GPX4 at the Crossroads of Lipid Homeostasis and Ferroptosis Giovanni C
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REVIEW GPX4 www.proteomics-journal.com GPX4 at the Crossroads of Lipid Homeostasis and Ferroptosis 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 proteins fall oxidation of lipids 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 selenoproteins that contain glutathione peroxidase 4 (GPX4) converts lipid hydroperoxides to lipid an essential selenocysteine in the enzyme + alcohols, and this process prevents the iron (Fe2 )-dependent formation of active site, while GPX5, 6 (in mouse and toxic lipid reactive oxygen species (ROS). Inhibition of GPX4 function leads to rats), 7, and 8 use an active site cysteine 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 enzymes 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 antioxidant 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 redox biochemical reactions. Glutathione peroxidases (GPXs) are active site selenocysteine.[18,20,21] GPX4 protein 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 gene on chromosome 19.[39] GPX4 transcription gree of unsaturation in membrane phospholipids[34] (Figure 3A).