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Study on the mechanism of nitrosothiol formation in biological systems

生物系统中亚硝基硫醇形成机制的研究

基本信息

批准号：
12670142
负责人：
MIYAMOTO Yoichi
金额：
$ 2.24万
依托单位：
Showa University (2002)Kumamoto University (2000-2001)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

项目摘要

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).

一氧化氮（NO）通过形成多种中间体来表现出多种生物作用。其中，我们发现亚硝基硫醇（RSNOs）是生物化合物SH部分和NO的加合物，可以通过血浆中主要含铜蛋白铜蓝蛋白催化NO的单电子氧化而高效生成。此外，我们还鉴定出s -氧化亚硝基谷胱甘肽[GS(O)NO]是谷胱甘肽和过氧亚硝酸盐（ONOO^-）的反应产物，过氧亚硝酸盐是NO和超氧化物的加合物。此外，GS(O)NO通过形成抗二硫苏糖的s -谷胱甘肽基MMPs激活基质金属蛋白酶（MMPs），表明ONOO^-通过形成s -氧亚硝基硫醇以及直接硝化或氧化生物分子来发挥其组织破坏作用。在这个项目的后半部分，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，包括类风湿关节炎（RA），其中NO的产生加速。我们可以检测到软骨细胞中铜蓝蛋白的表达，提示RA炎症灶可能形成亚硝基硫醇。另一方面，有报道称RA患者关节α_1-蛋白酶抑制剂（α_1 - pi）水平升高。正如我们之前报道的那样，α1PI很容易被NO s -硝基化，s -亚硝基化的α1PI在体内和体外都显示出组织保护作用。在本研究中，我们研究了蛋氨酸氧化α1PI [α_1PI-Met(O)]的亚硝基化反应以及该反应产物的生物活性，因为RA关节被认为处于高度氧化条件下。α_1PI-Met(O)的s -亚硝基化效果约为α1PI的80%。有趣的是，S-亚硝基化α1PI-Met(O)的体外抑菌活性是S-亚硝基化α_1PI的100倍。进一步的研究将阐明s -亚硝基化α_1 - pi - met (O)的病理生理作用。