Peroxiredoxin 6 as an Anti-oxidant Enzyme

该项目将评估一种新型谷胱甘肽过氧化物酶的性质，这种酶在肺中相当丰富。这种最近描述的蛋白质（称为1-cys Peroxiredoxin或Peroxiredoxin 6，Prdx 6）代表了唯一具有相对高比活性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的能力，不像经典的谷胱甘肽过氧化物酶，减少磷脂氢过氧化物。在目前作为R-01的资助支持期间，我们已经证明了在高氧应激期间对Prdx 6的诱导，并提供了该酶在细胞培养和整个动物研究中保护免受氧化应激的证据。此外，我们还开发了从大鼠和牛肺中分离Prdx 6蛋白的方法，并建立了产生活性表达的表达系统。 重组蛋白我们推测，氧化膜磷脂的还原是酶的抗氧化作用的基础。具体目的我将利用小鼠模型的改变Prdx 6的表达，继续我们的研究Prdx 6在抗氧化防御的作用。其中一个模型，Prdx 6基因敲除小鼠，是在目前的赠款支持期间开发的。第二个具体目标将研究Prdx 6表达的控制，特别强调抗氧化反应元件和Nrf 2。具体目标3将评价底物-蛋白质相互作用，特别关注蛋白质的磷脂结合，这是磷脂氢过氧化物酶活性的要求。这些研究将 利用基于从酶的晶体结构推导的定点诱变。具体目标4将利用细胞系统分析nGST在Prdx 6活性激活中的作用。拟议的研究将提供一个协调的努力，调查这种新的抗氧化酶在肺防御氧化应激的作用，并将提供有关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.