This program will evaluate the properties of a novel glutathione peroxidase enzyme that is considerably enriched in lungs, especially granular pneumocytes and bronchiolar epithelium. This recently described protein (called l-cysPeroxiredoxin or 1-cysPrx) does not contain selenium and thus represents the first non-selenium gluthathione peroxidase of relatively high specific activity. An important characteristic in the spectrum of activity for this enzyme is its ability to reduce phospholipid hydroperoxides, unlike classical glutathione peroxidase. We postulate that 1-cysPrx can reduce oxidized phospholipids in membranes and hypothesize that it is an important pulmonary antioxidant enzyme. We have previously described methods for isolation of l-cysPrx protein from rat and bovine lungs and have published the human, rat, mouse and bovine cDNA sequences. We have found that mutation of Cys 47 (the only conserved Cys residue) abolishes peroxidase activity with H2O2 and phospholipid hydroperoxide substrates compatible with this as the active site. An important preliminary funding is that site-directed mutagenesis of Ser 32 has no effect on reduction of H2O2 but abolishes activity with phospholipid hydroperoxide substrates indicating importance of secondary recognition/binding sites for phospholipid hydroperoxides. Based on this preliminary data and the crystal structure, we have developed a model of enzyme activity which requires a dimeric (or tetrameric) protein with a Ser 32 recognition site, a hydrophobic pocket for phospholipid substrate binding, and a Cys 47 active site.
Specific Aim 1 will evaluate physical properties of 1-cysPrx with special emphasis on the oligomeric state required for enzymatic activity.
Specific Aim 2 will evaluate our proposed model for substrate binding by testing hydroperoxide substrates and phospholipid liposomes with native and mutant proteins using tryptophan fluorescence and circular dichroism measurements.
Specific Aim 3 will evaluate specific activity of native and mutant enzyme preparations in order to test our predictions concerning the physical basis for enzymatic activity.
Specific Aim 4 will evaluate the role of 1-cysPrx in antioxidant defense using both in vitro and in vivo approaches. Cell lines that overexpress 1-cysPrx will be generated and evaluated for resistance to oxidative stress (H2O2, paraquat). To study tolerance in vivo, rats and mice will be exposed to hyperoxia; enzyme induction (mRNA, protein, activity) will be measured in control and selenium deficient rats exposed to 80 percent or 100 percent 02 and in mice with knock out of classical glutathione peroxidase. A mouse model with knock out of l-cysPrx currently under development will also be tested for oxygen tolerance. These studies will give insights into the function and antioxidant activity of this potentially important, newly described enzyme.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Lung Biology and Pathology Study Section (LBPA)
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Harabin, Andrea L
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University of Pennsylvania
Schools of Medicine
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Wang, Yan; Feinstein, Sheldon I; Fisher, Aron B (2008) Peroxiredoxin 6 as an antioxidant enzyme: protection of lung alveolar epithelial type II cells from H2O2-induced oxidative stress. J Cell Biochem 104:1274-85
Wang, Yan; Phelan, Shelley A; Manevich, Yefim et al. (2006) Transgenic mice overexpressing peroxiredoxin 6 show increased resistance to lung injury in hyperoxia. Am J Respir Cell Mol Biol 34:481-6
Wang, Yan; Feinstein, Sheldon I; Manevich, Yefim et al. (2006) Peroxiredoxin 6 gene-targeted mice show increased lung injury with paraquat-induced oxidative stress. Antioxid Redox Signal 8:229-37
Manevich, Yefim; Fisher, Aron B (2005) Peroxiredoxin 6, a 1-Cys peroxiredoxin, functions in antioxidant defense and lung phospholipid metabolism. Free Radic Biol Med 38:1422-32
Wang, Yan; Feinstein, Sheldon I; Manevich, Yefim et al. (2004) Lung injury and mortality with hyperoxia are increased in peroxiredoxin 6 gene-targeted mice. Free Radic Biol Med 37:1736-43
Wang, Yan; Manevich, Yefim; Feinstein, Sheldon I et al. (2004) Adenovirus-mediated transfer of the 1-cys peroxiredoxin gene to mouse lung protects against hyperoxic injury. Am J Physiol Lung Cell Mol Physiol 286:L1188-93
Manevich, Y; Feinstein, S I; Fisher, A B (2004) Activation of the antioxidant enzyme 1-CYS peroxiredoxin requires glutathionylation mediated by heterodimerization with pi GST. Proc Natl Acad Sci U S A 101:3780-5
Mo, Yiqun; Feinstein, Sheldon I; Manevich, Yefim et al. (2003) 1-Cys peroxiredoxin knock-out mice express mRNA but not protein for a highly related intronless gene. FEBS Lett 555:192-8
Kim, Han-Suk; Manevich, Yefim; Feinstein, Sheldon I et al. (2003) Induction of 1-cys peroxiredoxin expression by oxidative stress in lung epithelial cells. Am J Physiol Lung Cell Mol Physiol 285:L363-9
Pak, Jhang Ho; Manevich, Yefim; Kim, Han Suk et al. (2002) An antisense oligonucleotide to 1-cys peroxiredoxin causes lipid peroxidation and apoptosis in lung epithelial cells. J Biol Chem 277:49927-34

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