The Saccharomyces cerevisiae proteome of oxidized protein thiols: contrasted functions for the thioredoxin and glutathione pathways.
Protein thiol oxidation subserves important biological functions and constitutes a sequel of reactive oxygen species toxicity. We developed two distinct thiol-labeling approaches to identify oxidized cytoplasmic protein thiols in Saccharomyces cerevisiae. Inone approach, we used N-(6-(biotinamido)hexyl)-3'-(2'-pyridyldithio)-propionamide to purify oxidized protein thiols, and in the other, we used N-[(14)C]ethylmaleimide to quantify this oxidation. Both approaches showed a large number of the same proteins with oxidized thiols ( approximately 200), 64 of which were identified by mass spectrometry. We show that, irrespective of its mechanism, protein thiol oxidation is dependent upon molecular O(2). We also show that H(2)O(2) does not cause de novo protein thiol oxidation, but rather increases the oxidation state of a select group of proteins. Furthermore, our study reveals contrasted differences in the oxidized proteome of cells upon inactivation of the thioredoxin or GSH pathway suggestive of very distinct thiol redox control functions, assigning an exclusive role for thioredoxin in H(2)O(2) metabolism and the presumed thiol redox buffer function for GSH. Taken together, these results suggest the high selectivity of cytoplasmic protein thiol oxidation.
Le Moan
Natacha
N
Laboratoire Stress Oxydants et Cancer, Service de Biologie Moléculaire Systémique, Département de Biologie Joliot-Curie, CEA-Saclay, F-91191 Gif-sur-Yvette Cedex, France.
Clement
Gilles
G
Le Maout
Sophie
S
Tacnet
Frédérique
F
Toledano
Michel B
MB
D001710:Biotin / Q000737:chemistry; D015153:Blotting, Western; D002241:Carbohydrates / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism; D004220:Disulfides; D004229:Dithiothreitol / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D005978:Glutathione / Q000737:chemistry*; D019803:Glutathione Disulfide / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D008956:Models, Chemical; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D017382:Reactive Oxygen Species; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013438:Sulfhydryl Compounds; D013879:Thioredoxins / Q000737:chemistry*
D002241:Carbohydrates; D004220:Disulfides; D017382:Reactive Oxygen Species; D029701:Saccharomyces cerevisiae Proteins; D013438:Sulfhydryl Compounds; D013879:Thioredoxins; D001710:Biotin; D006861:Hydrogen Peroxide; D005978:Glutathione; D010100:Oxygen; D004229:Dithiothreitol; D019803:Glutathione Disulfide