title: Protein thiol oxidation and formation of S-glutathionylated cyclophilin A in cells exposed to chloramines and hypochlorous acid.
abstract: Neutrophil oxidants, including the myeloperoxidase products, HOCl and chloramines, have been linked to endothelial dysfunction in inflammatory diseases such as atherosclerosis. As they react preferentially with sulfur centers, thiol proteins are likely to be cellular targets. Our objectives were to establish whether there is selective protein oxidation in vascular endothelial cells treated with HOCl or chloramines, and to identify sensitive proteins. Cells were treated with HOCl, glycine chloramine and monochloramine, reversibly oxidized cysteines were labeled and separated by 1D or 2D SDS-PAGE, and proteins were characterized by mass spectrometry. Selective protein oxidation was observed, with chloramines and HOCl causing more changes than H(2)O(2). Cyclophilin A was one of the most sensitive targets, particularly with glycine chloramine. Cyclophilin A was also oxidized in Jurkat T cells where its identity was confirmed using a knockout cell line. The product was a mixed disulfide with glutathione, with glutathionylation at Cys-161. Glyceraldehyde-3-phosphate dehydrogenase, peroxiredoxins and cofilin were also highly sensitive to HOCl/chloramines. Cyclophilins are becoming recognized as redox regulatory proteins, and glutathionylation is an important mechanism for redox regulation. Cells lacking Cyclophilin A showed more glutathionylation of other proteins than wild-type cells, suggesting that cyclophilin-regulated deglutathionylation could contribute to redox changes in HOCl/chloramine-exposed cells.
authors.affiliation: Free Radical Research Group, Department of Pathology and the National Research Centre for Growth and Development, University of Otago Christchurch, P.O. Box 4345, Christchurch, New Zealand. stacey@lunenfeld.ca
authors.forename: Melissa M
authors.initials: MM
authors.lastname: Stacey
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Cuddihy
authors.forename: Mark B
authors.initials: MB
authors.lastname: Hampton
authors.forename: Christine C
authors.initials: CC
authors.lastname: Winterbourn
mesh_terms: D051339:Actin Depolymerizing Factors / Q000378:metabolism; D000595:Amino Acid Sequence; D002700:Chloramines / Q000378:metabolism*; D021984:Cyclophilin A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D006997:Hypochlorous Acid / Q000378:metabolism*; D019169:Jurkat Cells; D008969:Molecular Sequence Data; D016877:Oxidants / Q000378:metabolism; D010084:Oxidation-Reduction; D054464:Peroxiredoxins / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism*
chemical_list: D051339:Actin Depolymerizing Factors; D002700:Chloramines; D016877:Oxidants; D013438:Sulfhydryl Compounds; D006997:Hypochlorous Acid; D006861:Hydrogen Peroxide; D054464:Peroxiredoxins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D021984:Cyclophilin A; D005978:Glutathione