Subcellular analysis of the disulfide proteome in mammlian cells

由于正常的细胞代谢和细胞因子的炎症，我们体内的细胞不断暴露于潜在的有害分子活性氧（ROS）中。细胞检测ROS水平并以适当的方式作出反应是至关重要的。最近对酵母和细菌的研究表明，细胞内的某些蛋白质可以通过含硫氨基酸“感知”ROS，这些氨基酸可以被ROS直接氧化，并进行称为二硫键的化学连接。二硫键的形成导致关键蛋白质的激活，这些蛋白质可以增加基因的表达，这些基因要么参与保护性抗氧化反应，要么在活性氧水平高的情况下启动细胞死亡。新出现的证据表明，类似的二硫键控制激活系统也可能发生在哺乳动物细胞中。ROS产生于细胞膜和称为线粒体的特殊细胞器中，可以触发导致细胞核基因表达改变的信号事件。线粒体和细胞核内发生二硫键以响应ROS升高的确切蛋白质类型尚不清楚。利用模拟生理条件的小鼠细胞培养模型，结合一种新的蛋白质分离技术，我们将确定线粒体和细胞核中受ROS水平增加影响的二硫化物连接蛋白的光谱。最初的研究表明，一种主要的抗氧化蛋白可以通过二硫键与参与发育和DNA修复的核蛋白相互作用。鉴定和分析ROS传感蛋白将提供洞察氧化细胞条件如何影响正常和疾病相关的信号过程。

Cells in our bodies are continually exposed to potentially harmful molecules called reactive oxygen species (ROS) as a result of normal cellular metabolism and exposure to inflammatory factors called cytokines. It is essential for cells to detect ROS levels and respond in an appropriate manner. Recent studies in yeast and bacteria have shown that certain proteins within the cell can "sense" ROS by virtue of sulfur containing amino acids which can be directly oxidized by ROS and undergo a chemical linkage called a disulfide bond. The formation of disulfide bonds leads to activation of key proteins which can then function to increase expression of genes that either participate in a protective anti-oxidant response or, if the levels of ROS are high, initiate cell death. Emerging evidence indicates that a similar disulfide-bond controlled activation system may also occur in mammalian cells as well. ROS, produced at the cell membrane and in specialized organelles called mitochondria, can trigger signaling events leading to altered gene expression in the nucleus. The exact type of proteins within mitochondria and nuclei that undergo disulfide bonding in response to elevated ROS is poorly understood. Using mouse cell culture models that mimic physiological conditions, combined with a novel protein separation technique, we will determine the spectrum of disulfide linked proteins in mitochondria and nuclei that are affected by increased ROS levels. Initial studies have revealed that a major anti-oxidant protein can interact, via disulfide bonding, with nuclear proteins involved in development and DNA repair. Identification and analysis of ROS sensing proteins will provide insight into how oxidative cellular conditions influence both normal and disease-associated signaling processes.