Oxidative Thiol Modifications and Aging

描述(申请人提供)：衰老的自由基理论假设，活性氧和氮物种的积累与衰老生物体功能能力的逐渐下降有因果关系。我们现在已经开发了检验这一假设的工具。我们将检测、监测和明确衰老生物体中存在哪些氧化应激条件。我们将利用氧化蛋白质硫醇修饰的独特特性来实现这一点：对少量氧化剂的存在具有高敏感性，对不同的反应性氧化剂具有高度的特异性，以及在体外和体内的可逆性。我们能够在一次实验中定量描述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陈化过程中显著的硫醇修饰的蛋白质，并确定在老化过程中形成的活性氧或氮物种的类型(S)。酿酒酵母的野生型和长寿突变体将被用来研究细胞遇到的氧化应激的开始、类型(S)和程度与有机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质修饰。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。这些蛋白的功能变化可能与观察到的时间老化酵母细胞代谢和信号通路的变化有关。我们已经开发了基于荧光的在体氧化还原传感器，我们将使用它来检测酿酒酵母和线虫中的活性氧物种。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老化而在细胞和组织中发展的氧化应激条件。我们将从我们的研究中获得的知识可能会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.