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.

Public Health Relevance

We have characterized a novel enzyme, peroxiredoxin 6, that has three important activities that serve to protect the lungs against oxidant stress and also regulate metabolism of the lung surfactant. Our goals for the program are to evaluate these enzymatic activities to determine their relative importance in antioxidant defense, especially in the repair of cell membranes that have been damaged by lipid peroxidation during oxidant exposure. Understanding this role of peroxiredoxin 6 will indicate a potentially new target for increasing the ability of the lung cells to survive the oxidant stress that is a prominent feature f acute lung injury.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL102016-05A1
Application #
8816964
Study Section
Lung Injury, Repair, and Remodeling Study Section (LIRR)
Program Officer
Harabin, Andrea L
Project Start
2010-05-01
Project End
2019-03-31
Budget Start
2015-04-01
Budget End
2016-03-31
Support Year
5
Fiscal Year
2015
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Physiology
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Fisher, Aron B (2018) The phospholipase A2 activity of peroxiredoxin 6. J Lipid Res 59:1132-1147
Fisher, Aron B; Vasquez-Medina, Jose P; Dodia, Chandra et al. (2018) Peroxiredoxin 6 phospholipid hydroperoxidase activity in the repair of peroxidized cell membranes. Redox Biol 14:41-46
Fisher, Aron B (2017) Peroxiredoxin 6 in the repair of peroxidized cell membranes and cell signaling. Arch Biochem Biophys 617:68-83
Moawad, Adel R; Fernandez, Maria C; Scarlata, Eleonora et al. (2017) Deficiency of peroxiredoxin 6 or inhibition of its phospholipase A2 activity impair the in vitro sperm fertilizing competence in mice. Sci Rep 7:12994
Fisher, Aron B; Dodia, Chandra; Sorokina, Elena M et al. (2016) A novel lysophosphatidylcholine acyl transferase activity is expressed by peroxiredoxin 6. J Lipid Res 57:587-96
Zhou, Suiping; Sorokina, Elena M; Harper, Sandra et al. (2016) Peroxiredoxin 6 homodimerization and heterodimerization with glutathione S-transferase pi are required for its peroxidase but not phospholipase A2 activity. Free Radic Biol Med 94:145-56
Krishnaiah, Saikumari Y; Dodia, Chandra; Sorokina, Elena M et al. (2016) Binding sites for interaction of peroxiredoxin 6 with surfactant protein A. Biochim Biophys Acta 1864:419-25
Vázquez-Medina, José Pablo; Dodia, Chandra; Weng, Liwei et al. (2016) The phospholipase A2 activity of peroxiredoxin 6 modulates NADPH oxidase 2 activation via lysophosphatidic acid receptor signaling in the pulmonary endothelium and alveolar macrophages. FASEB J 30:2885-98
Sorokina, Elena M; Dodia, Chandra; Zhou, Suiping et al. (2016) Mutation of Serine 32 to Threonine in Peroxiredoxin 6 Preserves Its Structure and Enzymatic Function but Abolishes Its Trafficking to Lamellar Bodies. J Biol Chem 291:9268-80
Benipal, Bavneet; Feinstein, Sheldon I; Chatterjee, Shampa et al. (2015) Inhibition of the phospholipase A2 activity of peroxiredoxin 6 prevents lung damage with exposure to hyperoxia. Redox Biol 4:321-7

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