Role of Peroxiredoxin 6 in the Repair of Peroxidized Cell Membranes

描述（由申请人提供）：本次修订的续期申请是为了继续研究过氧化物还氧蛋白6 （Prdx6）在抗氧化防御中的作用。在即将到来的拨款期间，重点将放在Prdx6在氧化应激后过氧化膜磷脂修复中的作用。从理论上讲，膜修复可以通过两个过程发生，即直接还原过氧化磷脂（PLOOH）或需要磷脂酶A2 （PLA2）和溶血磷脂酰基转移酶（LPAT）顺序活性的重塑途径。我们之前已经证明，Prdx6通过利用谷胱甘肽的过氧化物酶活性和PLA2活性来表达减少PLOOH的能力，并且这两种活性在肺细胞抵抗氧化应激中发挥重要作用。我们最近发现Prdx6也表达LPAT活性。因此，Prdx6具有修复过氧化细胞膜所需的所有三种活性（过氧化物酶，PLA2， LPAT）。由于Prdx6的LPAT活性尚未在文献中描述，我们将研究这种新型酶活性的性质和调控。有人建议

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.