REACTIVE OXYGEN SPECIES AS MEDIATORS OF SIGNAL TRANSDUCTION

我们继续探索活性氧（ROS）在信号转导途径中的作用。我们之前已经表明，多种生长因子和细胞因子在配体结合后诱导ROS的产生。我们的研究表明，导致ROS生成的途径涉及小gtp结合蛋白rac1的激活。这促使我们探索这一蛋白质家族在氧化还原调节中的作用。我们还探讨了活性氧在细胞凋亡中的作用，以及最近在复制性衰老和衰老中的作用。去年，我们已经能够证明线粒体氧化剂也能刺激信号通路。我们提出线粒体氧化剂可能在生理上起调节细胞代谢的作用。在这种情况下，代谢增加刺激线粒体氧化剂的释放将激活一条通路，反过来，这将反馈和抑制代谢，从而抑制氧化剂的产生。我们还推导了一种基因组筛选策略，以分离调节线粒体氧化剂产生并可能调节细胞和生物体寿命的基因产物。此外，今年我们建立了一个体外系统来探索热量摄入与衰老之间的关系。最后，我们试图确定氧化应激的直接目标。我们已经开发了一种新的检测方法，可以快速分离谷胱甘肽以共价方式添加的蛋白质，并通过质谱进行后续鉴定。我们正试图了解谷胱甘肽在各种信号通路中的生理相关性。使用这个系统，我们已经证明凋亡和生长因子刺激导致一组离散的蛋白质的积累，其中活性半胱氨酸已被谷胱甘肽添加共价修饰。这些蛋白的进一步鉴定将为氧化信号的特异性提供重要信息。

We have continued to explore the role that reactive oxygen species (ROS)play in signal transduction pathways. We have previously shown that a variety of growth factors and cytokines induce the generation of ROS following ligand binding. Our studies suggest that the pathway leading to ROS generation involves the activation of the small GTP-binding proteins rac1. This has led us to explore the role of this family of proteins in redox regulation. We have also explored the role that ROS play in apoptosis, and more recently in replicative senescence and aging. In the last year we have been able to demonstrate that mitochondrial oxidants also stimulate signaling pathways. We have proposed that mitochondrial oxidants may function physiologically to regulate cellular metabolism. In this scenario the release of mitochondrial oxidants stimulated by an increase in metabolism would activate a pathway , that in turn , would feedback and inhibit metabolism, and hence oxidant generation. We have also derived a genomic screening strategy to isolate gene products that regulate mitochondrial oxidant production and potentially regulate cellular and organismal life span. In addition, this year we have establish a in vitro system to explore the relationship between caloric intake and aging. Finally, we have attempted to identify direct targets of oxidant stress. We have developed a new assay to allow for the rapid isolation and subsequent identification by mass spectroscopy of proteins in which glutathione has been added in a covalent fashion. We are attempting to understand the physiological relevance of glutathiolation in various signaling pathways. Using this system we have demonstrated that both apoptotic and growth factor stimulation leads to accumulation of a discrete set of proteins in which the reactive cysteine has been covalently modified by glutathione addition. The further identification of these proteins should provide important information on the specificity of oxidant signalling.