REACTIVE OXYGEN AND NITROGEN SPECIES IN RAW 2647 MACROPHAGE CELLS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。巨噬细胞在杀死细菌的过程中，通过活性氧和活性氮自由基修饰巨噬细胞蛋白，破坏入侵的病原体，在免疫系统中发挥重要作用。因此，我们在巨噬细胞中诱导自由基生成（激活）来观察氧化应激对细胞内蛋白质和蛋白质复合物的影响。我们在免疫印迹上观察到硝化蛋白的增加和随后的清除。为了验证我们在全蛋白裂解液中的发现，我们将一种纯硝化的标记蛋白引入系统，并回收蛋白质以光谱量化氧化和硝化的变化。我们发现在与裂解蛋白孵育后，纯蛋白中有大量的硝基酪氨酸损失。因此，巨噬细胞中自由基生成的诱导刺激了纯蛋白修饰清除的增加。使用12特斯拉FTICR的完整蛋白质质谱分析将使我们能够确定修饰清除的性质，无论是从硝基酪氨酸还原到酪氨酸，还原到氨基酪氨酸还是细胞降解机制的裂解。观察到的巨噬细胞中全局硝化蛋白的变化无疑会改变蛋白质复合物，并对细胞的生存能力产生影响。因此，深入表征与氧化应激相关的关键复合物的变化不仅将揭示巨噬细胞活化的机制，还将揭示细胞内蛋白质硝化的动力学。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Macrophages play an important role in the immune system by destroying invading pathogens with reactive oxygen and nitrogen radicals capable of modifying macrophage proteins in the process of killing bacteria. Thus we have induced radical generation in macrophages (activation) to look at the effects oxidative stress has on intracellular proteins and protein complexes. We observed an increase and subsequent clearance of nitrated proteins on immunoblot. To validate our findings in whole protein lysate we have introduced a pure nitrated his tagged protein into the system and recovered the protein to spectrally quantify the changes in oxidation and nitration. We find a substantial loss of nitrotyrosine in the pure protein post incubation with the lysate proteins. Thus, induction of radical generation in macrophages stimulates an increase in clearance of modifications to pure protein. Intact protein mass spectrometry with the 12 tesla FTICR will allow us to pinpoint the nature of the modification clearance, be it reversion from nitrotyrosine to tyrosine, reduction to amino tyrosine or cleavage by cellular degradation machinery. The observed changes in globally nitrated proteins in the macrophage will undoubtedly alter the protein complexes and have implications for the viability of the cell. Thus, an in depth characterization of the changes in key complexes associated with oxidative stress will shed light not only on the mechanisms of macrophage activation but also on dynamics of protein nitration within the cell.