1-Cys Peroxiredoxin, a Bifunctional Enzyme with Glutathione Peroxidase and Phospholipase A2 Activities

Abstract

This report provides definitive evidence that the protein 1-Cys peroxiredoxin is a bifunctional ("moonlighting") enzyme with two distinct active sites. We have previously shown that human, rat, and bovine lungs contain an acidic Ca 2؉ -independent phospholipase A 2 (aiPLA 2 ). The cDNA encoding aiPLA 2 was found to be identical to that of a non-selenium glutathione peroxidase (NSGPx). Protein expressed using a previously reported E. coli construct which has a His-tag and 50 additional amino acids at the NH 2 terminus, did not exhibit aiPLA 2 activity. A new construct which contains the His-tag plus two extra amino acids at the COOH terminus when expressed in Escherichia coli generated a protein that hydrolyzed the sn-2 acyl chain of phospholipids at pH 4, and exhibited NSGPx activity with H 2 O 2 at pH 8. The expressed 1-Cys peroxiredoxin has identical functional properties to the native lung enzyme: aiPLA 2 activity is inhibited by the serine protease inhibitor, diethyl p-nitrophenyl phosphate, by the tetrahedral mimic 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33), and by 1-Cys peroxiredoxin monoclonal antibody (mAb) 8H11 but these agents have no effect on NSGPx activity; NSGPx activity is inhibited by mercaptosuccinate and by 1-Cys peroxiredoxin mAb 8B3 antibody which have no effect on aiPLA 2 activity. Mutation of Ser 32 to Ala abolishes aiPLA 2 activity, yet the NSGPx activity remains unaffected; a Cys 47 to Ser mutant is devoid of peroxidase activity but aiPLA 2 activity remains intact. These results suggest that Ser 32 in the GDSWG consensus sequence provides the catalytic nucleophile for the hydrolase activity of aiPLA 2 , while Cys 47 in the PVCTTE consensus sequence is at the active site for peroxidase activity. The bifunctional catalytic properties of 1-Cys peroxiredoxin are compatible with a simultaneous role for the protein in the regulation of phospholipid turnover as well as in protection against oxidative injury.

Two glutathione peroxidases in the fungal pathogen Cryptococcus neoformans are expressed in the presence of specific substrates

“…Another possibility is that in the absence of a GPX, other antioxidant enzyme(s) are upregulated in vivo to compensate for the lack of Gpx activity. For example, in S. cerevisiae, 1-Cys peroxiredoxins have been shown to possess glutathione peroxidase activity (Chen et al, 2000). In addition, it has been reported that glutaredoxins, which are small glutathione-dependent oxidoreductases, can act as glutathione peroxidases and reduce peroxides in S. cerevisiae (Collinson et al, 2002).…”

Peroxiredoxin 6 interferes with TRAIL-induced death-inducing signaling complex formation by binding to death effector domain caspase

“…28,29 Interestingly, only Prx6 uses glutathione as a physiological reductant, while other Prxs use thioredoxin. 16 Then, how does Prx6 regulate TRAIL-mediated cell death through DED caspases? The antiapoptotic activity of Prx6 can be exerted mainly by two possible mechanisms.…”

“…Prx6 is the only member of 1-Cys Prx and has non-selenium glutathione peroxidase and phospholipase A 2 activities. 16 Upregulation of Prx6 suppresses intracellular enzyme inactivation, membrane phospholipid peroxidation, and cell death mediated by oxidative stress. 17,18 In addition, Prx6-deficient mice are susceptible to oxidative stress by hypoxia or paraquat treatment, leading to increase of mortality and lung injury.…”

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

“…Expression of the C47S Prdx6 mutant partially rescued (ϳ50%) both the agonist-induced PLA 2 activity and ROS generation (Table 4). On the other hand, there was no significant rescue of either PLA 2 activity or ROS generation in endothelial cells transfected with constructs that express S32A, H26A, or D140A mutant Prdx6; these 3 amino acids constitute the Prdx6 PLA 2 catalytic triad, and each is necessary for PLA 2 activity but not for H 2 O 2 peroxidase activity (21,22). These results for "rescue" show that the PLA 2 activity of Prdx6 is required for Ang II-mediated activation of NOX2 in mouse PMVEC.…”