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NO^● Represses the Oxygenation of Arachidonoyl PE by 15LOX/PEBP1: Mechanism and Role in Ferroptosis.

ABSTRACT: We recently discovered an anti-ferroptotic mechanism inherent to M1 macrophages whereby high levels of NO^● suppressed ferroptosis via inhibition of hydroperoxy-eicosatetraenoyl-phosphatidylethanolamine (HpETE-PE) production by 15-lipoxygenase (15LOX) complexed with PE-binding protein 1 (PEBP1). However, the mechanism of NO^● interference with 15LOX/PEBP1 activity remained unclear. Here, we use a biochemical model of recombinant 15LOX-2 complexed with PEBP1, LC-MS redox lipidomics, and structure-based modeling and simulations to uncover the mechanism through which NO^● suppresses ETE-PE oxidation. Our study reveals that O₂ and NO^● use the same entry pores and channels connecting to 15LOX-2 catalytic site, resulting in a competition for the catalytic site. We identified residues that direct O₂ and NO^● to the catalytic site, as well as those stabilizing the esterified ETE-PE phospholipid tail. The functional significance of these residues is supported by in silico saturation mutagenesis. We detected nitrosylated PE species in a biochemical system consisting of 15LOX-2/PEBP1 and NO^● donor and in RAW264.7 M2 macrophages treated with ferroptosis-inducer RSL3 in the presence of NO^●, in further support of the ability of NO^● to diffuse to, and react at, the 15LOX-2 catalytic site. The results provide first insights into the molecular mechanism of repression of the ferroptotic Hp-ETE-PE production by NO^●.

SUBMITTER: Mikulska-Ruminska K

PROVIDER: S-EPMC8156958 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Ferroptosis is an iron dependent form of cell death, that is triggered by the discoordination of iron, lipids, and thiols. Its unique signature that distinguishes it from other forms of cell death is the formation and accumulation of lipid hydroperoxides, particularly oxidized forms of polyunsaturated phosphatidylethanolamines (PEs), which drives cell death. These readily undergo iron-catalyzed secondary free radical reactions leading to truncated products which retain the signature PE headgroup and which can readily react with nucleophilic moieties in proteins via their truncated electrophilic acyl chains. Using a redox lipidomics approach, we have identified oxidatively-truncated PE species (trPEox) in enzymatic and non-enzymatic model systems. Further, using a model peptide we demonstrate adduct formation with Cys as the preferred nucleophilic residue and PE(26:2) +2 oxygens, as one of the most reactive truncated PE-electrophiles produced. In cells stimulated to undergo ferroptosis we identified PE-truncated species with sn-2 truncations ranging from 5 to 9 carbons. Taking advantage of the free PE headgroup, we have developed a new technology using the lantibiotic duramycin, to enrich and identify the PE-lipoxidated proteins. Our results indicate that several dozens of proteins for each cell type, are PE-lipoxidated in HT-22, MLE, and H9c2 cells and M2 macrophages after they were induced to undergo ferroptosis. Pretreatment of cells with the strong nucleophile, 2-mercaptoethanol, prevented the formation of PE-lipoxidated proteins and blocked ferroptotic death. Finally, our docking simulations showed that the truncated PE species bound at least as good to several of the lantibiotic-identified proteins, as compared to the non-truncated parent molecule, stearoyl-arachidonoyl PE (SAPE), indicating that these oxidatively-truncated species favor/promote the formation of PEox-protein adducts. The identification of PEox-protein adducts during ferroptosis suggests that they are participants in the ferroptotic process preventable by 2-mercaptoethanol and may contribute to a point of no return in the ferroptotic death process.

Project description:BackgroundAt present, the pathogenesis of depression is not fully understood, and nearly half of depression patients experience no obvious effects during treatment. This study aimed to establish a depression mouse model to explore the possible role of ferroptosis in the pathogenesis of depression, and observe the effects of Xiaoyaosan on PEBP1-GPX4-mediated ferroptosis in the hippocampus.MethodsForty-eight male C57BL/6 mice were randomly divided into a control group, CUMS group, Xiaoyaosan group and fluoxetine group, and the model was established by chronic unpredictable mild stress (CUMS) for a successive 6 weeks. The medication procedure was performed from the 4th to the 6th week of modeling. The behavioral evaluations were measured to evaluate depressive-like behaviors. The expressions of GPX4, FTH1, ACSL4 and COX2 were detected as ferroptosis-related indicators. Then, the total iron and ferrous content in the hippocampus were measured. The levels of PEBP1 and ERK1/2 were observed, and the expressions of GFAP and IBA1 were also detected to measure the functions of astrocytes and microglia in the hippocampus.ResultsEight herbs of Xiaoyaosan had 133 active ingredients which could regulate the 43 ferroptosis-related genes in depression. After 6 weeks of modeling, the data showed that mice in the CUMS group had obvious depressive-like behaviors, and medication with Xiaoyaosan or fluoxetine could significantly improve the behavioral changes. The expressions of GPX4, FTH1, ACSL4, COX2, PEBP1, ERK1/2, GFAP and IBA1 changed in the CUMS group mice, while the total iron and ferrous content also changed. Xiaoyaosan and fluoxetine had obvious curative effects that could significantly alleviate the above changes in the hippocampus.ConclusionOur results revealed that the activation of ferroptosis might exist in the hippocampi of CUMS-induced mice. The PEBP1-GPX4-mediated ferroptosis could be involved in the antidepressant mechanism of Xiaoyaosan. It also implied that ferroptosis could become a new target for research into the depression mechanism and antidepressant drugs.

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NO^● Represses the Oxygenation of Arachidonoyl PE by 15LOX/PEBP1: Mechanism and Role in Ferroptosis.

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NO● Represses the Oxygenation of Arachidonoyl PE by 15LOX/PEBP1: Mechanism and Role in Ferroptosis.

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OmicsDI is part of the ELIXIR infrastructure

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NO^● Represses the Oxygenation of Arachidonoyl PE by 15LOX/PEBP1: Mechanism and Role in Ferroptosis.