br Importance of iron and lipoxygenase in ferroptosis What

Importance of iron and lipoxygenase in ferroptosis What remains to be somewhat controversial is the role of iron and lipoxygenases (LOX) in the death process, specifically in lipid peroxidation. So far, iron chelators have been repeatedly shown to halt ferroptosis (hence its name) induced by chemical or genetic means [2], [13], and overloading of cells with iron indeed sensitizes tumor cells to ferroptosis [31]. Additionally, enzymes/proteins involved in iron metabolism [32], such as transferrin, transferrin receptor 1 and ferroportin [33], [34], as well as heme oxygenase-1 (HO-1) [35], [36] have been implicated in the death process. Ferritinophagy, an autophagic process leading to the degradation of cellular iron storage proteins including the iron binding protein ferritin and the ferritinophagy cargo receptor nuclear receptor coactivator 4 (NCOA4), was recently demonstrated to contribute to ferroptosis by increasing the cellular labile iron pool and increased oxygen radical formation [37]. Freely available iron is thus considered to contribute to the process of lipid peroxidation and cell death by attacking PUFA residues in lipid bilayers in a non-enzymatic manner [5]. Ever since the early recognition that GPX4 counteracts the activity of certain LOX by controlling the so-called cellular peroxide tone (reviewed in [38]), LOX have been surmised to contribute to ferroptotic death by introducing peroxides in fatty UK-5099 residues of phospholipids. The enzymatic peroxidation of PUFAs is predominantly catalyzed by LOX, which abstract protons from bis-allylic positions followed by the addition of molecular oxygen. In humans there are six different isoenzymes (ALOXe3/5/12/12/15/15b) harbouring a catalytic active iron – their nomenclature refers to the regional selectivity of hydroperoxide formation [39]. LOX are only able to bind molecular oxygen when iron is oxidized by peroxides to ferric iron [39]. Thus, lowering the peroxide tone of cells by GPX4 may indirectly impact lipoxygenase activity. Early findings indeed suggested that inhibition of 12-lipoxygenase prevents neuronal cell death in HT22 cells induced by GSH depletion and oxidative glutamate toxicity [40], and that lipoxygenase inhibitors rescued Gpx4 knockout induced cell death in mouse embryonic fibroblasts engineered to allow inducible deletion of GPX4 [24]. Conversely, genetic studies performed with Alox15 (the gene encoding 12/15-lipoxygenase in mice) knockout and conditional Gpx4 knockout mice taught us that the double mutant mice fail to rescue from acute renal failure in mice, ferroptosis in fibroblasts [13] and in CD8+ T cells [41], nor does it rescue the early embryonic lethal phenotype of Gpx4 embryos [42]. Only subfertility, as observed in mice expressing a dysfunctional GPX4 protein, was rescuable by systemic inactivation of Alox15 [43]. Oxi-lipidomics analysis of cell undergoing ferroptosis and studies with deuterated fatty acids, however, pointed towards a site-specific and thus enzyme-mediated oxidation of PUFA residues [30], [44], thereby contributing to the generation of proximate signals of ferroptotic cell death. Moreover, phosphatidylethanolamine binding protein-1 (PEBP1), a protein with high affinity to phosphatidylethanolamine and known to bind and to inhibit RAF1 kinase activity, was identified to associate and change the substrate specificity of 15-lipoxygenase [45]. This interaction with 15-lipoxygenase seemingly changes the substrate specificity of 15-lipxygenase thus yielding potentially hazardous, peroxidized phophatidylethanolamines, which, when not cleared by GPX4, contribute to ferroptosis. Along the same line, siRNA-mediated knockdown of individual or several lipoxygenases conferred resistance to erastin induced ferroptosis in different cell lines [44], indicating cell type specific contexts. A recent study by Pratt's group questioned the ostensibly outstanding importance of LOX in the ferroptotic process [46]. While many of the frequently used alleged isoform-selective LOX inhibitors suppress the ferroptotic cell death process not by inhibiting their respective LOX isoform, but rather by acting as unspecific radical trapping agents, overexpression studies with several human LOX isoforms (5-LOX, p12-LOX, and 15-LOX-1) clearly demonstrated that lipid autoxidation is the key driver of ferroptosis. The fact that many of the previous studies with animal models of disease were actually performed with these “specific” inhibitors questions whether these effects are actually mediated by LOX inhibition or by general “antioxidant” effects. In light of the still missing in vivo data that knockout of one or even several LOX isoforms clearly mitigates the effects induced by GPX4 loss, care needs to be taken that LOX indeed play a major role in ferroptosis [47].