Subcellular analysis of the disulfide proteome in mammlian cells

由于正常的细胞代谢和暴露在称为细胞因子的炎症因子中，我们体内的细胞不断地暴露在称为活性氧(ROS)的潜在有害分子中。对细胞来说，检测ROS水平并以适当的方式做出反应是至关重要的。最近在酵母和细菌中的研究表明，细胞内的某些蛋白质可以通过含硫的氨基酸“感觉”ROS，这些氨基酸可以被ROS直接氧化，并经历一种称为二硫键的化学键。二硫键的形成导致关键蛋白的激活，然后这些蛋白可以功能地增加基因的表达，这些基因要么参与保护性的抗氧化反应，要么如果ROS水平高，则启动细胞死亡。新出现的证据表明，在哺乳动物细胞中也可能存在类似的二硫键控制的激活系统。在细胞膜和称为线粒体的特殊细胞器中产生的ROS可以触发信号事件，导致细胞核中基因表达的改变。线粒体和细胞核中因ROS升高而发生二硫键反应的蛋白质的确切类型尚不清楚。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键连接蛋白的光谱。初步研究表明，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。ROS传感蛋白的鉴定和分析将有助于深入了解氧化细胞条件如何影响正常和疾病相关的信号传递过程。

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.