Study on the mechanism of nitrosothiol formation in biological systems

一氧化氮（NO）具有多种生物学作用，可通过形成多种NO衍生中间体而发挥作用。其中，我们发现血浆中主要的含铜蛋白铜蓝蛋白可催化NO单电子氧化生成亚硝基硫醇（RSNOs），RSNOs是生物化合物的巯基与NO的加合物。此外，我们还鉴定了S-氧代异亚硝基谷胱甘肽[GS（O）NO]是谷胱甘肽与过氧亚硝酸盐（ONOO^-）的反应产物，后者是NO与超氧化物的加合物。此外，GS（O）NO通过形成抗二硫苏糖醇的S-谷胱甘肽MMPs激活基质金属蛋白酶（MMPs），表明ONOO^-通过形成S-氧代-亚硝基硫醇以及生物分子的直接硝化或氧化发挥其组织破坏作用。在本项目的后半部分，我们研究了亚硝化和硝化应激在炎症性疾病中的生物学意义，包括类风湿性关节炎（RA），其中NO的产生加速。我们可以检测到铜蓝蛋白在软骨细胞中的表达，提示在RA的炎症灶中可能有亚硝基硫醇的形成。另一方面，有研究表明RA患者关节组织中α_1-蛋白酶抑制剂（α_1PI）水平升高。正如我们先前所报道的，α 1 PI很容易被NO S-亚硝基化，S-亚硝基化的α 1 PI在体外和体内都显示出组织保护作用。由于RA关节被认为处于高度氧化的环境中，本研究对甲硫氨酸氧化的α 1 PI [α 1 PI-Met（O）]的亚硝基化反应及其产物的生物活性进行了研究。α 1 PI Met（O）的S-亚硝基化效率约为α 1 PI的80%。有趣的是，S-亚硝基化的α_1PI-Met（O）的体外抗菌活性是S-亚硝基化的α_1PI的100倍。对S-亚硝基化α_1 PI-Met（O）的病理生理作用有待进一步研究。

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).