Peroxiredoxin 6 phospholipid hydroperoxidase activity in the repair of peroxidized cell membranes

Abstract

Although lipid peroxidation associated with oxidative stress can result in cellular death, sub-lethal lipid peroxidation can gradually resolve with return to the pre-exposure state. We have shown that resolution of lipid peroxidation is greatly delayed in lungs or cells that are null for peroxiredoxin 6 (Prdx6) and that both the phospholipase A₂ and the GSH peroxidase activities of Prdx6 are required for a maximal rate of recovery. Like other peroxiredoxins, Prdx6 can reduce H₂O₂ and short chain hydroperoxides, but in addition can directly reduce phospholipid hydroperoxides. This study evaluated the relative role of these two different peroxidase activities of Prdx6 in the repair of peroxidized cell membranes. The His26 residue in Prdx6 is an important component of the binding site for phospholipids. Thus, we evaluated the lungs from H26A-Prdx6 expressing mice and generated H26A-Prdx6 expressing pulmonary microvascular endothelial cells (PMVEC) by lentiviral infection of Prdx6 null cells to compare with wild type in the repair of lipid peroxidation. Isolated lungs and PMVEC were exposed to tert-butyl hydroperoxide and mice were exposed to hyperoxia (> 95% O₂). Assays for lipid peroxidation in wild type control and mutant lungs and cells showed ~4-fold increase at end-exposure. Control lungs and cells showed gradual resolution during a post-exposure recovery period. However, there was no recovery from lipid peroxidation by H26A-Prdx6 lungs or PMVEC. These studies confirm an important role for Prdx6 in recovery from membrane lipid peroxidation and indicate that reduction of H₂O₂ or short chain hydroperoxides does not play a role in the recovery process.

Keywords: Endothelial cells; Histidine mutation; Hyperoxia; Lipid peroxidation; Oxidant stress; Perfused lung.

Graphical abstract

Fig. 1

Repair of lung lipid peroxidation during perfusion of isolated mouse lungs.Lungs were perfused for 1 h (starting at −1) with medium containing 25 mM (WT) or 15 mM (Prdx6 null, H26A) t-BOOH followed by 2 h perfusion with fresh medium (recovery period). Experiments were terminated at intervals during the recovery period and lipid peroxidation in the lung homogenate was measured by assay of: A.TBARS; B. DPPP-oxide; and C. lipid OOH (FOX assay). Values are mean ± SE for n = 3 to 4.

Fig. 2

Repair of lipid peroxidation in pulmonary microvascular endothelial cells (PMVEC). PMVEC from Prdx6 null mice were infected with lentivirus to express vector alone (Prdx6 null), Prdx6 wild type (WT), or H26A-Prdx6.Cells were treated with 300 μM (WT) or 200 μM (null, H26A) t-BOOH for 4 h and then evaluated at intervals for recovery in medium free of t-BOOH by assay of: A. TBARS and B. DPPP-oxide.Values are mean ± SE for n = 3 to 4.

Fig. 3

Repair of lung lipid peroxidation during recovery in room air following hyperoxia. Wild type (WT), Prdx6 null, and H26A-Prdx6 “knock-in” mice were exposed to > 95% oxygen for 60 h followed by a 20 h recovery period.Mice were sacrificed at intervals for analysis of lung lipid peroxidation by assay of: A. TBARS and B.DPPP-oxide. Values are mean ± SE for n = 3 to 4.

Fig. 4

Schematic showing the role of Prdx6 in the pathways for oxidation and reduction of cell membrane phospholipids.The reactions are: 1) oxidation of the unsaturated fatty acid (FA) in phosphatidylcholine (PC) by Fe²⁺ catalyzed generation of ROS; 2) reduction (scavenging) of H₂O₂ by GSH-dependent GPx activity; 3) reduction by oxidized phospholipid by phospholipid hydroperoxide GPx (PHGPx) activity; 4) reduction of phospholipid alcohols by unspecified reductases; 5) hydrolysis of oxidized sn-2 FA (FAOOH) in PCOOH by PLA₂ to generate lysoPC (LPC) plus an oxidized free FA (not shown); 6) reacylation of LPC with free FA:CoA by LPC acyl transferase (LPCAT) activity (the importance of the Prdx6-LPCAT activity in cell membrane repair has not yet been demonstrated experimentally). The net result is the regeneration of reduced phospholipid following an oxidative event.