Thioredoxin and Lipoic Acid Catalyze the Denitrosation of Low Molecular Weight and ProteinS-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.

Thioredoxin is required for S-nitrosation of procaspase-3 and the inhibition of apoptosis in Jurkat cells

“…Future work is necessary to fully uncover the role of Trx in other transnitrosation reactions of biological significance. Trx is involved in the nitrosothiol content of endothelial cells (16) and HeLa cells (29), and thus we anticipate that Trx and related proteins such as protein disulfide isomerase (30-32) will mediate index. The data were fit to a two-state folding model (Kaleidagraph), and the ⌬G of folding in water (⌬G fold°) was calculated.…”

Glutathione disulfide and S‐nitrosoglutathione detoxification by Mycobacteriumtuberculosis thioredoxin system

“…Denitrosylation of GSNO by human thioredoxins has been studied by both the Holmgren and Stoyanovsky laboratories. Holmgren's group has reported a homolytic breakdown of GSNO generating GSH and NO [27], while Stoyanovsky has proposed a heterolytic breakdown of GSNO producing GSH and nitroxyl (HNO) [28]. However both groups suggest that other cellular nitrosylated proteins may serve as substrates for the thioredoxin system.…”

Thioredoxin-interacting Protein (Txnip) Is a Feedback Regulator of S-Nitrosylation

“…The first involves S-nitrosoglutathione (GSNO) reductase (GSNOR) (32, 33), an NADH-dependent oxidoreductase that specifically breaks down GSNO (protein-SNOs are not direct substrates). Because GSH/GSNO and protein-SH/protein-SNOs are in equilibrium via rapid transnitrosation reactions (34), GSNOR lowers the levels of protein S-nitrosothiols (35, 36) reactive toward GSH, including those that are S-nitrosylated under conditions of SNO signaling (5, 36) and nitrosative stress (35).It has recently been demonstrated that the thioredoxins (Trx) constitute a second class of broad spectrum denitrosylase (6,37,38). Maintained Trx activity requires thioredoxin reductase (TrxR) (thioredoxin system), an NADPH-dependent selenoprotein.…”

“…It has recently been demonstrated that the thioredoxins (Trx) constitute a second class of broad spectrum denitrosylase (6,37,38). Maintained Trx activity requires thioredoxin reductase (TrxR) (thioredoxin system), an NADPH-dependent selenoprotein.…”