Oxidative Thiol Modifications and Aging

描述（由申请人提供）：衰老的自由基理论假设活性氧和氮的积累与衰老生物体功能能力的逐渐下降有因果关系。我们现在已经开发出了测试这一假设的工具。我们将检测、监测并指定老化生物体中存在哪些氧化应激条件。我们将利用氧化蛋白硫醇修饰的独特特性来实现这一目标：对少量氧化剂的高敏感性，对不同活性氧化剂的高特异性以及体外和体内的可逆性。我们能够定量地描述在单一实验中数百种蛋白质的硫醇氧化状态的变化。这使我们能够识别在酵母老化过程中显著巯基修饰的蛋白质，并定义老化过程中产生的活性氧或氮的类型。酿酒葡萄球菌的野生型和长寿突变体将用于研究细胞遇到的氧化应激的发生、类型和程度与生物体的寿命之间是否存在联系。我们将定量描述酵母老化早期阶段的氧化蛋白修饰。这将揭示对细胞氧化还原状态的早期变化特别敏感的蛋白质。这些蛋白的功能改变可能是酵母细胞代谢和信号通路发生变化的原因。我们已经开发了基于荧光的体内氧化还原传感器，我们将使用它来检测酿酒葡萄球菌和秀丽隐杆线虫中的活性氧。这是一种非常新颖的方法，它为我们提供了一个独特的机会，可以实时了解随着生物体衰老，细胞和组织中氧化应激条件的变化。我们将从我们的研究中获得的知识可以为开发更具体的抗氧化剂奠定基础，这些抗氧化剂可以在体内对抗这些氧化剂，并可能延长寿命。

DESCRIPTION (provided by applicant): The free radical theory of aging postulates that the accumulation of reactive oxygen and nitrogen species is causally linked to the progressive decline in the functional capacity of aging organisms. We have now developed the tools to test this hypothesis. We will detect, monitor and specify which oxidative stress conditions exist in aging organisms. We will accomplish this by utilizing the unique characteristics of oxidative protein thiol modifications: high sensitivity to the presence of small amounts of oxidants, high specificity to distinct reactive oxidants and reversibility both in vitro and in vivo. We are able to quantitatively describe changes in the thiol oxidation status of hundreds of proteins in a single experiment. This allows us to identify proteins that are significantly thiol-modified during chronological yeast aging and to define the type(s) of reactive oxygen or nitrogen species that develop during aging. Wild type and longevity mutants of S. cerevisiae will be used to investigate if a link exists between the onset, type(s) and extent of oxidative stress that cells encounter, and the life span of the organism. We will quantitatively describe oxidative protein modifications at early stages of yeast aging. This will reveal proteins that are particularly sensitive to early changes in the cellular redox status. The functional alteration of these proteins might be responsible for changes observed in metabolic and signaling pathways of chronologically aging yeast cells. We have developed fluorescent-based in vivo redox sensors that we will use to detect reactive oxygen species in S. cerevisiae and C. elegans. This is a very novel approach, which provides us with the unique opportunity to obtain a real-time picture of the oxidative stress conditions that develop in cells and tissues as an organism ages. The knowledge that we will gain from our studies could form the basis for the development of more specific antioxidants that combat these oxidants in vivo and possibly extend longevity.