*2.2. Glutathione Peroxidase 4 and Ferroptosis*

Cells have several escape mechanisms against cell death [36,37]. In the ferroptotic process, one of the most important and most studied so far is the enzyme glutathione peroxidase 4 (GPx4), (also called Phospholipid Hydroperoxide Glutathione Peroxidase (PHGPx)) [38,39]. In the human organism, there are several isozymes of glutathione peroxidase, which vary in cell location and substrate specificity [40]. The GPx4 enzyme is a selenoprotein, with approximately 20–21 kDa, composed of 197 amino acids, and encoded by the GPx4 gene in chromosome 19 localization [41]. GPx4 has in its active site the amino acid selenocysteine, which is necessary for protection against ferroptosis [42]. The catalytic site of selenocysteine involves three different redox states: selenol, selenenic acid and seleninic acid. These different forms of the redox state allow the regulation of the catalytic efficiency of the peroxide reduction, which is dependent on the cellular redox state [43]. The enzymatic activity of GPx4 is vital to cells, since the enzyme can reduce H2O2 and is the only enzyme that can reduce phospholipid hydroperoxides [44].

In addition, by structural similarity, GPx4 can reduce both peroxidized fatty acids and esterified cholesterol hydroperoxides, as well as thymine hydroperoxide, a product of free radical attack on DNA. The reduction reaction can occur in membranes, in the cytoplasm and/or in lipoproteins [45,46]. In the antiferroptotic process, the GPx4 enzyme directly reduces toxic lipid peroxides (PL-OOH) to non-toxic lipid alcohols (PL-OH) using reduced glutathione (GSH) as a substrate [47–49]. The synthesis of GSH through the cystine/glutamate antiporter system xc− is a limiting step for the function of detoxification of lipid peroxides by GPx4 [50]. The rate-limiting compound of GSH synthesis is the non-essential amino acid cysteine. Cysteine can be imported into cells directly or in its oxidized form, cystine, through system xc−. Within the cell, cystine is reduced to cysteine by biosynthesis of GSH [51]. Figure 2 shows the complete GSH biosynthesis pathway.

GPx4 inhibitors, including ML210, ML162 and (1S), (3R)-RSL3 (RSL3), are used as specific ferroptosis inducers [52–55]. Moreover, the overexpression or silencing of the gene coding for 14-3-3 proteins controls the inactivation of GPx4 by RSL3 [56]. In addition, liproxstatin-1 is able to suppress ferroptosis in cells, inhibits mitochondrial lipid peroxidation, and restores the expression of GSH, GPX4 and ferroptosis suppressor protein 1 [57,58]. A variety of ferroptosis inducers can inhibit cystine absorption by inhibiting system xc−, such as: erastin, sulfasalazine and sorafenib, resulting in reduced GPx4 activity in different cells lines. Thus, there is no synthesis of GSH and the activity of GPx4 decreases. As a consequence, there is a reduction in the cell antioxidant capacity and hence increased L-ROS, ultimately leading to ferroptosis [59–67].

GSH biosynthesis is regulated by the ubiquitously expressed transcription factor nuclear factor erythroid-2 related factor 2 (Nrf2). In baseline conditions, Nrf2-dependent transcription is suppressed due to proteasomal degradation in the cytosol by Keap1 (kelch-like ECH-associated protein 1). However, due to the exposure to a variety of different stimuli, including oxidative stress, the ubiquitination and degradation of Nrf2 are blocked, leading to the stabilization and nuclear accumulation of Nrf2, where it induces dependent electrophilic response element (EpRE) gene expression to restore cellular redox homeostasis [72]. Nrf2 regulates several steps of GSH biosynthesis transcription enzymes, such as catalytic and regulatory subunits of glutamate cysteine ligase (GCL), GSH synthase, GPx2, GSH S-transferases (GSTs) and GSH reductase (GR) as well as the light-chain subunit of the xc- [72–75]. Nrf2 is also associated with the regulation of antioxidant enzymes, NADPH: quinine oxidoreductase-1 (NQO-1 and NQO-2) and nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2). In addition,

Nrf2 can regulate iron metabolism enzymes [76,77] and proteins associated with multiple drug resistance (ABCG2, MRP3, MRP4, glutathione S-transferase P (GSTP)) [72,78].

**Figure 2.** Glutathione (GSH) biosynthesis pathway. GSH is known as one of the small-molecule water-soluble antioxidants, the most important of somatic cells. GSH is a linear tripeptide formed by three amino acids: glutamic acid, cysteine and glycine. The thiol group present in the amino acid cysteine is considered the active site responsible for the antioxidant biochemical properties of glutathione. In biological systems, glutathione can be found in reduced form (GSH) or in oxidized form (GSSG). The oxidized form is a heterodimerization of the reduced form. The GSH/GSSG ratio is used to estimate the redox state of biological systems [51]. The rate-limiting compound of GSH synthesis is the non-essential amino acid cysteine. Cysteine can be imported into cells directly or in its oxidized form, cystine, through the cystine/glutamate antiporter system xc−. In humans, on chromosome 4, the SLC7A11 gene (solute carrier family 7A11) encodes the SLCA11 antiporter, which is part of a system called system xc−. The structure of this protein is heterodimeric and includes two chains: a specific light chain, xCT (SLCA11), and a heavy chain, 4F2hc (SLC3A2), which are linked by a disulfide bridge. The xCT chain has 12 transmembrane domains consisting of 501 amino acids, with the N and C terminal regions located intracellularly; it is not glycosylated and has a molecular mass of approximately 55 kDa. The heavy chain, 4F2hc, is a type II glycoprotein with a single transmembrane domain, an intracellular NH 2 terminal and a molecular weight of approximately 85 kDa. The 4F2hc chain is a subunit common to amino acid transport systems, while the xCT chain is unique to cystine/glutamate exchange. System xc− transports amino acids, independently of sodium and dependent on chloride, which are specific to import cystine and export glutamate at the same time through the plasma membrane. Both amino acids are transported in anionic form. The ratio of counter transport between cystine and glutamate is 1:1. Currently, it is known that system xc− is involved in (a) cystine uptake to maintain the extracellular balance of cysteine/redox cystine, (b) cysteine/cystine uptake for GSH synthesis and (c) non-vesicular glutamate export [68]. Within the cell, cystine is reduced to cysteine. This reduction reaction can be performed by intracellular GSH or by the enzyme thioredoxin reductase 1 (TRR1) [69]. The beginning of GSH synthesis is the formation of the γ-glutamylcysteine molecule, which is catalyzed by the enzyme glutamate cysteine ligase (GCL). GCL catalyzes the binding of glutamate and cysteine in the presence of adenosine triphosphate (ATP). Then, the enzyme GSH synthase (GS) catalyzes the formation of GSH through the link between γ-glutamylcysteine and glycine [69]. GSH reduces radicals (R•) non-enzymatically and organic hydroperoxides catalyzed by GSH peroxidase (GPx) and is thus

converted to GSH disulfide (GSSG). GSSG is recycled to GSH by GSH reductase (GR), a reaction that uses reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor [69]. GSH S-transferase (GST) forms GSH (GS-R) adducts from organic molecules (R) and GSH, which together with GSH and GSSG are exported from the cell by ABC transporters, mainly ABC-1 and ABC-G2 [70,71]. Extracellular GSH is metabolized by the γ-glutamyl transferase (GGT) ectoenzyme, which transfers the γ-glutamyl residue to different acceptor amino acids, leading to the formation of a dipeptide containing γ-glutamyl and the cysteine glycine dipeptide, which is cleaved by extracellular dipeptides to generate cysteine and glycine that can be taken up by cells, starting the glutathione biosynthesis cycle [69].

Recently, Doll et al. [79] described an in vitro model, a parallel pathway that included FSP1-CoQ10-NADPH, which cooperates with GPx4 and the glutathione system to suppress lipid peroxidation of phospholipids. Ferroptosis suppressor protein 1 (FSP1) provides protection against ferroptosis induced by the deletion of the GPx4 enzyme via RSL3. This effect is mediated by coenzyme Q10 (CoQ10). The reduced form of the enzyme, ubiquinol, captures lipid peroxyl radicals that mediate lipid peroxidation, while FSP1 catalyzes the regeneration of CoQ10 using NADPH as a cofactor. Moreover, the authors described that the antiferroptotic function of FSP1 is independent of cellular glutathione concentration, GPx4 activity, ACSL4 expression and oxidizable fatty acid content [79]. Coenzyme Q10 is an endogenous lipophilic antioxidant produced in the mevalonate pathway, as well as a part of the mitochondrial respiratory chain, and from the metabolism of fatty acid and pyrimidine [80,81]. Indeed, the homologous proteins MDM2 and MDMX, negative regulators of the tumor suppressor p53, promote ferroptosis by regulating lipid peroxidation by altering PPARα activity. MDM2–MDMX complex inhibition increased the levels of both FSP1 proteins and coenzyme Q10 [82].
