Peroxiredoxin 6 as an Anti-oxidant Enzyme

该项目将评估一种在肺部相当丰富的新型谷胱甘肽过氧化物酶的性质。这种最近被发现的蛋白质(称为1-半胱氨酸过氧化还蛋白或过氧化还蛋白6，PRDX 6)是唯一一种比活性相对较高的非硒谷胱甘肽过氧化物酶。这种酶的光谱活性的一个重要特征是它不同于经典的谷胱甘肽过氧化物酶，它有能力还原磷脂过氧化氢。在目前作为R-01的赠款支持期间，我们已经证明了在高氧应激期间PRDX 6的诱导，并在细胞培养和整体动物研究中提供了该酶保护免受氧化应激的证据。此外，我们开发了从大鼠和牛肺中分离PrDX 6蛋白的方法，并建立了产生活性蛋白的表达系统。 重组蛋白。我们推测，氧化膜磷脂的还原是该酶抗氧化作用的基础。具体目的I将利用PRDX 6表达改变的小鼠模型来继续我们对PRDX 6在抗氧化防御中的作用的研究。其中一种模型，PRDX 6基因敲除小鼠，是在目前的赠款支持期间培育出来的。第二个特定目标将研究PRDX 6表达的控制，特别强调抗氧化反应元件和NRF 2。特定目标3将评估底物与蛋白质的相互作用，特别关注蛋白质与磷脂的结合作为磷脂氢过氧化物酶活性的要求。这些研究将 利用基于酶晶体结构推断的定点突变。特殊目的4将利用细胞系统分析nGST在激活PRDX 6活性中的作用。这些研究将为研究这种新的抗氧化酶在肺对氧化应激防御中的作用提供协调的努力，并将提供关于其酶活性的生化调节的新信息。

This project will evaluate the properties of a novel glutathione peroxidase enzyme that is considerably enriched in lungs. This recently described protein (called 1-cys Peroxiredoxin or Peroxiredoxin 6, Prdx 6) represents the only non-selenium glutathione peroxidase of relatively high specific activity. An important characteristic in the spectrum activity for this enzyme is its ability, unlike classical glutathione peroxidase, to reduce phospholipid hydroperoxides. During the present grant Support as an R-01, we have demonstrated induction of Prdx 6 during hyperoxic stress and provided evidence that the enzyme protects against oxidant stress in cell culture and whole animal studies. Further, we have developed methods for isolation of Prdx 6 protein from rat and bovine lungs and have established expression systems for generation of active recombinant protein. We postulate that reduction of oxidized membrane phospholipids represents the basis for the anti-oxidant effect of the enzyme. Specific Aim I will utilize mouse models of altered Prdx 6 expression to continue our studies of the role of Prdx 6 in anti-oxidant defense. One of the models, Prdx 6 knock-out mice, was developed during the present period of grant support. The second Specific Aim will investigate control of Prdx 6 expression with special emphasis on the anti-oxidant response element and Nrf 2. Specific Aim 3 will evaluate substrate-protein interactions with a special focus on phospholipid binding by the protein as a requirement for the phospholipid hydroperoxide peroxidase activity. These studies will utilize site directed mutagenesis based on deduction from the crystal structure of the enzyme. Specific Aim 4 will utilize cellular systems for analysis of the role of nGST in activation of Prdx 6 activity. The proposed studies will provide a coordinated effort to investigate the role of this novel anti-oxidant enzyme in lung defense against oxidant stress and will provide new information concerning the biochemical regulation of its enzymatic activity.