Cloning and expression of rat lung acidic Ca 2+ -independent PLA 2 and its organ distribution

Proc. Natl. Acad. Sci. U.S.A.

Fisher

2004

322

266

1-cys peroxiredoxin (1-cysPrx), a member of the peroxiredoxin superfamily, can protect cells against membrane oxidation through glutathione (GSH)-dependent reduction of phospholipid hydroperoxides to corresponding alcohols. However, purified native or recombinant enzyme in vitro generally lacks GSH peroxidase (GPx) activity because of oxidation of its single conserved cysteine. Reduction of the resultant oxidized cysteine is difficult because of its protected location within the homodimer formed by the oxidized protein monomers. Partial purification of 1-cysPrx from bovine lung revealed the presence of GST in an active preparation, while purification to homogeneity yielded enzyme that inactivated with time. We show that heterodimerization of 1-cysPrx with GSH-saturated GST results in glutathionylation of the oxidized cysteine in 1-cysPrx followed by subsequent spontaneous reduction of the mixed disulfide and restoration of enzymatic activity. Maximum activation of 1-cysPrx occurred with a 1:1 molar ratio of GSH-saturated GST and a 2:1 molar ratio of GSH to 1-cysPrx. Liposome-mediated delivery of oxidized recombinant enzyme into NCI-H441 cells that lack 1-cysPrx but express GST resulted in 1-cysPrx activation, whereas activation in MCF7 cells required co-delivery of GST. Our data indicate a physiological mechanism for glutathionylation of the oxidized catalytic cysteine of 1-cysPrx by its heterodimerization with GST followed by its GSH-mediated reduction and enzyme activation. P eroxiredoxins are a superfamily of nonheme and nonselenium peroxidases that are widely distributed throughout all phyla (1-4). Of the six mammalian peroxiredoxins, five (Prx I-V) contain two conserved cysteines that participate in intramolecular disulfide͞sulfhydryl redox cycling with thioredoxin resulting in reduction of H 2 O 2 and organic hydroperoxides into corresponding alcohols (2). By contrast, 1-cys peroxiredoxin (1-cysPrx or Prx VI) † has a single conserved cysteine (5) and does not use thioredoxin as reductant (5, 6). This peroxiredoxin is expressed in all tissues but at particularly high levels in brain, eye, testes, and lung (2,7,8). Expression of 1-cysPrx protein or mRNA is decreased in a mouse that is susceptible to experimental atherosclerosis (9) and is elevated in brains of patients with Parkinsonian dementia (10), sporadic Creutzfeldt-Jacob disease (11), and Pick disease (12), in lungs from newborns (13), in malignant mesothelioma (14), in the healing edge of skin wounds (15), and in experimental cellular premature senescence (16). 1-cysPrx can reduce phospholipid and other hydroperoxides (6) and protects against cellular membrane damage (17, 18). This enzyme has phospholipase A 2 activity (19), participates in the activation of neutrophil NADPH oxidase through its interaction with p67 phox (20), and prevent methemoglobin formation in erythrocyte hemolysates (21).1-cysPrx catalysis results in the peroxide-mediated oxidation of its Cys-47 to sulfenic acid as deduced from 1-cysPrx crystallization (22). Reduction of the sul...

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Activation of the antioxidant enzyme 1-CYS peroxiredoxin requires glutathionylation mediated by heterodimerization with πGST

Manevich

Proc. Natl. Acad. Sci. U.S.A.

Fisher

2004

322

266

“…Expression of a green fluorophore (either ZsGreen1 or GFP) was used as a transfection efficiency control, and hydroethidine (red) was used to detect ROS. The pIRES-ZsGreen vector encoded mouse Prdx6, whereas the GFP-C1 vector encoded rat Prdx6; the deduced Prdx6 protein sequences for the two species show 94% amino acid identity (20). Transfection efficiency in these cells using the Amaxa electroporation system was ϳ70%.…”

Section: Ros Production With Ang II Treatment Of Lung Endothelium In mentioning

confidence: 99%

Peroxiredoxin 6 Phosphorylation and Subsequent Phospholipase A2 Activity Are Required for Agonist-mediated Activation of NADPH Oxidase in Mouse Pulmonary Microvascular Endothelium and Alveolar Macrophages

Chatterjee

Journal of Biological Chemistry

Dodia

et al. 2011

Self Cite

126

198

Peroxiredoxin 6 (Prdx6), a bifunctional enzyme with glutathione peroxidase and phospholipase A2 (PLA 2 ) activities, participates in the activation of NADPH oxidase 2 (NOX2) in neutrophils, but the mechanism for this effect is not known. We now demonstrate that Prdx6 is required for agonist-induced NOX2 activation in pulmonary microvascular endothelial cells (PMVEC) and that the effect requires the PLA 2 activity of Prdx6. Generation of reactive oxygen species (ROS) in response to angiotensin II (Ang II) or phorbol 12-myristate 13-acetate was markedly reduced in perfused lungs and isolated PMVEC from Prdx6 null mice. Rac1 and p47phox , cytosolic components of NOX2, translocated to the endothelial cell membrane after Ang II treatment in wild-type but not Prdx6 null PMVEC. MJ33, an inhibitor of Prdx6 PLA 2 activity, blocked agonist-induced PLA 2 activity and ROS generation in PMVEC by >80%, whereas inhibitors of other PLA 2 s were ineffective. Transfection of Prx6 null cells with wild-type and C47S mutant Prdx6, but not with mutants of the PLA 2 active site (S32A, H26A, and D140A), "rescued" Ang II-induced PLA 2 activity and ROS generation. Ang II treatment of wild-type cells resulted in phosphorylation of Prdx6 and its subsequent translocation from the cytosol to the cell membrane. Phosphorylation as well as PLA 2 activity and ROS generation were markedly reduced by the MAPK inhibitor, U0126. Thus, agonist-induced MAPK activation leads to Prdx6 phosphorylation and translocation to the cell membrane, where its PLA 2 activity facilitates assembly of the NOX2 complex and activation of the oxidase.

“…5, A and B). DISCUSSION 1-cysPrx is a recently described antioxidant enzyme member of the Prx family that is expressed at relatively high levels in the lung (14). In lung epithelial cell lines, overexpression of 1-cysPrx protein was shown to increase the ability of these cells to degrade H 2 O 2 and to protect them against oxidantinduced plasma membrane damage (17), whereas treatment with an antisense oligonucleotide to 1-cysPrx resulted in lipid peroxidation and apoptosis (20).…”

Section: Lung Distribution Of Gene Transfermentioning

confidence: 99%

“…Peroxiredoxin VI or 1-cys peroxiredoxin (1-cysPrx) has a single redox-active cysteine and utilizes GSH to catalyze the reduction of H 2 O 2 and organic hydroperoxides including phospholipid hydroperoxides (8). 1-cysPrx is highly enriched in the lung compared with other organs and is especially expressed in Clara and alveolar epithelial type II cells (14). Previous studies have demonstrated that overexpression of 1-cysPrx in NIH 3T3 cells enhances their ability to reduce H 2 O 2 and protects their glutamine synthetase against H 2 O 2 -mediated inactivation (12).…”

mentioning

confidence: 99%

Adenovirus-mediated transfer of the 1-cys peroxiredoxin gene to mouse lung protects against hyperoxic injury

Wang

Manevich

American Journal of Physiology-Lung Cellular and Molecular Phys

et al. 2004

Self Cite

1-Cys peroxiredoxin (1-cysPrx) is a novel antioxidant enzyme that has been shown to reduce a broad spectrum of peroxides including phospholipid hydroperoxides. We tested the hypothesis that adenovirus-mediated transfer of the 1-cysPrx gene can protect lungs of mice from oxidant injury. Mice infected with AdLacZ/AdNull were used as a control (AdCon). X-galactosidase staining revealed widespread expression of the LacZ gene in airways and lung alveoli. Compared with AdCon, 1-cysPrx expression was increased about twofold at 3 days after adenovirus infection. Mice with increased Prx expression showed less loss of body weight and longer survival during exposure to 100% O2 or to 85% O2 for 4 days followed by 100% O2. At 72 h of 100% O2 exposure, AdPrx infection protected mouse lungs from injury as indicated by less pleural effusion, lower lung wet/dry weight, less protein and fewer nucleated cells in bronchoalveolar lavage fluid, and lower content of thiobarbituric acid-reactive substances and protein carbonyls in lung homogenate. These findings show that increased expression of 1-cysPrx through adenovirus-mediated gene transfer protects mouse lungs from hyperoxic injury and delays death.