Identification of S-glutathionylated cellular proteins during oxidative stress and constitutive metabolism by affinity purification and proteomic analysis.
Redox modification of proteins is proposed to play a central role in regulating cellular function. However, high-throughput techniques for the analysis of the redox status of individual proteins in complex mixtures are lacking. The aim was thus to develop a suitable technique to rapidly identify proteins undergoing oxidation of critical thiols by S-glutathionylation. The method is based on the specific reduction of mixed disulfides by glutaredoxin, their reaction with N-ethylmaleimide-biotin, affinity purification of tagged proteins, and identification by proteomic analysis. The method unequivocally identified 43 mostly novel cellular protein substrates for S-glutathionylation. These include protein chaperones, cytoskeletal proteins, cell cycle regulators, and enzymes of intermediate metabolism. Comparisons of the patterns of S-glutathionylated proteins extracted from cells undergoing diamide-induced oxidative stress and during constitutive metabolism reveal both common protein substrates and substrates failing to undergo enhanced S-glutathionylation during oxidative stress. The ability to chemically tag, select, and identify S-glutathionylated proteins, particularly during constitutive metabolism, will greatly enhance efforts to establish posttranslational redox modification of cellular proteins as an important biochemical control mechanism in coordinating cellular function.
D002460:Cell Line; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D003545:Cysteine / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D054477:Glutaredoxins; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000502:physiology*; D010088:Oxidoreductases; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D020543:Proteome; D014471:Umbilical Veins / Q000187:drug effects / Q000378:metabolism
D054477:Glutaredoxins; D011506:Proteins; D020543:Proteome; D010088:Oxidoreductases; D005978:Glutathione; D003545:Cysteine; D005033:Ethylmaleimide