Study on the mechanism of nitrosothiol formation in biological systems

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Nitric oxide (NO) exhibits multiple biological actions through formation of various intermediates derived from NO. Among them, we found that nitrosothiols (RSNOs), adducts of SH moiety of biological compounds and NO, could be formed efficiently via one-electron oxidation of NO catalyzed by ceruloplasmin, a major copper-containing protein in plasma. In addition, we identified S-oxiso-nitrosoglutathione [GS(O)NO] as a reaction product of glutathione and peroxynitrite (ONOO^-), an adduct of NO and superoxide. Furthermore, GS(O)NO activated matrix metalloproteinases (MMPs) through formation of dithiothreitol-resistant S-glutathionyl MMPs, indicating that ONOO^- exerts its tissue destructive effects via formation of S-oxo-nitrosothiols as well as direct nitration or oxidation of biological molecules. In the latter part of this project, we investigated the biological significance of nitrosative and nitrative stresses in inflammatory disorders including rheumatoid arthritis (RA), where NO production is accelerated. We could detect the expression of ceruloplasmin in chondrocytes, suggests the possible formation of nitrosothiols in the inflammatory foci of RA. On the other hand, it is reported that α_1-protease inhibitor (α_1PI) level in joint is increased in RA patients. As we reported earlier, α1PI is readily S-niotrosylated by NO and S-nitrosylated α1PI shows tissue protective effects in vitro and in vivo. In this study we studied nitrosylation of methionine-oxidized α1PI [α_1PI-Met(O)] and the biological activities of this reaction products, because RA joints are thought to be under highly oxidative conditions. The efficacy of S-nitrosylation of α_1PI-Met(O) was around 80% of that of α1PI. Interestingly, antibacterial activity of S- nitrosylated α1PI-Met(O) in vitro was 100 times more potent than that of S-nitrosylated α_1PI. The further study will be performed to clarify the pathophysiological roles of S-nitrosylated α_1PI-Met(O).