The F508delCFTR protein, resultant from the mutant gene found in 70% of CF chromosomes, is aberrantly folded, mostly blocked for maturation and degraded in the ER by the ubiquitin proteosome pathway. This mutant still functions as a cAMPdependent Cl channel to some degree if the protein is directed to the plasma membrane by some strategy that corrects its cellular location. The properties of wild-type and mutant CFTR proteins are studied extensively using different biochemical and biophysical approaches. Although these studies are being developed for more than 15 years, many dark areas in the research field of CFTR protein still need to be unmasked. For example, the production of specific antiCFTR antibodies was one of the main problems for Western immunoprecipitation and immunocytochemistry experiments. Today, the evaluation of existing antibodies shows that only few anti-CFTR antibodies are suitable for native tissue analysis, but several can be used for cell line experiments. The applicability of existing anti-CFTR antibodies is discussed here [1], as well as the basic biochemical techniques that are currently used for the study of CFTR protein produced in vivo [2] and the techniques for its in vitro study [3]. New studies based on proteomic approaches are reviewed here [4]. These are being developed to get insights on proteins differentially expressed in CF versus non-CF cells/tissues or on proteins interacting with CFTR/F508delCFTR that may be involved in the traffic/degradation and or activity of CFTR/F508del-CFTR. The recent elucidation of murine CFTR nucleotide binding domain 1 (NBD1) called for a discussion and review of the CFTR structural aspects [5]. The role of normal and mutant CFTR on the glycosylation pattern of other proteins
and its consequences for host – bacterial interactions in the light of recently published data are also discussed in this section [6]. Novel insights on lipidic alterations in CF and possible consequences for CF deserve our attention and are also reported from a methodological point of view [7]. We believe that we were able to gather in this section (often as authors in one joint manuscript) key groups in each subarea dealt with. All articles are outlines/reviews of various protocols available at the European Working Group on CFTR Expression website [8] that should be used extensively as supplementary material. Our aim was to provide a series of methodologies that hopefully will be useful to the novice and experienced researcher alike by reducing effort and research time of all with an interest in this field.
References [1] Mendes F, Farinha CM, Roxo-Rosa M, Fanen P, Edelman A, Dormer RL, et al. Antibodies for CFTR studies. J Cyst Fibros 2004;3:69 – 72. [2] Farinha CM, Penque D, Roxo-Rosa M, Lukacs G, Dormer RL, McPherson M, et al. Biochemical methods to assess CFTR expression and membrane localization. J Cyst Fibros 2004;3:73 – 7. [3] Benos DJ, Berdiev BK, Ismailov II, Ostedgaard L, Kogan I, Li C, et al. Methods to study CFTR protein in vitro. J Cyst Fibros 2004;3:79 – 83. [4] Roxo-Rosa M, Davezac N, Bensalem N, Majumder M, Heda GD, Simas A, et al. Proteomics techniques for cystic fibrosis research. J Cyst Fibros 2004;3:85 – 9. [5] Dorwart M, Thibodeau P, Thomas P. Cystic fibrosis: recent structural insights. J Cyst Fibros 2004;3:91 – 4. [6] Rhim AD, Stoykova LI, Trindade AJ, Glick MC, Scanlin TF. Altered terminal glycosylation and the pathophysiology of CF lung disease. J Cyst Fibros 2004;3:95 – 6. [7] Ollero M. Methods for the study of lipid metabolites in cystic fibrosis. J Cyst Fibros 2004;3:97 – 8. [8] European Working Group on CFTR Expression. The Online Virtual Repository of Methods and Reagents for CFTR Expression and Functional Studies (section C). http://central.igc.gulbenkian.pt/cftr/ vr/biochemistry.html.
* Corresponding author. Tel.: +33-1-40-61-56-21; fax: +33-1-40-6155-91. E-mail addresses: [email protected] (A. Edelman), [email protected] (M.D. Amaral). 1 Tel.: +351-21-751-92-33; fax: +351-21-750-00-88. 1569-1993/$ - see front matter D 2004 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jcf.2004.05.015