Unraveling the mechanism of ferroptosis

Ferroptosis, a form of regulated cell death (RCD), has been recently reported to be primed by missing activity of the ubiquitous selenoperoxidase glutathione peroxidase 4 (GPx4). GPx4 catalyzes a glutathione (GSH)-dependent reduction of membrane hydroperoxides to corresponding alcohols. Since ferroptosis is executed by lipid peroxidation, oxygen, polyunsaturated fatty acids (PUFA), and iron are necessary constraints. Furthermore, since phospholipid hydroperoxides (PL-OOH) have a key role in membrane lipid peroxidation –i.e. their decomposition generates radicals in the membranes- GPx4-mediated regulation of ferroptosis is consistent with both, the mechanisms of lipid peroxidation and protection which have been finely elucidated in vitro in the last forty years of the XX century. In this study, we used as a suitable model of ferroptotic cell death, that primed by erastin in HT1080 cells. Erastin, by preventing cystine import, in these cells decreases the cellular level of GSH and, consequently, GPx4 activity. On this model, we disclosed a critical constraint permitting membrane lipid oxidation and therefore ferroptosis execution. Unexpectedly, we observed that modulating the electron flow through the mitochondria respiratory chain, is indeed unsuccessful in modulating ferroptosis sensitivity. For this indispensable function we identified, instead, the activity of mitochondrial alpha-keto acid dehydrogenases. The deduced mechanism we propose is the formation of O2•- during re-oxidation of dihydrolipoate in the last step of the oxidative decarboxylation, a reaction, already observed in isolated mitochondria. In relation to ferroptosis, we propose that it is the HO2• that generates a carbon centered radical in a PUFA, which, following oxygen addiction and stabilization by hydrogen transfer, forms a PL-OOH. We also studied the mechanisms underlying the inhibition of GPx4 by a prototype of electrophile: (1S,3R) RSL3. This study allowed to disclose the protein 14-3-3 epsilon as an adaptor protein necessary to achieve GPx4 inactivation by (1S,3R) RSL3. By these observations, we clarified the requirements for GPx4 inactivation by electrophiles, which, in vivo is supposed to have a regulatory role. Further, we provided evidence that the ‘inactivation-permitting activity’ of 14-3-3 epsilon on GPx4 is redox regulated by thiol-disulphide transition. This links inhibition by electrophiles to intracellular redox status. This set of new information contributes to the view of cell death by ferroptosis as intrinsically connected to aerobic life through a fine modulation of specific metabolic events. It is an amazing thought that electron transitions between oxygen and iron permits in aerobic organism both, life and death. The latter, therefore, comes up as a fundamental component of aerobic life.