Effect of protein nitration on peri-gingivitis and inflammatory regulation mechanism involving nitration and de-nitration.

为了阐明蛋白质硝化在炎症中的作用，我们用脂多糖、肿瘤坏死因子和过氧亚硝基阴离子刺激培养的巨噬细胞系（RAW 267），发现类固醇激素受体超家族之一的过氧化物酶体增殖物激活受体γ（PPARγ）被强烈硝化。此外，在配体依赖性机制中，硝化受体不能作为转录因子进入细胞核。此外，在炎症状态下，如炎症诱导的关节炎和缺血再灌注诱导的结肠炎，甚至在肝炎引起的人类肝脏中，这种受体在动物模型的各种组织中被硝化。另一方面，炎症过程中的硝化蛋白与细胞死亡（凋亡）有关。我们发现钒酸盐能抑制过氧亚硝基阴离子诱导的SH-SY 5 Y细胞死亡;抗炎药双氯芬酸通过抑制细胞生长和分化而导致神经干细胞凋亡;新发现的钙结合蛋白对凋亡最后阶段的caspase-3起调控作用。这些数据表明磷酸化和硝化系统之间存在相互作用。我们还探索了通过去硝化恢复受损蛋白质功能的机制，并发现硝化蛋白质可能是酶促消失的，尽管它可能是催化硝化氨基酸特定序列的肽酶之一。因此，尽管由于限制，我们无法获得牙龈炎的人类样本，阐明了硝化作用参与炎症过程中的调节系统，并可能是调节炎症的药物的可能靶点。

In order to clarify the involvement of protein nitration in inflammation, we stimulated cultured cell lines from macrophages (RAW267) by lipopolysaccharide, tumor necrosis factor and peroxynitrite and found that peroxisome proliferator-activated receptor γ(PPARγ), one of steroid hormone receptor superfamily is strongly nitrated. Moreover, the nitrated receptors fail to move into nucleus as a transcription factor in the ligand-dependent mechanism. In addition, this receptor is nitrated in various tissues of animal models during inflammatory condition such as adjuvant-induced arthritis and ischemia-reperfusion induced intestinitis, and even in human liver from hepatitis.On the other hand, nitrated proteins during inflammation are related to cell death (apoptosis). We found that vanadate inhibits peroxynitrite-induced cell death of SH-SY5Y, that the treatment of neural stem cells with diclofenac, one of anti-inflammatory agents, results in apoptosis by inhibition of cell growth and differentiation, and that caspase-3, which works at the final stage of apoptosis, is regulated by newly found calcium binding proteins. These data indicate the existence of interaction between phosphorylation and nitration systems.We also searched the mechanism to restore the impaired function of protein by de-nitration and revealed that there is an presumably enzymatic disappearance of nitrated proteins, although it may be one of peptidases which catalyze the specific sequence of nitrated amino acids.Therefore, although we could not obtain human samples of gingivitis due to the restriction, the nitration is clarified to be involved in the regulatory system during inflammation and may be a possible target of drugs to regulate inflammation.