Role of Peroxiredoxin 6 in the Repair of Peroxidized Cell Membranes

描述（由申请人提供）：本修订后的更新申请旨在继续研究过氧化物氧还蛋白6（Prdx 6）在抗氧化防御中的作用。在接下来的资助期间，重点将放在Prdx 6在氧化应激后过氧化膜磷脂修复中的作用上。理论上，膜修复可以通过两个过程发生，直接还原过氧化磷脂（PLOOH）或需要磷脂酶A2（PLA 2）和溶血磷脂酰基转移酶（LPAT）连续活性的重塑途径。我们先前已经表明，Prdx 6表达通过利用谷胱甘肽的过氧化物酶活性以及PLA 2活性来还原PLOOH的能力，并且这两种活性在肺细胞中抗氧化应激中起重要作用。我们最近发现Prdx 6也表达LPAT活性。因此，Prdx 6具有所有三种活性（过氧化物酶、PLA 2、LPAT），这三种活性被认为是修复过氧化细胞膜所需的。由于Prdx 6的LPAT活性尚未在文献中描述，因此我们将研究这种新型酶活性的性质和调节。拟议将 膜脂质过氧化的修复是与氧化应激相关的细胞存活的关键决定因素，并且表达其3种重要活性的Prdx 6起关键作用。我们已经开发出Prdx 6无效或通过“敲入”技术表达Prdx 6的过氧化物酶或PLA 2/LPAT活性的小鼠模型，并且我们已经从它们的肺中分离出突变细胞。将通过Prdx 6缺失细胞的慢病毒感染产生另外的突变体。我们将研究氧化应激在离体肺细胞（肺泡II型上皮细胞，肺泡巨噬细胞微血管内皮细胞），离体灌注肺，和完整的小鼠，以确定需要Prdx 6在修复过氧化的肺细胞膜和直接还原和reacylation途径的相对作用。肺和肺细胞中的脂质过氧化将通过质谱或生物化学测定来测量，并且将在去除氧化应激后确定修复。对于肺和细胞，应激源将是叔丁基过氧化氢或Cu 2 +/抗坏血酸盐，而对于小鼠，应激源将是高氧。我们还将对小鼠施用博来霉素，以研究急性氧化应激后膜修复与随后肺纤维化发展的关系。这些研究应提供重要的新信息，有关恢复肺氧化应激，并可能导致治疗方式，以增加恢复过程。

DESCRIPTION (provided by applicant): This revised renewal application is to continue studies of the role of peroxiredoxin 6 (Prdx6) in antioxidant defense. The focus during the coming grant period will be on the role of Prdx6 in the repair of peroxidized membrane phospholipids following oxidant stress. Theoretically, membrane repair can occur through two processes, direct reduction of peroxidized phospholipid (PLOOH) or a remodeling pathway requiring the sequential activity of phospholipase A2 (PLA2) and lysophospholipid acyl transferase (LPAT). We have shown previously that Prdx6 expresses both the ability to reduce PLOOH by a peroxidase activity utilizing glutathione as well as a PLA2 activity, and that both of these activities play an important role in resistance to oxidative stress in lung cells. We have recently discovered that Prdx6 also expresses LPAT activity. Therefore, Prdx6 has all three activities (peroxidase, PLA2, LPAT) that are assumed to be required for repair of peroxidized cell membranes. As the LPAT activity of Prdx6 is as yet undescribed in the literature, we will investigate the properties and regulation of this novel enzymatic activity. It is proposed that the repair of membrane lipid peroxidation is a key determinant for cell survival associated with oxidant stress and that Prdx6 expressing its 3 important activities plays a crucial role. We have developed mouse models that are Prdx6 null or, through "knock-in" technology, express either the peroxidase or the PLA2/LPAT activities of Prdx6 and we have isolated mutant cells from their lungs. Additional mutants will be generated by lentivirus infection of Prdx6 null cells. We will study oxidant stress in isolated lung cells (alveolar type II epithelial, alveolar macrophages microvascular endothelial cells), isolated perfused lungs, and intact mice in order to determine the requirement for Prdx6 in repair of peroxidized lung cell membranes and the relative roles of the direct reduction and the reacylation pathways. Lipid peroxidation in lungs and lung cells will be measured by mass spectroscopy or biochemical assay and repair will be determined following removal of the oxidant stress. The stressors will be tert-butyl hydroperoxide or Cu2+/ ascorbate for lungs and cells and hyperoxia for mice. We also will administer bleomycin to mice to investigate the relationship of membrane repair following acute oxidant stress to the subsequent development of lung fibrosis. These studies should provide important novel information related to recovery of lungs from oxidative stress and could lead to therapeutic modalities to augment the recovery process.