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 不同的活性氧物种及其在衰老和氧化中的作用 骨骼细胞内钙稳态的应激相关变化 和心肌。我们将重点放在(I)详细调查 两种主要活性氧在体外和体内的氧化 参与细胞钙调节的蛋白质：SR、Ca-ATPase和 钙调素，以及(Ii)关于分子机制的表征 哪些活性氧物种与特定的蛋白质结构域发生反应 溶剂可及性、金属结合倾向和 邻近氨基酸所产生的锚定效应。具体来说，我们 目的：(1)表征体外氧化的分子产物 羟基自由基、超氧阴离子对Ca-ATPase和CaM的影响 自由基，过氧化氢，单氧，过氧基，氧自由基， 一氧化氮、二氧化氮和过氧亚硝酸盐(过氧硝酸盐)； 蛋白质体外氧化产物模式的比较 从沙门氏菌分离蛋白中获得体内氧化产物的模式 不同定义年龄的大鼠；(3)表征 蛋白质，特别是钙调蛋白，作为溶液结构和 与目标蛋白质结合；(4)详细研究为什么某些蛋白质 结构域比其他结构域更容易被氧化。这两种技术的结合 AIMS(1)和(2)将提供关于是否和哪种反应的信息 氧物种是衰老过程中的破坏性蛋白质 有机体。这是一个重要的目标，因为不同的活性氧物种 都隐含在“衰老的自由基理论”中，但基于化学物质 研究表明，它们与靶蛋白的反应方式可能截然不同。 形成不同的分子产物。目标(3)和(4)的结合 将提供有关蛋白质序列和结构如何对 暴露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种，以及 蛋白质产物的高效液-质联用和核磁共振分析。通过这些研究，我们 设想对氧化应激的基本理解做出贡献并 在分子水平上的衰老，这一方法应该会使更多的 合理开发氧化饮食或药物疗法 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.