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. 2018 Dec 26;140(51):17835-17839.
doi: 10.1021/jacs.8b09913. Epub 2018 Dec 17.

Empowerment of 15-Lipoxygenase Catalytic Competence in Selective Oxidation of Membrane ETE-PE to Ferroptotic Death Signals, HpETE-PE

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Empowerment of 15-Lipoxygenase Catalytic Competence in Selective Oxidation of Membrane ETE-PE to Ferroptotic Death Signals, HpETE-PE

Tamil S Anthonymuthu et al. J Am Chem Soc. .
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Abstract

sn2-15-Hydroperoxy-eicasotetraenoyl-phosphatidylethanolamines ( sn2-15-HpETE-PE) generated by mammalian 15-lipoxygenase/phosphatidylethanolamine binding protein-1 (15-LO/PEBP1) complex is a death signal in a recently identified type of programmed cell demise, ferroptosis. How the enzymatic complex selects sn2-ETE-PE as the substrate among 1 of ∼100 total oxidizable membrane PUFA phospholipids is a central, yet unresolved question. To unearth the highly selective and specific mechanisms of catalytic competence, we used a combination of redox lipidomics, mutational and computational structural analysis to show they stem from (i) reactivity toward readily accessible hexagonally organized membrane sn2-ETE-PEs, (ii) relative preponderance of sn2-ETE-PE species vs other sn2-ETE-PLs, and (iii) allosteric modification of the enzyme in the complex with PEBP1. This emphasizes the role of enzymatic vs random stochastic free radical reactions in ferroptotic death signaling.

Figures

Figure 1.
Figure 1.
(A, B) Accumulation of HOO-PL products in pro-ferroptotic cells (RSL3 treatment) and tissue (acute kidney injury) compared to normal conditions. (refer Table S1 for the species list). (C, D). Fragmentation of sn1-18:0/sn2-20:4-OOH showing sn2-15-HpETE-PE formation in pro-ferroptotic conditions. (E) Venn diagram of significant signals from pro and anti ferroptotic conditions showing “surviving” PE-OOH products
Figure 2.
Figure 2.
(A) Oxidation rates of various PLs by 15-LO2 in liposomes. (B) PA oxidation in liposomes with variable sn1–18:1/sn2–18:1-PC:sn1–18:0/sn2–20:4-PA ratios compared to liposomes with constant sn1–18:1/sn2–18:1-PC:sn1–18:0/sn2–20:4-PA ratio. *p<0.05 vs. liposome of variable sn1–18:1/sn2–18:1-PC:sn1–18:0/sn2–20:4-PA ratio. (C) Ternary plot showing PS oxidation in liposomes with varying content of non-oxidizable PA and PC.
Figure 3.
Figure 3.
(A) Oxidation rates of phospholipids by 15-LO2 and 15-LO2/PEBP1 complex in liposomes prepared from a phospholipid mixture. *p<0.05 vs. 15-LO2. (B) ANM structure of 15-LO2 showing the movement of lid helix upon binding with PEBP1. (C) Images showing the reduction in the size of the active site entrance in 15-LO2 (left) and 15-LO2/PEBP1 complex (right).
Figure 4.
Figure 4.
(A) Percentage of oxidation occurring at each oxidizable carbon position of ETE esterified to PE. (B) Distance of all carbon atoms of sn2-ETE present in various PL from 15-LO2 catalytic iron. (C) Docking of sn1-18:0/sn2-20:4-PE on 15-LO2 with (red) and without (green) PEBP1, showing the change in the nearest carbon to the catalytic iron.
Figure 5.
Figure 5.
(A) PE oxidation by complexes of 15-LO2 with PEBP1 mutants. N=3, *p<0.05 vs 15-LO2 alone, #p<0.05 vs 15-LO2/PEBP1 (WT). (B) Effect of WT and mutant (H86E) PEBP1 overexpression on RSL3 induced ferroptosis. *p<0.05 vs pCDNA only, #p<0.05 vs pCDNA1-PEBP1(WT).

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