Peroxiredoxin 6 (1-cys peroxiredoxin, Prdx6) is an atypical peroxiredoxin widely expressed throughout the body with the lungs, brain, and testes showing the highest levels [1]. In contrast to other peroxiredoxins, Prdx6 lacks a resolving cysteine and uses glutathione (GSH) to complete its peroxidatic reaction [[2], [3], [4], [5]]. Besides its GSH peroxidase activity, Prdx6 expresses acidic, calcium-independent phospholipase A2 (aiPLA2) [[6], [7], [8]] and lysophosphatidylcholine acyltransferase (Prdx6-LPCAT) activities [9] in separate catalytic sites. The PLA2, LPCAT, and GSH peroxidase activities of Prdx6 are differentially regulated by the subcellular distribution of the protein, substrate binding, and post-translational modifications including hyperoxidation at cysteine 47 and phosphorylation at threonine 177 [[10], [11], [12], [13], [14], [15], [16]].

Prdx6 exhibits maximal PLA2 activity at acidic pH and maximal GSH peroxidase activity at neutral pH under unstimulated conditions [12]. Hence, when localized to lysosomal-type organelles such as lamellar bodies of type II pneumocytes, Prdx6 functions as a PLA2 [12]. In contrast, cytosolic Prdx6 functions mainly as a GSH peroxidase [12]. Notably, aiPLA2 increases with enzyme phosphorylation and in the presence of oxidized phospholipid substrate at neutral pH [6]. Moreover, aiPLA2 and the LPCAT activities of Prdx6 appear to represent a two-step coupled reaction without the release of the intermediate lysophosphatidylcholine product [9]. Prdx6 binds to phospholipids with an oxidized fatty acid in the sn-2 position at cytosolic pH [6,11,[17], [18], [19]]. Hence, the translocation of Prdx6 to cell membranes under oxidant stress has several biologically relevant effects involving its phospholipid hydroperoxide GSH peroxidase, aiPLA2, and LPCAT activities [9,13,[20], [21], [22]].

Oxidant stress induced by paraquat, hyperoxia or organic peroxides promotes the translocation of Prdx6 to cell membranes [15,19,[21], [22], [23]]. In peroxidized membranes, Prdx6 reduces oxidized phospholipids through its phospholipid hydroperoxide GSH peroxidase activity and replaces the oxidized sn-2 fatty acyl group through hydrolysis/reacylation by aiPLA2 and Prdx6-LPCAT [13]. Prdx6 is, therefore, a complete system for the repair of peroxidized cell membranes [13]. Mice and lung cells lacking Prdx6 (Prdx6 KO) or carrying a mutation that prevents Prdx6 from binding to phospholipids (Prdx6-H26A) are unable to repair peroxidized cell membranes after exposure to hyperoxia or treatment with organic peroxides [[19], [20], [21], [22]]. Furthermore, mice with mutations that inactivate either aiPLA2 (Prdx6-D140A) or the GSH peroxidase activity of Prdx6 (Prdx6-C47S) alone show incomplete repair of peroxidized membranes [[19], [20], [21], [22]].

The unique role of Prdx6 as a suppressor of lung lipid peroxidation suggests that this enzyme regulates ferroptosis, and evidence of such effect in cancer cell lines with silenced Prdx6 expression is available [24], although genetic confirmation in cells with full Prdx6 depletion (KO) is still lacking. Similarly, the role of the specific activities of Prdx6 and its relevance in primary cells is unknown. Early work shows that both a PLA2 and a phospholipid hydroperoxidase activity are necessary to repair peroxidized lipids [[25], [26], [27]]. Ferroptosis is a newly described form of regulated cell death driven by an iron-dependent accumulation of phospholipid hydroperoxides [[28], [29], [30]]. The selenoprotein glutathione peroxidase 4 (GPx4) suppresses ferroptosis through its ability to reduce phospholipid hydroperoxides [31,32]. To date, no other enzymes that regulate the canonical GSH-dependent lipid peroxidation suppression pathway during ferroptosis have been identified [33], but alternative systems that cooperate with GPx4 to suppress the propagation of lipid peroxidation such as FSP1 (ferroptosis suppressor protein 1) and DHODH (dihydroorotate dehydrogenase) are well known [[34], [35], [36]]. Similarly, enzymes that modulate cellular lipid composition including acyl-CoA synthetase long-chain family member 4 (ACSL4) [[37], [38], [39], [40], [41]], lysophosphatidylcholine acyltransferase 3 (LPCAT3) [37,42], and other calcium-independent PLA2s [[43], [44], [45]] have been shown to regulate ferroptosis.

Here we show that Prdx6 is widely expressed in pulmonary endothelial and epithelial cells and that Prdx6 deletion or knockdown increases erastin-induced, ferrostatin-1-sensitive lipid peroxidation and cell death in murine and human lung endothelial cells in primary culture. Using cells derived from animals harboring single-point mutations that inactivate either aiPLA2 (Prdx6-D140A) or Prdx6-peroxidase (Prdx6-C47S) we also show that both activities participate in the anti-ferroptotic effect of Prdx6. Moreover, we found that Prdx6 deficiency induces transcriptional signatures associated with mitochondrial function and selenoamino acid metabolism and corroborated that Prdx6 depletion blunts mitochondrial function and upregulates GPx4 expression and abundance, while GPx4 depletion has the opposite effect. Furthermore, using in situ proximity ligation, we found that Prdx6 interacts with GPx4, suggesting that both proteins cooperate to counteract lipid peroxidation. Hence, Prdx6 suppresses ferroptosis by limiting lipid peroxidation through both aiPLA2 and Prdx6-peroxidase and likely by supporting mitochondrial function and modulating other cytoprotective pathways in the pulmonary endothelium.