REVIEW GPX4 www.proteomics-journal.com GPX4 at the Crossroads of Homeostasis and Giovanni C. Forcina and Scott J. Dixon*

formation of toxic radicals (e.g., R-O•).[5] Oxygen is necessary for aerobic metabolism but can cause the harmful The eight mammalian GPX fall oxidation of and other macromolecules. Oxidation of cholesterol and into three clades based on amino acid phospholipids containing polyunsaturated fatty acyl chains can lead to lipid sequence similarity: GPX1 and GPX2; peroxidation, membrane damage, and cell death. Lipid hydroperoxides are key GPX3, GPX5, and GPX6; and GPX4, GPX7, and GPX8.[6] GPX1–4 and 6 (in intermediates in the process of lipid peroxidation. The lipid hydroperoxidase humans) are that contain 4 (GPX4) converts lipid hydroperoxides to lipid an essential in the + alcohols, and this process prevents the iron (Fe2 )-dependent formation of , while GPX5, 6 (in mouse and toxic lipid (ROS). Inhibition of GPX4 function leads to rats), 7, and 8 use an active site lipid peroxidation and can result in the induction of ferroptosis, an instead. Unlike other family members, GPX4 (PHGPx) can act as a phospholipid iron-dependent, non-apoptotic form of cell death. This review describes the hydroperoxidase to reduce lipid perox- formation of reactive lipid species, the function of GPX4 in preventing ides to lipid alcohols.[7,8] Thus,GPX4ac- oxidative lipid damage, and the link between GPX4 dysfunction, lipid tivity is essential to maintain lipid home- oxidation, and the induction of ferroptosis. ostasis in the cell, prevent the accumula- tion of toxic lipid ROS and thereby block the onset of an oxidative, iron-dependent, non-apoptotic mode of cell death termed 1. Introduction ferroptosis.[9,10] Cell death can be executed by a number of regulated pathways Oxygenation of the planet, which began roughly 2.3 billion years including apoptosis and the non-apoptotic processes of necrop- ago, paved the way for the evolution of complex, multicellular tosis, pyroptosis, and ferroptosis.[11–13] Compared to apoptosis, life.[1,2] The O -dependent oxidation of carbon fuels in the mi- 2 necroptosis, and other forms of non-apoptotic cell death, fer- tochondria allows for the generation of abundant ATP, but also roptosis is unique in the central involvement of iron-dependent gives rise to partially reduced oxygen species (i.e., reactive oxy- lipid ROS accumulation (Figure 2).[14,15] Ferroptosis can be trig- gen species [ROS]). At low levels, ROS are important signaling [3,4] gered by small molecules that block GSH synthesis or GPX4 ac- molecules, but at higher levels these species can be toxic. To − tivity. Erastin inhibits system x -mediated cystine import.[9,16] defend against ROS toxicity, aerobic cells and organisms have c Within the cell, cystine is reduced to cysteine, which is rate- evolved a multifaceted network of and metabolites to − limiting for GSH synthesis in many cells.[17] Thus, system x protect against oxidative damage and to prevent the onset of cell c inhibition leads to GSH depletion and inactivation of GPX4 death.[4] (and presumably other GPX enzymes). Direct inhibition of GSH One essential axis of defense conserved in eukary- biosynthesis, or genetic manipulations that lead to lower steady- otes is dependent on the non-ribosomal tripeptide glutathione state intracellular GSH levels, can also trigger or sensitize to (Figure 1). Glutathione can cycle between reduced (GSH) and ferroptosis.[18,19] By contrast, 1S,3R-RSL3 (hereafter RSL3), and oxidized (GSSG) states, enabling this metabolite to participate in related molecules, covalently inactivate GPX4 by binding to the biochemical reactions. Glutathione (GPXs) are active site selenocysteine.[18,20,21] GPX4 expression and/or evolutionarily highly conserved enzymes that use GSH as a cofac- activity can also be inhibited indirectly by the small molecules tor to reduce peroxides (e.g., R–OOH) to their corresponding al- FIN56 and FINO , through mechanisms that remain only par- cohols (R–OH), thereby limiting the transition metal-dependent 2 tially characterized.[22–24] In susceptible cells, indirect or direct GPX4 inactiva- tion unleashes a lethal lipid peroxidation process centered G. C. Forcina, Dr. S. J. Dixon on the oxidative destruction of membrane polyunsaturated Department of Biology phospholipids.[20,25] This process can be induced in certain Stanford University Stanford, CA 94305, USA cancer cell populations, potentially in a controlled manner as E-mail: [email protected] a therapeutic approach; it is also a mechanism of pathologi- cal cell death in mammals.[10] Ferroptosis, or closely related The ORCID identification number(s) for the author(s) of this article iron-dependent, oxidative processes, may also be initiated in can be found under https://doi.org/10.1002/pmic.201800311 diverse organisms including plants, fish, and protozoa.[26–28] It is DOI: 10.1002/pmic.201800311 therefore of great interest to understand how this process can be

Proteomics 2019, 1800311 1800311 (1 of 11) C 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.advancedsciencenews.com www.proteomics-journal.com opposed by glutathione peroxidases such as GPX4 which prevent Giovanni C. Forcina completed his lipid peroxidation. Here, we review the molecular mechanisms B.Sc. in molecular biophysics and that link lipid peroxidation, GPX4 and ferroptosis, and describe biochemistry at Yale University. He is the burgeoning set of conditions where this lethal process currently a Ph.D. candidate at Stan- contributes to cell death. ford University in the Department of Biology and the Chemical/Biology Interface training program. 2. Lipid Peroxide Formation and Propagation The membranes of mammalian cells are composed mainly of Scott J. Dixon received a Ph.D. in med- phospholipids and sterols.[29] Lipid peroxidation is the process ical genetics from the University of through which lipids that are capable of non-enzymatic oxidation Torontoin 2007. He then completed a (i.e., autoxidizable) react with free radicals to propagate a radical postdoctoral fellowship with Dr. Brent chain reaction. Lipid peroxidation occurs in three steps (Scheme Stockwell at Columbia University in 1; Figure 3A)[30]: 2013. He opened his lab at Stanford University in 2014, and is currently an · · L − H + R → L + R − H(1)assistant professor in the Department of Biology and a fellow of the ChEM-H (Chemistry, Engineer- · · ing & Medicine for Human Health) initiative. L + O2 → LOO (2a)

· · LOO + L − H → L + L − OOH (2b) Cholesterol and other sterols are also potential targets for − + 2+ → · + − + 3+ L OOH Fe LO OH Fe (2c) autoxidation.[36] Canonical oxidation of cholesterol occurs when a free radical species abstracts a hydrogen atom from the C- · · LOO + LOO → Nonradical breakdown products (3) 7 position of cholesterol. This C─H bond has a BDE of ࣈ78– 83 kcal mol−1, in between the BDE of motile hydrogen atoms The first step is the initiation reaction which occurs when a on PUFAs and MUFAs.[36,37] Though the C─H bond is slightly hydrogen atom is abstracted from a lipid species (L) by a reac- stronger in cholesterol than in PUFAs, the abundance of choles- • • tive species (R , e.g., soluble hydroxyl radical, OH ), generating a terol in membranes, making up to 50% molar lipid content in • lipid radical (L ). The second step, comprising three substeps, is some cell types,[38] makes this species an important source of au- the propagation reaction. The lipid radical can react with diatomic toxidizable lipids in the lipid peroxidation reaction. After hydro- • oxygen (O2) to form a lipid peroxyl radical (L–OO ). These radi- gen atom abstraction, the C-7 position bears a free radical that cals can then abstract hydrogen atoms from other lipid species, can react with diatomic oxygen and generate a cholesterol per- forming lipid peroxides (L–OOH) while propagating the radical oxide. Analogous to the behavior of polyunsaturated hydroper- process to adjacent lipid molecules. Redox-active transition met- oxides, reactive metals like Fe2+ can generate cholesterol per- + als such as ferrous iron (Fe2 ) can then catalyze the generation oxide radicals that can further propagate the lipid peroxidation • of highly reactive lipid alkoxyl radicals (L-O ) from lipid hydroper- reaction (Figure 3B). Cholesterol can also be oxidized at the C- oxides via Fenton chemistry.[31] The last step of the peroxidation 4, C-5, and C-6 positions to yield a variety of different oxidized reaction is termination, where enough radical species sponta- cholesterol species that contribute to the peroxidation reaction.[36] neously react to yield non-radical breakdown products. Whether cholesterol oxidation contributes to ferroptosis is not Not all lipids are equally susceptible to oxidation. Saturated and known. monounsaturated fatty acids (MUFAs) contain only alkyl- and al- lylic carbon–hydrogen bonds, which have bond dissociation ener- −1 −1 [32] gies (BDEs) of 101 kcal mol and 88 kcal mol , respectively. 3. GPX4 Structure and Function In contrast, polyunsaturated fatty acyl chains, which contain bis- allylic carbon–hydrogen bonds, have weaker C─HBDEsofabout GPX4 reduces lipid hydroperoxides to non-toxic lipid alcohols, 75 kcal mol−1.[33] Hydrogen atom abstraction from PUFAs is thereby limiting the propagation of lipid peroxidation within the therefore more favorable than from monounsaturated or satu- membrane. Compared to other GPX enzymes, GPX4 is unique rated fatty acyl chains. Indeed, the higher the number of bis- for its ability to reduce larger organic peroxides, including allylic hydrogen atoms in a given acyl chain, the more likely it is to polyunsaturated lipids and sterols.[7,8] In humans, GPX4 exists be oxidizable, and the rate of lipid peroxidation scales with the de- as a single copy on 19.[39] GPX4 transcription gree of unsaturation in membrane phospholipids[34] (Figure 3A). is regulated by stimulating proteins 1 and 3 (SP1/3) and nuclear While less energetically favorable, monounsaturated fatty acyl factor Y (NF-Y), which are regulated by several other transcrip- chains can also be oxidized in response to small molecule ROS tion factors including cAMP-response element modulator-tau inducers and physiological stimuli (e.g., see ref.35). While a role (CREM-tau), early growth response protein 1 (EGR1), nuclear for PUFA oxidation in ferroptosis is established, whether the oxi- factor κB(NF-κB), and sterol regulatory-binding element 1 dation of monounsaturated phospholipids contributes to ferrop- (SREBP1).[40] SREBP1 is a master transcriptional regulator of tosis is unclear but appears unlikely (see further). lipid biosynthetic , potentially coordinating lipid synthesis

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R-OOH R-OH

GPX enzyme

O

HO SH HN O OO O OO NH H H 2 N S N OH HO N OH HO N S H H NH O NH O O 2 2 O NH OH

O

Reduced glutathione (GSH) Oxidized glutathione (GSSG) Figure 1. Glutathione and its utilization by glutathione peroxidases. Glutathione can exist in the monomeric reduced (GSH) state, or the oxidized (GSSG) disulfide dimer state. Glutathione peroxidases are a family of enzymes that use two molecules of GSH per cycle of catalysis to reduce organic and inorganic peroxides (R-OOH) to alcohols (R-OH), producing one molecule of GSSG. and repair processes.[41] The GPX4 mRNA contains a seleno- sential for the full activity of GPX4. Mutation of selenocysteine cysteine insertion sequence (SECIS) element found in the 3’ to cysteine diminishes GPX4 activity by 90% and heightens sen- untranslated region that encodes an active site selenocysteine sitivity to redox stress.[50] The inability of cysteine to compensate [42] (U46) via a UGA opal codon. Because the UGA codon is for selenocysteine is likely because of differences in pKa between typically read as a stop codon, a distinct set of proteins is needed selenocysteine (5.2) and cysteine (8.2).[48] Since the active site of to direct the incorporation of selenocysteine into GPX4 (and GPX4 is near the protein surface, selenocysteine is more likely other selenoproteins). GPX4 expression is therefore regulated to be in a deprotonated state at neutral pH, which is necessary by the availability of .[43,44] Selenocysteine tRNA is also for its catalytic function. In the context of GPX4, selenocysteine an essential part of this translation machinery, and for efficient is also less likely to become irreversibly oxidized and inactivated selenocysteine addition, this tRNA must first be activated by the than cysteine.[50] addition of an isopentenyl lipid group, a product of the meval- The catalytic cycle of all GPX proteins occurs in two distinct onate (MVA) pathway.[45] This may explain how disruption of the stages following a ping-pong mechanism, whereby the enzyme MVA pathway by statins can cause reduced GPX4 expression active site shuttles between an oxidized and reduced state. The and increased lipid peroxidation and ferroptosis in some cells.[46] first stage involves the reduction of a peroxide species by the Crystal structures of a mutant (U46C) GPX4 and a recombi- active site selenocysteine or cysteine, which is concomitantly nant selenium-containing GPX4 have been solved to 1.55 and oxidized. The second stage replenishes the active site residues 1.3 A,˚ respectively.[47,48] GPX4 is composed of a mo- through the use of a reducing substrate (e.g., GSH), whereby tif made of four solvent-exposed alpha helices and seven beta the active site residues are reduced and the reducing substrate is strands, five of which create a central beta sheet. The multi- oxidized.[6] Intriguingly, GPX proteins do not follow Michaelis– meric glutathione peroxidases (i.e., GPX1–3, 5, 6) contain a sim- Menton reaction kinetics, and it has been proposed that this is ilar thioredoxin motif as well as a solvent-exposed loop that may because the rate-limiting step in peroxide reduction is not the serve as the structural basis for the exclusion of larger organic decay of the GPX–peroxide complex, but rather by the initial substrates from the active site.[47] The active site of GPX4 is sim- binding of the peroxide to the enzyme active site.[51] GPX4 ilar to other selenium-containing glutathione peroxidases and catalyzes the reduction of large organic hydroperoxides through utilizes a conserved near the surface of the pro- the oxidation of the active site selenol (Se–H) to selenenic acid tein consisting of selenocysteine (U46), glutamine (Q81), and (Se–OH), using GSH to reduce the selenenic acid back to the tryptophan (W136). Mutation of any of these residues results in active selenol (Scheme 2).[6] greatly diminished GPX4 function.[47] In addition to these three − residues, N136 may be important for catalysis, as mutation of this GPX4 − Se + L − OOH → GPX4 − SeOH + L − OH (4) residue to alanine, histidine, and aspartate results in impaired en- zymatic activity in Drosophila GPX4 (N137 in the human GPX4), − − + + + → − − + and this residue is conserved across more than 420 GPX primary GPX4 SeO H GSH GPX4 Se SG H2O(5) sequences.[49] Thus, the active site of GPX4 is better described as − a catalytic tetrad. The active site selenocysteine residue is also es- GPX4 − Se − SG + GSH → GPX4 − Se + H + GSSG (6)

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Erastin Sulfsalazine Transferrin-iron Sorafenib Cys2 Glu System x - Glutamate c REDUCTION TFRC Cys Glu

GCL GSS Gly Lysosomal Iron Pools

PUFAs Reduced Oxidized ACSL4 GSH GSSG RSL3 PUFAs** ML162 GPX4 ML210 FINO LPCAT3 2

PUFA PLs PUFA L-OOH ROS, O ACSL3 2 Fe2+ LOX? MUFAs MUFAs**

Ferrostatin-1 L-ROS Liproxstatin-1 − Figure 2. Overview of ferroptosis. Cystine is imported into the cell through the action of system xc to support the synthesis of reduced glutathione (GSH) through the actions of GCL and GSS enzymes. GSH, in turn, is used as a cosubstrate for GPX4 cycling between a reduced and oxidized state to restore the active site selenocysteine of GPX4. GPX4 reduces lipid hydroperoxides to their corresponding alcohols. Ferroptosis can be initiated by compounds that − inhibit system xc or GPX4, and leads to the accumulation of toxic lipid ROS. Lipid ROS is formed by iron-mediated Fenton chemistry which produces alkoxyl radicals on PUFA hydroperoxides, and other reactive species. Intracellular iron may be stored in lysosomes after receptor mediated endocytosis of transferrin–iron complexes by the transferrin receptor (TFRC). PUFAs are incorporated into membrane PLs by the actions of ACSL4 and LPCAT3. MUFA activated by ACSL3 compete with PUFAs activated by ACSL4 for incorporation into PLs. Lipophilic and iron chelators suppress ferroptosis by blocking lipid ROS accumulation. Abbreviations: ACSL3, acyl–CoA synthetase long-chain family member 3; ACSL4, acyl–CoA synthetase long-chain family member 4; GCL, glutathione cysteine ; GPX4, 4; GSS, glutathione synthetase; LPCAT3, lysophosphatidylcholine acyltransferase 3; L–OOH, lipid hydroperoxide; L–ROS, lipid reactive oxygen species; MUFA, monounsaturated fatty acid; MUFA**, activated MUFA; PL, phospholipid; PUFA, polyunsaturated fatty acid; PUFA**, activated PUFA; TFRC, transferrin receptor.

A crystal structure of seleno–GPX4 observed the presence of a essential for ferroptosis. At a more detailed level, it is important seleninic acid (Se–OO—) in the enzyme active site.[48] It is there- to understand which lipids are modified and to pinpoint the fore possible that GPX4 goes through low oxidation cycles of se- nature of these modifications. In erastin-treated HT-1080 cells, lenol to selenenic acid and high oxidation cycles of selenenic acid numerous PUFA free fatty acids, including eicosapentenoate (a to seleninic acid, depending on the burden of a derivative of arachidonic acid), linoleate, linolenate, and docosa- cell. These two catalytic states could have differing biological ac- hexaenoate, are depleted.[53] All of these changes are attenuated tivities and serve as an adaptive mechanism to increased oxidative by cotreatmemt with ferrostatin-1, indicating that they occur stress, but this remains to be verified experimentally. downstream of lipid peroxidation.[53] In mouse embryonic fi- broblasts (MEFs) treated with RSL3, or where Gpx4 is genetically inactivated, a specific subset of phosphatidylethanolamines (PEs) 4. Lipid Peroxidation and Ferroptosis esterified with arachidonoyl (C20:4, AA) and adrenoyl (C20:5, AdA) acyl chains are primary targets for peroxidation.[54,55] These The death of cells treated with erastin or RSL3 can be potently oxidized AA and AdA PE phospholipids (i.e., PUFA-PLs) were suppressed by lipophilic radical-trapping antioxidants including doubly oxidized at the C-15 position while other AA and AdA ferrostatin-1, liproxstatin-1, and α-tocopherol.[9,46–50] Feeding PEs were triply oxidized (e.g., peroxidation at C-15 with an cells PUFA free fatty acids where bis-allylic hydrogens have been additional hydroxyl group at another site on the acyl chain). replaced with deuterium atoms, which are less susceptible to Direct supplementation of PE–AA–OOH to RSL3-treated cells oxidation, renders these cells less susceptible to ferroptosis.[20,52] enhanced ferroptosis, suggesting that oxidation of these species Together, these data demonstrate that lipid peroxidation is is important for cell death.[54] The oxidation of polyunsaturated

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A H H RO PUFA Propagation O - Initiation ROS (e.g. O2 , HOO∙) • RO O2 RO O O •OO O2 RO

O HOO Fe2+ Termination RO Lipid Breakdown Products O •O

Biological Membrane B

2+ ROS O2 Fe

6 • HO 5 HO HO OOH HO O• Cholesterol

Oxysterol Breakdown Products

Figure 3. Overview of lipid peroxidation. A) Polyunsaturated fatty acids (PUFAs) contain bis-allylic hydrogen atoms (red) that can be abstracted by reactive species to yield lipid radical species. Lipid peroxidation is initiated when ROS abstract one of these motile H atoms. In the presence of oxygen, lipid radical species can be oxidized to lipid hydroperoxides. Hydroperoxides can react with ferrous iron to produce reactive alkoxyl radicals via Fenton chemistry. Lipid alkoxyl or peroxyl radicals propagate a free radical chain reaction by abstracting other motile H atoms. Termination occurs when enough radicals are generated such that they spontaneously react to yield non-radical breakdown products. B) Reactive species can modify cholesterol at the 5,6 carbon–carbon double bond (red). In the presence of oxygen, cholesterol can be made into a peroxide at the 4, 5, 6, or 7 position. This cholesterol peroxide can be further oxidized by ferrous iron to generate a reactive species that can propagate a free radical chain reaction. Cholesterol oxidation can occur at several positions, but for simplicity, only oxidation at C-7 is shown.

PEs specifically on the sn-2 acyl chain can be regulated by the genetic disruption of acyl–CoA synthetase long-chain family protein phosphatidylethanolamine-binding protein 1 (PEBP1), member 4 (ACSL4) and lysophosphatidylcholine acyltransferase which complexes with PEs and the pro-oxidant enzyme 15- 3(LPCAT3) conferred resistance to ferroptosis.[55,58] ACSL4 lipoxygenase (15-LOX; see also further).[56,57] PEBP1 genetic preferentially activates PUFAs, like AA, for incorporation into silencing leads to ferroptosis resistance while its overexpression lipids while LPCAT3 preferentially incorporates PUFA–CoA increases ferroptosis sensitivity in cultured cells.[57] The impor- into membrane phospholipids[59]; disruption of ACSL4 and tance of PUFA-PL oxidation in driving ferroptosis is further LPCAT3 therefore decreases the membrane load of oxidizable substantiated by genetic suppressor screens of ferroptosis where PUFA-PLs.

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GPX4 not only reduces PUFA hydroperoxides, but it can function by altering the physicochemical properties of phospho- also act to reduce oxidized cholesterol and its esters. In- lipid acyl chains (e.g., oxidized acyl chains may “float” to the creased levels of oxidized cholesterols are observed in erastin- surface of the membrane to interact with the aqueous phase); treated cells,[53] but whether cholesterol oxidation is required computational modeling of the plasma membrane with oxidized to promote ferroptosis and how this might interact with the lipids suggests that this causes dramatic changes to membrane oxidation of polyunsaturated phospholipids is unclear. Inter- shape, curvature, and accessibility to oxidants.[66,67] Oxidized acyl estingly, the proferroptotic compound FIN56 activates squa- chains are preferred substrates for certain lipases, and signifi- lene synthase/farnesyl–diphosphate:farnesyl–diphosphate far- cant increases in lysophospholipids are observed in ferroptotic nesyl (SQS/FDFT1), a downstream enzyme in cells.[18] A phospholipase A2 inhibitor can prevent ferroptosis the mevalonate pathway leading to cholesterol synthesis.[22] in inducible Gpx4 null cells, which suggests that the cleavage Activation of this pathway could sensitize to ferroptosis in sev- of oxidized acyl chains could contribute directly to membrane eral ways. Activation of SQS could deplete isopentenyl pyrophos- damage and cell death. Just prior to the onset of membrane per- phate, which would reduce the levels of lipidated Sec tRNA,[45] meabilization, cells about to undergo ferroptosis display the for- and consequently limit the availability of selenocysteine for in- mation of membrane “blisters” that could be the result from corporation into GPX4.[22] SQS activation could also enhance the oxidation of membrane phospholipids and excess genera- flux through the squalene synthesis pathway, reducing levels tion of lysophospholipids.[64] Numerous aldehydic compounds of the upstream intermediate (farnesyl pyrophosphate) used can also arise from non-radical breakdown products and frag- [22] [68] to synthesize the isoprenoid coenzyme Q10. Outside of its mented lipids produced by the peroxidation reaction. The best- canonical functions in electron transport chain, CoQ10 moon- studied examples of aldehydic lipid breakdown products are 4- lights as a lipophilic antioxidant, and depletion of CoQ10 could hydroxynonenal (4-HNE) and malondialdehyde (MDA), which potentially induce or sensitize to ferroptosis.[22,53] Yet another can form mutagenic DNA adducts and also covalently modify cys- consequence of activating SQS could be increasing cholesterol teine, histidine, and lysine residues within proteins.[69,70] Chemo- synthesis. Higher cholesterol levels may expand the pool of oxi- proteomic profiling of proteins modified by lipid-derived elec- dizable lipids and contribute to lipid peroxidation during ferrop- trophiles revealed over 400 modified proteins in cells undergoing tosis. Of note, in ALK+ large cell lymphomas, the accumulation ferroptosis, though the functional significance of these modifi- of squalene due to the loss of squalene monooxygenase expres- cations is not known.[71] The upregulation of AKR1C-family en- sion inhibits lipid peroxidation and the onset of ferroptosis.[60] zymes that can detoxify lipid aldehydes is correlated with partial It is therefore possible that different MVA intermediates and resistance to ferroptosis.[16,72] However, the fact that high levels products (e.g., cholesterol) will have cell type-specific roles in of gene upregulation are not associated with complete ferropto- modulating ferroptosis sensitivity. sis resistance suggests that once a membrane has reached the Where lipid ROS accumulate within the cell to cause stage where lipid aldehydes are formed, overall membrane in- ferroptosis is an active area of investigation. Oxidation of tegrity may already be fatally compromised. lipid ROS-sensitive probes in cells undergoing ferroptosis has been observed at lysosomes, the endoplasmic reticulum and mitochondria.[54,61,62] Most recently, confocal microscopy com- 5.TheRoleofIroninLipidPeroxidationand bined with the redox membrane dye C11-BODIPY 581/591 has Ferroptosis identified “rings” of lipid oxidation surrounding ferroptotic cells that overlap with plasma membrane markers.[63,64] Interestingly, During ferroptosis, lipid peroxidation occurs in an iron- in cells supplemented with exogenous monounsaturated fatty dependent manner. Accordingly, lipid peroxidation and fer- acids (MUFAs), lipid oxidation at the plasma membrane is roptotic cell death induced by perturbation of glutathione blocked and ferroptosis is inhibited, indicating that oxidation metabolism or GPX4 activity are fully suppressed by iron of plasma membrane lipids may be necessary for the execu- chelators like deferoxamine (DFO) and ciclopirox (CPX).[9,73,74] tion of ferroptosis.[64] This protective effect of exogenous MU- Disruption of the iron-sulfur cluster biogenesis pathway enzyme FAs requires free fatty acid activation by ACSL3, suggesting cysteine desulfurase NFS1 sensitizes to ferroptosis in cultured that competition between ACSL4-driven incorporation of highly cancer cells and in xenograft tumor models, most likely, by oxidizable PUFAs versus ACSL3-driven incorporation of less increasing the pool of free iron within the cell.[75] Normally, iron oxidizable MUFAs into phospholipids shapes the sensitivity of is imported into the cell via the transferrin/transferrin receptor the plasma membrane to oxidation. A different enzyme, fatty acid system and liberated from transferrin within lysosomes. Immun- desaturase 2 (FADS2), which converts the saturated fatty acid odepletion of transferrin or genetic silencing of transferrin recep- palmitate (C16:0) to the MUFA sapienate (cis-6-C16:1), can poten- tor inhibits ferroptosis.[74,76] Iron storage protein, the ferritins, tially promote ferroptosis sensitivity by limiting the incorporation are also degraded in lysosomes, and disruption of this process of other protective MUFAs into membrane phospholipids.[65] can inhibit ferroptosis.[61,77,78] Erastin treatment results in iron ac- Further investigation is required to understand how different cumulation in the lysosome and, to a lesser extent, the endoplas- MUFAs modulate ferroptosis sensitivity. mic reticulum.[79] However, treatment with How lipid peroxidation leads to frank membrane permeabi- (H2O2), which promotes Fenton chemistry within lysosomes and lization during ferroptosis remains unclear. Normally, the acyl causes lysosomal membrane permeabilization, does not trigger a chains of membrane phospholipids are well ordered within the ferrostatin-1-sensitive ferroptosis phenotype.[53] Thus, it may be hydrophobic inner leaflet of the bilayer. However, the forma- that iron transits through the lysosome, but then acts at another tion of lipid peroxides may distort membrane structure and site to promote ferroptosis. By contrast, disruption of processes

Proteomics 2019, 1800311 1800311 (6 of 11) C 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.advancedsciencenews.com www.proteomics-journal.com that act as “sinks” for free intracellular iron sensitize to ferrop- tive site of Gpx4 in place of Sec is compatible with embryonic tosis by increasing the basal intracellular levels of this metal. development, but leads to fatal epileptic seizures following birth Exactly how iron promotes lipid peroxidation and ferropto- due to loss of parvalbumin positive interneurons.[50] Inherited sis is controversial. Iron is required for the activity of lipoxy- mutations in GPX4 in humans are associated with Sedaghatian- genase (LOX) enzymes that generate PUFA hydroperoxides type spondylometaphyseal chondrodysplasia, a lethal rare disease as part of their normal function (e.g., generating signaling that results in skeletal, heart, and brain abnormalities plausibly eicosanoids.[80]). Iron chelation could therefore potentially in- linked to increased cell death.[98] activate LOX enzymes. As noted above, complex formation Of note, GPX4 has three isoforms: mitochondrial (mGPX4), between 15-LOX and PEBP1 may act in some models to non-mitochondrial (also known as cytosolic, cGPX4), and nu- specifically promote the proferroptotic oxidation of polyunsatu- clear (nGPX4 or snGPX4).[99,100] All three isoforms are ubiqui- rated phosphatidylethanolamines.[54,57] LOX orthologs secreted tously expressed in all tissues, but mGPX4 and nGPX4 are ex- by Pseudomonas aeruginosa can also trigger PUFA-PE oxidation pressed at lower levels than cGPX4 in all tissues except the and “theft ferroptosis” in host epithelial cells.[81] It has also been testes.[100,101] Mitochondrial function does not appear to be es- proposed that vitamin E may inhibit ferroptosis not merely as a sential for ferroptosis[9,102] (but see refs.62,103). Thus, cGPX4 is general lipophilic antioxidant, but as a specific inhibitor of 15- likely sufficient to suppress ferroptosis, but this has not been LOX enzyme activity.[54,82] The small molecule LOX inhibitors definitively shown. Collectively, the above findings suggest that baicalein and nordihydroguaiaretic acid (NDGA) can also inhibit many cells exist on the verge of ferroptosis, and that inactivation ferroptosis in various models,[83,84] and chemical activators of of GPX4 tips them over the edge into cell death. LOX enzyme function can enhance ferroptosis.[84,85] However, An extensive literature, predating by decades the description genetic deletion of individual Alox genes does not prevent fer- of ferroptosis in 2012, links lipid peroxidation to cell death, roptosis in mice lacking Gpx4, cells that express no endoge- tissue damage, and pathology (e.g., refs.15,104–107). More nous LOX enzymes are still capable of undergoing ferroptosis, recently, the induction of ferroptosis has been more specifically and a number of commonly employed small molecule LOX in- linked to specific pathological processes thanks in large part hibitors can act directly as radical-trapping antioxidants, provid- to the development of potent and specific inhibitors such as ing a LOX-independent explanation for their ability to inhibit ferrostatin-1 and liproxstatin-1. High concentrations of extracel- ferroptosis.[52,86,87] Thus, LOX enzyme activity may not be uni- lular glutamate induce cell death in ex vivo rat hippocampal brain versally essential for ferroptosis, but more selectively required in slices that was partially suppressed by ferrostatin-1 and the iron certain cells or tissues and/or accelerate this process. chelator ciclopirox, providing the first evidence that ferroptosis In the absence of LOX enzyme activity, it is possible that labile could be involved in neuronal cell death.[9] Ferrostatin-1-sensitive (i.e., free) intracellular iron is sufficient to promote ferroptosis cell death can also be induced in cell models of Huntington’s by catalyzing Fenton chemistry on soluble peroxides or lipid per- disease, periventricular leukomalacia, and Sertoli cell death oxides to generate toxic hydroxyl or lipid alkoxyl radicals, which induced by combined oxygen and glucose deprivation.[53,108] can then propagate lipid peroxidation (see Scheme 1). This would Ischemia-reperfusion injury (IRI), occurring when a tissue is explain how cells that do not express LOX enzymes remain sen- deprived of blood or oxygen for an extended period and then sitive to ferroptosis, and is consistent with evidence that elevat- reperfused (e.g., after stroke), can trigger ferroptosis in the ing the concentration of intracellular iron can itself be sufficient small intestine, testes, liver, heart, kidney, and brain that can be to trigger ferroptosis in certain contexts[88] (but see also ref.9). prevented by known ferroptosis inhibitors or genetic inhibition Where free iron may act to promote ferroptosis is unclear. Re- of ACSL4 expression.[76,109–113] Inhibition of ferroptosis can cently, it was shown that lipid peroxidation occurring locally at also improve brain cell survival and neurological outcomes the plasma membrane is necessary for ferroptosis.[64] Some evi- after hemorrhagic stroke and traumatic brain injury.[113–115] dence suggests that iron can bind directly to free fatty acids and Intriguingly, important features of ferroptosis, including the phospholipids.[89,90] Speculatively, a pool of iron localized in this loss of GSH, increased ROS, and lipid peroxidation, have been manner to the plasma membrane could promote lipid peroxida- observed in models of Alzheimer’s disease and Parkinson’s tion at this site. disease, suggesting a potential link between these diseases and ferroptosis.[116–120] Characterizing the specific GPX4-regulated lipid peroxidation events in diseases states—pioneered by the 6. GPX4, Lipid Peroxidation, and Ferroptosis in Kagan laboratory for asthma, acute kidney injury, and traumatic [57] Development and Disease brain injury —remains an important goal for other conditions where ferroptosis is implicated. Gpx4 is an essential gene required for mouse embryonic develop- The induction of ferroptosis may also be a promising anti- ment, with genetic inactivation or silencing of Gpx4 expression cancer strategy. Erastin, RSL3, and related molecules are useful resulting in death around embryonic day 7.5, likely from defects tool compounds, but until very recently have not proven to be in brain development.[50,91,92] Inactivation of Gpx4 in post-natal especially effective in vivo due to poor drug-like properties. One mice is sufficient to promote lethal, acute renal failure, suggest- agent that can trigger ferroptosis in xenograft tumors in vivo are ing an important homeostatic role for this protein in the devel- engineered nanoparticles that deliver high concentrations of iron oping kidney.[93] Tissue-specific disruption of Gpx4 can also lead into the cell.[88] Enzymatic depletion of plasma cysteine and cys- to the destruction of muscle, neuronal and other cells, indicat- tine using an engineered enzyme, cyst(e)inase, can also reduce ing a broad requirement for Gpx4 in the survival of many adult tumor growth in various xenograft models of cancer in mice, but cells.[94–97] Genetic engineering of mice to express Cys at the ac- does not result in regressions.[121,122] Whether these two agents

Proteomics 2019, 1800311 1800311 (7 of 11) C 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.advancedsciencenews.com www.proteomics-journal.com cause GPX4 inactivation and how lipid peroxidation is affected at null mice, Prdx6 null mice are viable with relatively restricted the molecular level are not clear. Most recently, treatment with the degenerative phenotypes,[135] suggesting that GPX4 may be bioavailable erastin analogue imidazole ketone erastin (IKE) em- more important than PRDX6 in phospholipid repair and the bedded within a biodegradable polyethylene glycol-poly(lactic-co- prevention of ferroptosis in most tissues, but that some degree glycolic acid) carrier reduced tumor burden in a xenograft model of partially overlapping function is also possible. Until these of diffuse large B cell lymphoma (DLBCL).[123] Protein adducts possibilities are examined more fully, GPX4 appears to be the key characteristic of enhanced lipid peroxidation were detected in tu- enzyme sitting at the crossroads of oxidative lipid homeostasis mors treated with this agent, consistent with increased lipid per- and ferroptosis. A better understanding of how inactivation of oxidation. Metabolically stable small molecule GPX4 inhibitors GPX4 leads to lipid peroxidation and ferroptosis should yield suitable for in vivo do not exist, but preclinical investigations us- valuable new insights into the regulation of cell health and may ing cell lines engineered to lack GPX4 suggest that targeting this yield novel therapies for a slew of important human diseases. protein may be especially effective for cells that may undergone an epithelial-to-mesenchymal transition or that exhibit tolerance or resistance to targeted or cytotoxic chemotherapies.[46,124,125] Acknowledgements How these different cell states re-wire intracellular metabolism or GPX4 function to increase sensitivity to lipid peroxidation- The authors thank Leslie Magtanong for comments on the manuscript. dependent cell death remains only partially understood. G.C.F. is supported by the Stanford ChEM-H Chemistry/Biology In- terface predoctoral training program. S.J.D. is supported by the NIM (1R01GM122923). 7. Summary and Future Directions Inactivation of GPX4 leads to the accumulation of lipid per- Conflict of Interest oxides and ferroptotic cell death. The activity of GPX4 is re- S.J.D. is on the scientific advisory board of Ferro Therapeutics. quired to prevent excess lipid hydroperoxide accumulation by converting these species into non-toxic lipid alcohols. GPX4 is therefore reminiscent of metabolite repair enzymes that Keywords prevent the accumulation of potentially harmful byproducts of cellular metabolism.[126] However, signaling lipids such as cell death, ferroptosis, glutathione, GPX4, iron, lipids, reactive oxygen eicosanoids are derived from enzymatically oxygenated PUFAs species and play important signaling roles, particularly in mediating inflammation.[127] Thus, GPX4 serves as both a guardian against Received: December 7, 2018 oxidative lipid damage as well as a key regulator of physiologi- Revised: February 27, 2019 Published online: cally important signaling lipids, and these two functions must be carefully balanced to maintain homeostasis.[50] There is some evi- dence that GPX4 can be post-translationally modified by tyrosine [128,129] , N-glycosylation, and possibly acylation. [1] T. W. Lyons, C. T. Reinhard, N. J. Planavsky, Nature 2014, 506, 307. GPX4 protein levels can also be directly regulated by chaperone- [2] D. 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