Lipid peroxidation and ferroptosis: The role of GSH and GPx4
Graphical abstract
Introduction
Following the historical distinction between “accidental” and “regulated” cell death, a continuously growing list of regulated cell death (RCD) routines had been compiled. Agonists, inhibitors, metabolic pathways and genetic regulation of different forms of non-accidental cell death have been identified and classified by the Nomenclature Committee of Cell Death. Ferroptosis (FPT) is a newly described form of regulated cell death, defined as the result of missing or insufficient activity of the selenoperoxidase Glutathione Peroxidase 4 (GPx4), which causes a specific form of cell death operated by membrane lipid peroxidation (LPO) [1].
Introducing the concept of FPT, we recall the fundamental axiom of oxygen metabolism and toxicity stating that the high thermodynamic oxidative potential of molecular oxygen is mitigated by the kinetic sluggishness, due to incompatibility between the ground spin status of molecular oxygen and the reduced carbon of organic compounds [2]. To get fire, a spark is used to provide the energy exceeding the high energetic barrier of the transition state of the reaction between oxygen and fuel. In the case of the biological reactions of oxygen, the spin restriction can be bypassed at low temperature, when molecular oxygen interacts –accepting a single electron-with free radicals or transition metals [3]. Thus, the membrane damaging LPO, although thermodynamically favorable, is kinetically controlled and can only initiate when the spin barrier of oxygen is circumvented.
Oxidative damage has been for decades the leitmotif of studies on accidental free radical damage associated to spontaneous or experimental diseases. The remarkable insight, recently brought by the discovery of FPT, is framed in the notion that LPO is proposed today as a “regulated” event.
LPO has been identified as the executor of FPT from the results obtained by pharmacologic manipulations leading to either GSH depletion or GPx4 inhibition in cells [4,5]. These results agree with the earlier observations that whole gene deletion of GPx4 is embryonically lethal [6], and, in the adult or in a specific tissue, its depletion results in specific cell death phenotypes [[7], [8], [9], [10], [11], [12], [13]].
This set of evidence supports the notion that FPT is not the outcome of a death pathway sparked by a specific agonist, but, rather, a kind of distortion of the integrated mechanisms controlling oxygen metabolism and toxicity. The implicit fall-out is that these mechanisms must be continuously operating for keeping low the steady state between reactions permitting and inhibiting LPO.
In this review, we will critically discuss the specific aspects and the constraints of the relationship between GPx4 activity, LPO and FPT as schematically outlined in Fig. 1.
Our proposal is that the traces of hydroperoxide derivatives of lipids (LOOH), unavoidably produced as a consequence of oxygen activation during aerobic metabolism, are continuously reduced by GPx4 in the presence of GSH. Not until the GPx4 reaction becomes limiting, ferrous iron initiates LPO by LOOH decomposition, leading to cell death by FPT. This features the Janus face of the oxygen molecule and highlights the proposed notion of a threshold that is exceeded when not only the indispensable constraints of LPO are fulfilled, and GPx4 activity is insufficient.
Section snippets
Lipid autoxidation and peroxidation
Both terms, “autoxidation” and “peroxidation”, indicate the formation of LOOH and their decomposition leading to a series of products, including reactive electrophiles. Although these terms are indifferently used in the scientific literature on oxidative degradation of lipids, here we will refer to this semantic distinction solely to point out the peculiar analogy and difference between the slow oxidative degradation of organic matter, typically occurring in polymers, foods or oils
How is the “first” LOOH required to initiate oxidative chain reactions produced?
The energetic barrier of the reaction between molecular oxygen and carbon, due to the ground electron status of carbon (singlet) and di-oxygen (triplet), is escaped when either oxygen reacts with a free radical or it is reduced by an uneven number of electrons unveiling the reactivity as free radical itself. The species produced by monovalent reduction of molecular oxygen is the superoxide anion (O2•—).
The major biological sources of O2•—, are NADPH oxidases (NOXs) [27], and mitochondria, which
Role of lipoxygenases
Besides the above mechanisms, LOOH can be produced also by some dioxygenase homologs, namely lipoxygenases (LOXs) [44]. Evidence has been produced for their role in FPT [45,46], and thus, since antioxidants suppress LOXs activity [47], a relevant question emerges whether FPT inhibition by antioxidants could be due to LOXs inhibition [48]. Against there is the observation that ferrostatin and liprostatin 1, the two paradigmatic FPT inhibitors, are poor inhibitors of 12/15 LOX but excellent
From microsomal lipid peroxidation to GPx4
The rancidity of fats, a typical consequence of autoxidation, was known from antiquity, and carpenters and painters used oxidation-polymerized linseed oil for centuries. The first scientific description dates back to early XIX century when Théodore de Saussure provided manometric and gravimetric evidence for the spontaneous addition of oxygen to walnut oil [57]. Since then, the process of oxidative degradation, and the procedures to prevent it, attracted the attention primarily of rubber
The products of lipid peroxidation and ferroptosis
The occurrence of LPO leading to FPT, although inferred a priori from the missed anti-peroxidant activity of GPx4, is still far from being evaluated with rigorous quantitative precision.
In the early studies on LPO, which were carried out on lipid dispersions or subcellular fractions, the analytical procedures were borrowed from oil and food analysis. Diene conjugation, colorimetric determination of malondialdehyde or titration of hydroperoxides have been measured for decades. In our experience,
Role of glutathione
GSH, the reducing substrate of GPx4 activity, is indispensable for preventing FPT. Consistently, GSH is indispensable for life as the silencing of γ-Glu-Cys ligase (GCL), the rate-limiting enzyme of GSH synthesis, is lethal [73,74]. Cell death is seemingly due to LPO, although other mechanisms limiting the maintenance of vital homeostasis are also possible.
More generally, the relationship between GSH redox status and regulated cell death –originally cumulatively referred to as
Role of iron
Iron has an indispensable function in LPO, and it is the cellular pool of redox-active free Fe2+ – i.e. the so called “labile iron pool” (LIP) [97] - having the key role of initiating LPO from LOOH.
Iron is ingested with food and is continuously recycled among tissues, leaving the organism only by bleeding, or by cell loss from intestine or skin. Intracellular concentration and biological availability of iron is carefully controlled, resulting from the rates of uptake, utilization, protein
Role of PUFAs in membrane phospholipids
As outlined above, LPO requires polyunsaturated phospholipids (see paragraph 2). It can be anticipated, therefore, that phospholipid PUFA content is a major indispensable constraint of FPT.
In cells, long chain free fatty acids (FFA) are trapped and channeled to the different metabolic pathways upon activation by acyl-CoA synthetases (ACSLs). Mammalian cells are endowed of five homologs of these enzymes, which select FFA, although specificity is not strict [104]. Specifically, ACSL4 exhibits a
Transcriptional and translational regulation of GPx4 expression
GPx4 activity is obviously regulated by selenium availability. Besides its intrinsic role in enzyme catalysis, Se affects the level of the different isoforms of the tRNA[Ser]Sec [107] and, for some glutathione peroxidases, which in this respect are the most studied among selenoproteins, also mRNA stability [108]. In addition, the 3’ UTR of the mRNA encoding for selenoproteins, which is indispensable in recoding the UGA termination codon for selenocysteine insertion, also modulates Sec
Inactivation of GPx4
FPT was identified as a novel form of RCD by screening cancer-specific drug candidates whose suggested molecular mechanism was inhibition of cellular GPx4 activity.
The observation that, in vitro, 1S, 3R-RSL3 does not inhibit purified GPx4 was an intriguing, unexpected evidence [120]. Since inhibition required cytosol, presence of an “inhibition permitting activity” was postulated and the protein accounting for it purified. This protein, 14.3.3ε, interacts with GPx4 and permits the alkylation of
Conclusions and perspectives
Congruence between enzymology and cell biology is a desirable achievement for the accurate understanding of a (patho)physiological event. This is the case of GPx4, first described in vitro as a unique anti-peroxidant, fully characterized enzyme, and re-discovered decades later as the controller of survival or death operated by FPT. It was known from decades that missing GPx4 activity leads to LPO, and what additionally we learned today is that this has a major impact in neurodegenerative
Acknowledgements
This article did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
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