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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)通过其衍生的各种中间体的形成而发挥多种生物学作用。其中，我们发现血浆中主要的含铜蛋白质铜蓝蛋白催化NO单电子氧化生成亚硝硫醇(RSNO)，它是生物化合物的SH部分与NO的加合物。此外，我们还鉴定了S-氧代-亚硝基-谷胱甘肽[GS(O)NO]是谷胱甘肽和过氧亚硝酸根(ONOO^-)的反应产物，过氧亚硝酸根是NO和超氧化物的加合物。此外，GS(O)NO通过形成抗二硫苏糖醇的S-谷胱甘肽基金属蛋白酶(MMPs)来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来发挥其组织破坏作用。在这个项目的后半部分，我们调查了亚硝化和亚硝化应激在包括类风湿性关节炎(RA)在内的炎症性疾病中的生物学意义，在这些疾病中，NO的产生被加速。在软骨细胞中可检测到铜蓝蛋白的表达，提示类风湿关节炎炎症灶中可能存在亚硝硫醇。另一方面，有研究表明类风湿关节炎患者关节中α_1-蛋白水解酶抑制物(α_1PI)水平升高。正如我们之前报道的那样，α1PI很容易被NO S硝化，而S硝化的α1PI在体外和体内都显示出组织保护作用。在这项研究中，我们研究了蛋氨酸氧化的α1PI[α_1PI-Met(O)]的亚硝化反应及其产物的生物活性，因为RA关节被认为是在高度氧化的条件下。α_1PI-蛋氨酸(O)的S亚硝化效果约为α_1PI的80%。有趣的是，S硝化的α1PI-蛋氨酸(O)的体外抗菌活性是S硝化的α_1PI的100倍。S硝化α_1PI-蛋氨酸(O)的病理生理作用有待进一步研究。