REACTIVE OXYGEN AND NITROGEN SPECIES IN RAW 2647 MACROPHAGE CELLS

原始 2647 巨噬细胞中的活性氧和氮物种

• 批准号: 7359114
• 负责人: HEATHER S SMALLWOOD
• 金额: $ 4.2万
• 依托单位: BATTELLE PACIFIC NORTHWEST LABORATORIES
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7359114
• 关键词: REACTIVE; OXYGEN; NITROGEN; SPECIES; RAW

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.

该子项目是利用 NIH/NCRR 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中得到体现。列出的机构是中心的机构，不一定是研究者的机构。巨噬细胞在免疫系统中发挥重要作用，通过在杀死细菌的过程中改变巨噬细胞蛋白的活性氧和氮自由基来消灭入侵的病原体。因此，我们诱导了巨噬细胞中自由基的产生（激活），以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物中的发现，我们将纯硝化his标记蛋白引入系统中，并回收该蛋白以对氧化和硝化的变化进行光谱定量。我们发现与裂解物蛋白一起孵育后纯蛋白中硝基酪氨酸大量损失。因此，巨噬细胞中自由基生成的诱导刺激了纯蛋白质修饰清除的增加。使用 12 特斯拉 FTICR 的完整蛋白质质谱分析将使我们能够查明修饰清除的性质，无论是从硝基酪氨酸逆转为酪氨酸、还原为氨基酪氨酸还是细胞降解机制造成的裂解。观察到的巨噬细胞中整体硝化蛋白质的变化无疑会改变蛋白质复合物，并对细胞的活力产生影响。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，而且还将揭示细胞内蛋白质硝化的动态。