H2O2-dependent Hyperoxidation of Peroxiredoxin 6 (Prdx6) Plays a Role in Cellular Toxicity via Up-regulation of iPLA2 Activity

Abstract

Peroxiredoxin 6 (Prdx6) is a bifunctional enzyme with peroxidase activity and Ca 2؉ -independent phospholipase A2 (iPLA2) activity. Here, we report that H 2 O 2 -induced cellular toxicity acts through Prdx6 hyperoxidation. Under high concentrations of H 2 O 2 (>100 M), Prdx6, and 2-Cys Prdxs were hyperoxidized. Contrary to hyperoxidation of 2-Cys Prdxs, hyperoxidation of Prdx6 was irreversible in vivo. Surprisingly, H 2 O 2 -induced cell cycle arrest at the G2/M transition correlated with hyperoxidation and increased iPLA2 activity of Prdx6. This arrest was also associated with up-regulation of p53 and p21 and with downregulation of cyclin B1. Furthermore, the H 2 O 2 -mediated increase in iPLA2 activity was dramatically abolished in a hyperoxidation mutant (C47A), an iPLA2 mutant (S32A), and a double mutant (C47A/S32A) of Prdx6, demonstrating the essential requirement of Prdx6 C47 hyperoxidation for its iPLA2 activity. Together, our results demonstrate that H 2 O 2 -mediated hyperoxidation of Prdx6 induces cell cycle arrest at the G2/M transition through up-regulation of iPLA2 activity. Peroxiredoxins (Prdxs)2 are a family of peroxidases that reduce mainly hydrogen peroxide (H 2 O 2 ) and alkyl hydroperoxides to water and alcohol, respectively (1, 2). Prdxs are classified as either 1-Cys or 2-Cys Prdxs, based on whether the protein contains one or two conserved cysteine residues, respectively. In mammals, six members of the Prdx family have been described. Five of these (Prdx1, Prdx2, Prdx3, Prdx4, and Prdx5) are 2-Cys enzymes that use thioredoxin as the electron donor of their catalytic cycle (3, 4). In contrast, Prdx6, the sole mammalian 1-Cys Prdx, does not use thioredoxin as a reductant.In addition to peroxidase activity, Prdx6 has a Ca 2ϩ -independent phospholipase A2 (iPLA2) activity (5). The peroxidase activity of Prdx6 has been widely studied in cells and animal models for its antioxidant properties that provide protection against the harmful consequence of oxidative stress (6 -8). However, the iPLA2 activity of Prdx6 remains poorly understood. Considering the many functions of iPLA2 activity, including cell cycle progression, apoptosis, and tumorigenesis (9), it may also play an important role in either H 2 O 2 -mediated signaling or H 2 O 2 -related cellular events.Prdxs like Prdx6 use cysteine as a catalytic center, rather than selenocysteine which characterizes the glutathione peroxidases (GPx) (2, 3). The N-terminal conserved cysteines (Cys 51 of Prdx1) of Prdx1-5 are selectively oxidized by H 2 O 2 to Cys-SOH (10). The unstable Cys 51 -SOH reacts with Cys 172-SH of another Prdx molecule, creating a homodimer through an intermolecular disulfide bond. The disulfide is then reduced back to the Prdx active thiol form by the thioredoxin-thioredoxin reductase system (11-16). Under oxidative stress condition, however, the sulfenic intermediate is susceptible to hyperoxidation and thus occasionally hyperoxidized by H 2 O 2 , leading to the formation of sulfinic acid (Cys-SO 2 H) or sulfonic acid (Cys-SO 3...

Calcium-independent phospholipases A2 and their roles in biological processes and diseases

“…The ability of iPLA 2 ␤ to promote cell proliferation becomes prominent in the context of tumorigenesis. Several in vitro studies reveal higher expression of iPLA 2 ␤ in stimulated immortal cell lines and that chemical inhibition or siRNAs targeted against iPLA 2 ␤ reduces proliferation and promotes apoptosis of the cells ( 97, 108,125,[356][357][358][359][360][361][362][363][364]. Subsequent studies targeting specifi c cancers suggest that iPLA 2 ␤ promotes cancer cell growth via signal transduction pathways involving epidermal growth factor receptors, MAPKs, E3 ubiquitin-protein ligase mdm2, tumor suppressor protein p53, and cell cycle regulator p21 (365)(366)(367).…”

Hydrogen Peroxide Induces Cell Cycle Arrest in Cardiomyoblast H9c2 Cells, Which Is Related to Hypertrophy

“…Kim et al showed that hyperoxidation of peroxiredoxin 6 induces cell cycle arrest in H 2 O 2 -treated Hela cells, with an increase in p21 expression. 14) Magenta et al demonstrated that in human umbilical vein endothelial cell (HUVEC) exogenous H 2 O 2 induces activation of protein phosphatase 2A, which arrests cell cycle via RB activation. 15) H 2 O 2 induces various cellular events involving cell cycle arrest.…”

Phospholipase A2 of peroxiredoxin 6 has a critical role in tumor necrosis factor-induced apoptosis

“…We recently reported that Prdx6 is involved in H 2 O 2 -induced cellular toxicity through the hyperoxidation and upregulation of the iPLA 2 activity of Prdx6. 6 Our current work suggests that Prdx6 has dual functions in apoptotic cell death that are dependent on its peroxidase and PLA 2 activities. The knockdown of Prdx6 rendered cells highly sensitive to H 2 O 2 -induced apoptosis but resistant to TNF-a/CHX-induced apoptosis.…”

“…5 The peroxidase activity of Prdx6 has been widely studied in cells and animal models for its antioxidant properties, which provides protection against the harmful consequences of oxidative stress. [6][7][8] However, the iPLA 2 activity of Prdx6 remains poorly understood. We recently reported that Prdx6 is involved in H 2 O 2 -induced cellular toxicity through the hyperoxidation and upregulation of the iPLA 2 activity of Prdx6.…”

“…iPLA 2 activities were measured as described, 6 according to the manufacturer's recommendations (Cayman Chemicals, Ann Arbor, MI, USA). The 2 0 ,7 0 -dichlorodihydrofluorescein (DCFH) staining was performed as described.…”