Strikingly High Activity of 15‐Lipoxygenase Towards Di‐Polyunsaturated Arachidonoyl/Adrenoyl‐Phosphatidylethanolamines Generates Peroxidation Signals of Ferroptotic Cell Death

The vast majority of membrane phospholipids (PLs) include two asymmetrically positioned fatty acyls: oxidizable polyunsaturated fatty acids (PUFA) attached predominantly at the sn2 position, and non‐oxidizable saturated/monounsaturated acids (SFA/MUFA) localized at the sn1 position. The peroxidation of PUFA‐PLs, particularly sn2‐arachidonoyl(AA)‐ and sn2‐adrenoyl(AdA)‐containing phosphatidylethanolamines (PE), has been associated with the execution of ferroptosis, a program of regulated cell death. There is a minor subpopulation (≈1–2 mol %) of doubly PUFA‐acylated phospholipids (di‐PUFA‐PLs) whose role in ferroptosis remains enigmatic. Here we report that 15‐lipoxygenase (15LOX) exhibits unexpectedly high pro‐ferroptotic peroxidation activity towards di‐PUFA‐PEs. We revealed that peroxidation of several molecular species of di‐PUFA‐PEs occurred early in ferroptosis. Ferrostatin‐1, a typical ferroptosis inhibitor, effectively prevented peroxidation of di‐PUFA‐PEs. Furthermore, co‐incubation of cells with di‐AA‐PE and 15LOX produced PUFA‐PE peroxidation and induced ferroptotic death. The decreased contents of di‐PUFA‐PEs in ACSL4 KO A375 cells was associated with lower levels of di‐PUFA‐PE peroxidation and enhanced resistance to ferroptosis. Thus, di‐PUFA‐PE species are newly identified phospholipid peroxidation substrates and regulators of ferroptosis, representing a promising therapeutic target for many diseases related to ferroptotic death. Doubly polyunsaturated phospholipids are present in biomembranes, albeit at very low abundancies. Their specific function is not known. We discovered that 15‐lipoxygenase catalyzes oxygenation of these phospholipids at the same high rate as free fatty acids and yield 15‐hydroperoxy‐products which act as pro‐ferroptotic signals of cell death.