title: Regulation of redox-sensitive exofacial protein thiols in CHO cells.
abstract: Thiols affect a variety of cell functions, an effect known as redox regulation, largely attributed to modification of transcription factors and intracellular signaling mechanisms. Since exofacial protein thiols are more exposed to redox-acting molecules used in cell culture and may represent sensors of the redox state of the environment, we investigated their susceptibility to redox regulation. Exofacial protein thiols were measured using cell-impermeable Ellman's reagent [5,5'-dithiobis(2-nitrobenzoic acid), DTNB]. For quantification, we also set up an ELISA assay based on the cell-impermeable biotinylated SH reagent, N-(biotinoyl)-N-(iodoacetyl) ethylendiamine (BIAM). Exposure of CHO cells to H(2)O(2) induces oxidation of surface thiols at concentrations not affecting intracellular GSH. Depletion (50%) of GSH decreases surface thiols by 88%. Surface thiols are also highly sensitive to thiol antioxidants, since exposure to 5 mM N-acetyl-L-cysteine (NAC) for 2 h augmented their expression without increasing GSH levels. Using BIAM labeling and two-dimensional gel electrophoresis, we show that this increase in surface thiols is due to the reduction of specific membrane proteins. Peptide mass fingerprinting by MALDI mass spectrometry allowed us to identify two of these proteins as Erp57 and vimentin.
authors.affiliation: Istituto di Ricerche Farmacologiche Mario Negri, I-20157 Milan, Italy.
authors.forename: Teresa
authors.initials: T
authors.lastname: Laragione
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Gianazza
authors.forename: Rossella
authors.initials: R
authors.lastname: Tonelli
authors.forename: Paolo
authors.initials: P
authors.lastname: Bigini
authors.forename: Tiziana
authors.initials: T
authors.lastname: Mennini
authors.forename: Filippo
authors.initials: F
authors.lastname: Casoni
authors.forename: Tania
authors.initials: T
authors.lastname: Massignan
authors.forename: Valentina
authors.initials: V
authors.lastname: Bonetto
authors.forename: Pietro
authors.initials: P
authors.lastname: Ghezzi
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D005978:Glutathione / Q000378:metabolism; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D011506:Proteins / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism*
chemical_list: D000975:Antioxidants; D011506:Proteins; D013438:Sulfhydryl Compounds; D005978:Glutathione