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●.

Ferroptosis: A Critical Moderator in the Life Cycle of Immune Cells

“…It has been shown to have higher resistance to ferroptosis induced by GPX4 deletion than M2 ( 50 ). In terms of mechanism, M1 phenotype produces NO free radicals (NO•), which causes the inhibition of secondary ALOX15 activity, while iNOS expression and NO• production in M2 phenotype are inhibited ( 51 ). In addition to this, the expression of iron metabolism-related proteins in M2 phenotypes is different.…”

A tale of two lipids: Lipid unsaturation commands ferroptosis sensitivity

“…A similar function may be performed by the flavoenzyme dihydroorotate dehydrogenase (DHODH), which can suppress ferroptosis by reducing CoQ 10 to CoQ 10 -H2, although this is debated [31,32]. The inducible nitric oxide synthase (iNOS) system also suppresses ferroptosis independently of GPX4 by nitrosylation of PLs and inhibition of PL peroxidation [12,33,34]. The next level of metabolic regulation includes synthesis of reducing agents like NADPH and thioredoxin, which function as upstream regulators of lipid peroxide formation and ferroptosis sensitivity [35,36].…”

Ferroptosis and tumor immunotherapy: A promising combination therapy for tumors

“…The number and distribution of TAMs may be associated with the prognosis of underlying malignant disease ( 107 ). M1-like macrophages have higher nitric oxide radicals due to higher intracellular inducible NO synthase (iNOS) content than M2-like macrophages, thus inhibiting lipid peroxidation; in contrast, M2-like macrophages have a less inhibitory effect on lipid peroxidation due to lower iNOS content and less production of nitric oxide radicals ( 108 – 110 ). Therefore, RSL3 could not induce ferroptosis in M1-like macrophages but could induce ferroptosis in M2-like macrophages ( 111 ).…”