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Both Peroxidase and Phospholipase A2 Activities of Peroxiredoxin 6 Play a Role in its Protection against Oxidative Stress
Author(s) -
Lien YuChin,
Gao Ling,
Gonder Daniel S,
Feinstein Sheldon I,
Fisher Aron B
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.617.14
Subject(s) - transfection , oxidative stress , peroxiredoxin , peroxidase , phospholipase a2 , antioxidant , microbiology and biotechnology , mutant , biochemistry , chemistry , cytotoxicity , biology , enzyme , gene , in vitro
Peroxiredoxin 6 (Prdx6) is a bifunctional antioxidant enzyme enriched in lung. The goal of our experiments is to determine whether the ability of Prdx6 to protect against oxidative stress is due solely to the active cysteine of the peroxidase or also requires the active center of the phospholipase A 2 . Our studies are designed to test the ability of Prdx6 containing mutations in these sites to rescue cells from injury due to oxidative stress. We cloned the wild‐type mouse Prdx6 into pIRES2‐ZsGreen1 vector (Promega) that expresses ZsGreen from the same transcript as the cloned gene, using an internal ribosome entry site, and prepared several mutants using site‐directed mutagenesis. We were able to transfect up to 20‐40% of the MLE12 cells with our mouse constructs using Fugene 6 with no visible cytotoxicity. We then tested transfected MLE12 cells for sensitivity to tert‐butyl hydroperoxide, using MTT and neutral red assays. With exposure to 500 uM tert‐butyl hydroperoxide, the survival of wild‐type Prdx6 transfected cells was 3 to 4 fold higher than that of vector transfected cells. The C47S‐Prdx6 (without peroxidase activity) increased survival by less than 2‐fold. Prdx6 constructs with peroxidase but no PLA 2 activity showed more of a protective effect. The results suggest that while the peroxidase activity is critical for the antioxidant effect of Prdx6, the PLA 2 activity may also contribute. Supported by HL P01‐75587.