Role of Peroxiredoxin 6 in the Repair of Peroxidized Cell Membranes

过氧化还原蛋白 6 在过氧化细胞膜修复中的作用

• 批准号: 8816964
• 负责人: Aron B. FISHER
• 金额: $ 48.72万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8816964
• 关键词: Active Sites; Acute; Acute Lung Injury; Alveolar Macrophages; Antioxidants; Binding; Biochemical; Biological Assay; Bleomycin; Cell Survival; Cell membrane; Cells; Endothelial Cells; Enzymes; Epithelial; Excision; Fatty Acids; Fibrosis; Glutathione; Glutathione Disulfide; Goals; Grant; Hydrogen Peroxide; Hyperoxia; Kinetics; Knock-in Mouse; Lead; Lentivirus Infections; Lipid Peroxidation; Literature; Lung; Lysophospholipids; Mass Spectrum Analysis; Measures; Membrane; Membrane Lipids; Metabolism; Modality; Modeling; Mus; Mutant Strains Mice; Natural regeneration; Normal Cell; Null Lymphocytes; Oxidants; Oxidative Stress; Oxygen; Pathway interactions; Peroxidases; Phase; Phospholipase A2; Phospholipids; Phosphorylation; Play; Process; Property; Proteins; Pulmonary Surfactants; Reaction; Recovery; Regulation; Relative (related person); Research; Resistance; Role; Scheme; Site-Directed Mutagenesis; Substrate Specificity; Technology; Testing; Therapeutic; Transferase; alveolar type II cell; ascorbate; base; follow-up; lung development; lung injury; mouse model; mutant; novel; oxidant stress; peroxidation; peroxiredoxin; peroxiredoxin I; prevent; programs; public health relevance; repaired; stressor; tert-Butylhydroperoxide

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.

描述（由申请人提供）：修订后的续签应用是继续研究过氧蛋白6（PRDX6）在抗氧化剂防御中的作用。在即将到来的赠款期间的重点将是PRDX6在氧化剂应激后的过氧化膜磷脂修复中的作用。从理论上讲，可以通过两个过程进行膜修复，即直接降低过氧化的磷脂（PLOOH）或需要磷脂酶A2（PLA2）和溶血磷脂酰基酰基酰基转移酶（LPAT）顺序活性的重塑途径。我们先前已经证明，PRDX6既表达了使用谷胱甘肽和PLA2活性通过过氧化物酶活性降低PLOOH的能力，并且这两种活动在肺细胞中抗氧化应激的抗性中都起着重要作用。我们最近发现PRDX6也表达LPAT活性。因此，PRDX6具有所有三种活性（过氧化物酶，PLA2，LPAT），这些活性被认为是修复过氧化细胞膜所必需的。由于文献中尚未描述PRDX6的LPAT活性，我们将研究这种新型酶促活性的特性和调节。有人提出 修复膜脂质过氧化是与氧化剂应激相关的细胞存活的关键决定因素，而表达其3种重要活动的PRDX6起着至关重要的作用。我们开发了PRDX6 NULL或通过“敲入”技术的小鼠模型表达PRDX6的过氧化物酶或PLA2/LPAT活性，并且我们从其肺部具有分离的突变细胞。 PRDX6无效细胞的慢病毒感染将产生其他突变体。我们将研究分离的肺细胞中的氧化应激（肺泡II型上皮，肺泡巨噬细胞微血管内皮细胞），孤立的灌注肺和完整的小鼠，以确定PRDX6在修复过氧化的肺细胞膜和直接还原途径和重新降低的肺部相对角色中对PRDX6的需求。肺和肺细胞中的脂质过氧化将通过质谱法或生化测定法测量，并且在去除氧化剂应激后将确定。应力源是小鼠的肺和细胞和细胞的TERT叔丁基氢过氧化物或Cu2+/抗坏血酸。我们还将对小鼠进行博来霉素的施用，以研究急性氧化剂应激与随后的肺纤维化发展后膜修复的关系。这些研究应提供与从氧化应激中恢复肺有关的重要新信息，并可能导致治疗方式增加恢复过程。