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: 9237295
• 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/9237295
• 关键词: Active Sites; Acute; Acute Lung Injury; Alpha Cell; Alveolar; Alveolar Macrophages; Antioxidants; Binding; Biochemical; Biological Assay; Bleomycin; Cell Survival; Cell membrane; Cells; Endothelial Cells; Enzymes; Epithelial; Excision; Fatty Acids; Glutathione; Glutathione Disulfide; Goals; Grant; Hydrogen Peroxide; Hyperoxia; Kinetics; Knock-in; Lead; Lentivirus Infections; Lipid Peroxidation; Lipid Peroxides; 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; Oxides; Oxygen; Pathway interactions; Peroxidases; Peroxides; Phase; Phospholipase A2; Phospholipases A; Phospholipids; Phosphorylation; Play; Process; Property; Proteins; Pulmonary Fibrosis; Pulmonary Surfactants; Reaction; Recovery; Regulation; Research; Resistance; Role; Scheme; Site-Directed Mutagenesis; Substrate Specificity; Technology; Testing; Therapeutic; Transferase; ascorbate; base; follow-up; lung development; lung injury; mouse model; mutant; novel; oxidant stress; peroxidation; peroxiredoxin; 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）：本次修订后的续展申请旨在继续研究过氧化还原蛋白 6 (Prdx6) 在抗氧化防御中的作用。即将到来的拨款期间的重点将是 Prdx6 在氧化应激后修复过氧化膜磷脂中的作用。理论上，膜修复可以通过两个过程进行：过氧化磷脂 (PLOOH) 的直接还原或需要磷脂酶 A2 (PLA2) 和溶血磷脂酰基转移酶 (LPAT) 连续活性的重塑途径。我们之前已经表明，Prdx6 表达通过利用谷胱甘肽的过氧化物酶活性以及 PLA2 活性来还原 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全部三种活性（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特性和调节。建议 膜脂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，而表达其 3 个重要活性的 Prdx6 发挥着至关重要的作用。我们开发了 Prdx6 无效的小鼠模型，或者通过“敲入”技术表达 Prdx6 的过氧化物酶或 PLA2/LPAT 活性，并且我们从它们的肺部分离了突变细胞。通过慢病毒感染 Prdx6 无效细胞将产生额外的突变体。我们将研究离体肺细胞（肺泡 II 型上皮细胞、肺泡巨噬细胞、微血管内皮细胞）、离体灌注肺和完整小鼠的氧化应激，以确定修复过氧化肺细胞膜对 Prdx6 的需求以及直接还原和再酰化途径的相对作用。将通过质谱或生化测定来测量肺和肺细胞中的脂质过氧化，并在去除氧化应激后确定修复。对于肺和细胞来说，压力源是叔丁基过氧化氢或 Cu2+/抗坏血酸，对于小鼠来说，压力源是高氧。我们还将给小鼠注射博来霉素，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。这些研究应该提供与肺部从氧化应激中恢复相关的重要新信息，并可能导致促进恢复过程的治疗方式。