PROTEIN OXIDATION BY REACTIVE OXYGEN SPECIES--RELATION TO AGING

我们的长期目标是了解蛋白质氧化的机制， 不同的活性氧，以及它们在老化和氧化过程中的作用 骨骼肌细胞内钙稳态应激性变化 和心肌。 我们将集中于（i）详细调查 体外和体内两种主要的活性氧氧化 参与细胞钙调节的蛋白质：SR Ca-ATPase和 钙调素，和（ii）对表征的分子机制， 其中活性氧物质与特定的蛋白质结构域反应， 溶剂可及性、金属结合倾向和 由相邻的氨基酸产生的近聚效应。 我们特别 目的：（1）对体外氧化的分子产物进行表征 羟自由基、超氧阴离子对Ca-ATP酶和钙调素的影响 自由基，氢过氧化物，单氧，过氧自由基，氧自由基， 一氧化氮、二氧化氮和过氧硝酸盐（oxoperoxonitrate）;（2） 比较蛋白质体外氧化产物模式 从分离的蛋白质体内获得的氧化产物模式 各种规定年龄的大鼠;（3）表征氧化的 蛋白质，特别是钙调蛋白，作为溶液结构的函数， 与靶蛋白结合;（4）详细研究为什么某些蛋白质 域比其它域更容易氧化。 的组合 目标（1）和（2）将产生关于是否以及哪些反应的信息， 氧物种在老化过程中破坏蛋白质， 有机体 这是一个重要的目标，因为各种活性氧物质 在“衰老的自由基理论”中暗示，但是，基于化学 研究表明，它们与靶蛋白的反应非常不同。 形成不同的分子产物。 目标（3）和（4）的结合 将产生蛋白质序列和结构如何有助于 蛋白质的氧化敏感性和产物特异性 氧化应激 在实验上，这些研究将涉及 通过化学定义的方法产生活性氧物质， 通过HPLC/MS和NMR分析蛋白质产物。 通过这些研究，我们 设想有助于对氧化应激的基本理解， 在分子水平上老化，这种方法应该有利于更多的人， 合理开发饮食或药物疗法的氧化 压力和/或年龄相关的功能障碍。

Our long-term goal is to understand the mechanisms of protein oxidation by distinct reactive oxygen species, and their role in age- and oxidative stress-related changes of intracellular calcium homeostasis in skeletal and cardiac muscle. We shall focus on (i) a detailed investigation of the in vitro and in vivo oxidation by reactive oxygen species of two major proteins involved in cellular calcium regulation: SR Ca-ATPase and calmodulin, and (ii) on the characterization of the molecular mechanism by which reactive oxygen species react with particular protein domains as a function of solvent accessibility, metal-binding propensities, and anchimeric effects exerted by neighboring amino acids. Specifically, we aim to: (1) characterize the molecular products of the in vitro oxidation of Ca-ATPase and calmodulin by hydroxyl radicals, superoxide anion radical, hydroperoxide, single oxygen, peroxyl radicals, oxyl radicals, nitric oxide, nitrogen dioxide and peroxynitrite (oxoperoxonitrate); (2) compare the product patterns of the in vitro oxidation with protein oxidation product patterns obtained in vivo from proteins isolated from rats of various defined ages; (3) characterize the oxidation of the proteins, particularly calmodulin, as a function of solution structure and binding to target proteins; (4) investigate in detail why certain protein domains are more susceptible to oxidation than others. The combination of aims (1) and (2) will yield information on whether and which reactive oxygen species are damaging proteins in the course of aging of an organism. This is an important goal since various reactive oxygen species are implied in the "free radical theory of aging," but, based on chemical studies, they are expected to react very differently with target proteins forming different molecular products. The combination of aims (3) and (4) will yield information on how protein sequence and structure contribute to the oxidation sensitivity and product specificity in proteins exposed to oxidative stress. Experimentally, these studies will involve the generation of reactive oxygen species by chemically defined means, and the analysis of protein products by HPLC/MS and NMR. With these studies, we envisage a contribution to the basic understanding of oxidative stress and aging on the molecular level, an approach which should benefit a more rational development of dietary or pharmaceutical therapies of oxidative stress- and/or age-related dysfunctions.