title: Thioredoxin and lipoic acid catalyze the denitrosation of low molecular weight and protein S-nitrosothiols.
abstract: The nitrosation of cellular thiols has attracted much interest as a regulatory mechanism that mediates some of the pathophysiological effects of nitric oxide (NO). In cells, virtually all enzymes contain cysteine residues that can be subjected to S-nitrosation, whereby this process often acts as an activity switch. Nitrosation of biological thiols is believed to be mediated by N2O3, metal-nitrosyl complexes, and peroxynitrite. To date, however, enzymatic pathways for S-denitrosation of proteins have not been identified. Herein, we present experimental evidence that two ubiquitous cellular dithiols, thioredoxin and dihydrolipoic acid, catalyze the denitrosation of S-nitrosoglutathione, S-nitrosocaspase 3, S-nitrosoalbumin, and S-nitrosometallothionenin to their reduced state with concomitant generation of nitroxyl (HNO), the one-electron reduction product of NO. In these reactions, formation of NO and HNO was assessed by ESR spectrometry, potentiometric measurements, and quantification of hydroxylamine and sodium nitrite as end reaction products. Nitrosation and denitrosation of caspase 3 was correlated with its proteolytic activity. We also report that thioredoxin-deficient HeLa cells with mutated thioredoxin reductase denitrosate S-nitrosothiols less efficiently. We conclude that both thioredoxin and dihydrolipoic acid may be involved in the regulation of cellular S-nitrosothiols.
authors.affiliation: Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA. stoyanovskyd@upmc.edu
authors.forename: Detcho A
authors.initials: DA
authors.lastname: Stoyanovsky
authors.forename: Yulia Y
authors.initials: YY
authors.lastname: Tyurina
authors.forename: Vladimir A
authors.initials: VA
authors.lastname: Tyurin
authors.forename: Deepthi
authors.initials: D
authors.lastname: Anand
authors.forename: Dhara N
authors.initials: DN
authors.lastname: Mandavia
authors.forename: David
authors.initials: D
authors.lastname: Gius
authors.forename: Juliana
authors.initials: J
authors.lastname: Ivanova
authors.forename: Bruce
authors.initials: B
authors.lastname: Pitt
authors.forename: Timothy R
authors.initials: TR
authors.lastname: Billiar
authors.forename: Valerian E
authors.initials: VE
authors.lastname: Kagan
mesh_terms: D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002384:Catalysis; D004578:Electron Spin Resonance Spectroscopy; D006367:HeLa Cells; D006801:Humans; D019811:Hydroxylamine / Q000032:analysis; D008970:Molecular Weight; D009154:Mutation; D009569:Nitric Oxide / Q000378:metabolism; D009589:Nitrogen Oxides / Q000378:metabolism; D015538:Nitrosation; D009603:Nitroso Compounds / Q000378:metabolism; D011199:Potentiometry; D011506:Proteins / Q000378:metabolism*; D026422:S-Nitrosoglutathione / Q000378:metabolism; D026403:S-Nitrosothiols / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000378:metabolism; D012977:Sodium Nitrite / Q000032:analysis; D008063:Thioctic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D013880:Thioredoxin-Disulfide Reductase / Q000235:genetics / Q000378:metabolism; D013879:Thioredoxins / Q000378:metabolism*
chemical_list: D009589:Nitrogen Oxides; D009603:Nitroso Compounds; D011506:Proteins; D026403:S-Nitrosothiols; C091665:S-nitrosoalbumin; D012710:Serum Albumin, Bovine; D019811:Hydroxylamine; D009569:Nitric Oxide; D013879:Thioredoxins; D026422:S-Nitrosoglutathione; D008063:Thioctic Acid; C015144:dihydrolipoic acid; D013880:Thioredoxin-Disulfide Reductase; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; C039900:nitroxyl; D012977:Sodium Nitrite