Achieving Life through Death: Redox Biology of Lipid Peroxidation in Ferroptosis

Abstract

Redox balance is essential for normal brain, hence dis-coordinated oxidative reactions leading to neuronal death, including programs of regulated death, are commonly viewed as an inevitable pathogenic penalty for acute neuro-injury and neurodegenerative diseases. Ferroptosis is one of these programs triggered by dyshomeostasis of three metabolic pillars: iron, thiols, and polyunsaturated phospholipids. This review focuses on: (1) lipid peroxidation (LPO) as the major instrument of cell demise, (2) iron as its catalytic mechanism, and (3) thiols as regulators of pro-ferroptotic signals, hydroperoxy lipids. Given the central role of LPO, we discuss the engagement of selective and specific enzymatic pathways versus random free radical chemical reactions in the context of the phospholipid substrates, their biosynthesis, intracellular location, and related oxygenating machinery as participants in ferroptotic cascades. These concepts are discussed in the light of emerging neuro-therapeutic approaches controlling intracellular production of pro-ferroptotic phospholipid signals and their non-cell-autonomous spreading, leading to ferroptosis-associated necroinflammation.

Keywords: cerebral hemorrhage; cerebral ischemia; glutathione peroxidase 4; lipoxygenase; neurodegeneration; phospholipid; redox lipidomics; regulated cell death; traumatic brain injury.

Fig. 1.. Lipoxygenases (LOXs) catalyze the formation of essential lipid mediators with pro- and anti-inflammatory propensities.

In cells, essential signaling molecules, lipid mediators, are formed by lipoxygenases (LOX) from free PUFA (w-6 and w-3 series). These oxidation products coordinate many metabolic processes and cell responses, including inflammation. 12-LOX and 5-LOX are involved in the generation of pro-inflammatory mediator leukotriene B4 (LTB4) from arachidonic acid (AA). The first step in biosynthesis of hepoxilins A3 and B3 (HXA3 and HXB3) is catalyzed by 12-LOX. 15-LOX is implicated in biosynthesis of anti-inflammatory lipid mediators such as docosahexaenoic acid (DHA) derived resolvins (RvD1 and RVD5), docosapentaenoic acid (DPA) derived protectin D1, AA-derived lipoxins (LXA3). Maresin R1 (MaR1), a DHA-derived lipid mediator generated by 12-LOX, stimulates switch of macrophages from M1 to M2 type. Dysregulated production of equivocal lipid signal by LOXs may lead to the triggering of a specialized program of cell death, ferroptosis, in dis-coordinated cells aberrantly producing confusing signals. Dis-regulation of lipid mediators biosynthesis leads to the assembly and engagement of 15-LOX/PEBP1 enzymatic complexes that start producing pro-ferroptotic death signals. PEBP1 (Phosphatidylethanolamine Binding Protein 1), a scaffold protein, an inhibitor of protein kinase (RAF-K), complexes with 15-LOX, and changes the substrate selectivity of the dioxygenase (AA-PE instead of free AA) and catalyzes a specific oxygenation generating 15-HOO-AA-PE as a ferroptotic cell death signal.

Fig. 2.. Iron chaperone limits the chemical reactivity of cytosolic labile iron pool.

Non-transferrin-bound iron is reduced to Fe(II) by ferrireductases at the plasma membrane surface and transported into the cytosol via divalent metal transporter, DMT1, or Zip14. Circulating iron in the form of Fe(III)₂-transferrin (Tf) binds to transferrin receptor 1 (Tfr1) on the plasma membrane to form Tf-Tfr1 complex, which is endocytosed. At the low pH of the endocytic vesicle, Fe(III) is released from Tf and ferrireductases (e.g., STEAP3) reduce Fe(III) to Fe(II), which is translocated into the cytosol by DMT1. If it remains free, Fe(II) reacts with hydrogen peroxide (H₂O₂) to produce cytotoxic hydroxyl radicals (OH^•) via the Fenton reaction. Cytosolic Fe(II) is coordinated by reduced glutathione (GSH) in the cytosolic labile iron pool. PCBP1 and PCBP2 are iron chaperones that play integral roles in intracellular iron trafficking. Structurally PCBPs contain three tandem K-homology domains (KH1–3) that can bind GSH coordinated Fe(II) with high affinity in 1:1 ratio. Under physiological conditions a significant proportion (>90%) of the labile iron pool may be coordinated by PCBPs. PCBP2 binds iron loaded DMT1 to facilitate iron influx, binds heme degrading heme oxygenase to facilitate iron redistribution, and binds Ferroportin (Fpn1) to facilitate iron efflux. PCBP1 (to a lesser extent, PCBP2) binds and delivers iron to ferritin and mono/di-nuclear iron proteins and Fe-S carrier proteins. Ferritin binds to Nuclear Receptor Coactivator 4 (NcoA4) to undergo lysosomal degradation, process referred to as ferritinophagy. Ferritin iron released in the lysosome can be transferred back to the cytosol or transported to mitochondria. PCBPs may also metalate di-iron containing fatty acid desaturases (e.g. FADS2) and mono-iron containing lipoxygenases (e.g. 15-LOX), which are integral in lipid metabolism. FADS2 contributes to the formation of polyunsaturated fatty acids (PUFAs) and 15-LOX catalyzes the dioxygenation of PUFAs to generate phospholipid hydroperoxide (PL-OOH). Both PUFA and PL-OOH are the targets of the peroxidation reactions associated with ferroptosis. System X_C⁻ imports cystine, which is reduced to cysteine and used to synthesize GSH. Glutathione peroxidase 4 (GPX4) uses GSH to eliminate lipid peroxides formed in phospholipids containing PUFAs. Arrows indicate promotion; broad arrow indicate higher extent; dotted arrow indicates lesser extent.

Fig. 3.. Principle schemas of the reaction mechanism and specific features of enzymatic vs random non-enzymatic peroxidation of PUFA phospholipids.

Both enzymatic - selective (left panel) and non-enzymatic - non-selective (right panel) lipid peroxidation begins with the abstraction of bis-allylic hydrogen, rearrangement of the resonance radical structure, addition of the molecular oxygen leading to the generation of peroxyl radical and the formation of the primary molecular product, hydroperoxy-lipid. During random oxidation all PUFA containing phospholipids undergo oxidation whereby the rates are proportional to the number of readily abstractable bis-allylic H, resulting in the accumulation of a highly diversifies pattern of oxidation products with the dominance of oxygenated PUFA-PLs with 6, 5, 4, 3, and 2 double bonds. LOX-driven oxidation results in the preferential generation of specific AA-PE oxidation yielding 15-HOO-AA-PE. The selectivity and specificity of the reaction is due to the organization of the catalytic site in 15-LOX/PEBP1 complex (Wenzel et al., 2017) resulting in highly selective, site-specific and stereo-selective product- 15-HOO-AA-PE. The substrate radical rearrangement is accompanied by the addition of molecular oxygen delivered via a special channel in the protein. Non-enzymatic lipid peroxidation proceeds by a free radical chain reaction. This process is not specific and non-selective.

Fig. 4.. Most commonly used assays for indirect assessments of different “peroxidation/peroxidase activities associated with the execution of ferroptosis. Note that only Liperfluo assay detects hydroperoxyl-lipids.

i) BODIPY® 581/591 detects “peroxidase activity” resulting in changed fluorescence characteristics of the non-lipidic BODIPY chromophore. Oxidation of butadiene portion (circled in red) of the BODIPY® 581/591 results in a shift of fluorescence emission peak from 590 nm to 510 nm. ii) linoleate-based Click-iT® LAA (linoleamide alkyne) assay. This assay is designed to detect lipid-peroxidation derived modification of protein in fixed cells. Incorporated into cellular membranes Click-iT® LAA can undergo lipid peroxidation resulting in production of 9- and 13-hydroperoxy linoleic acid that further decomposes to unsaturated aldehydes which can modify proteins. These modified proteins are detected by Click-iT® chemistry. iii) Liperfluo fluorogenic assay of hydroperoxy-containing groups. Liperfluo, a perylene derivative containing oligooxyethylene, detects L-OOH. LiperFluo can react with L-OOH and its fluorescence reliably reports intracellular sites of L-OOH accumulation by a fluorescence microscopy. Both free PUFA-OOH and PUFA-OOH esterified into phospholipids display high reactivity toward LiperFluo. The results in the robust fluorescence response of oxidatively modified LiperFluo in cells exposed to pro-ferroptotic stimuli) are shown on the right panel. Chemically, Liperfluo reduces L-OOH to the respective alcohols similarly to the reaction catalyzed by GPX4.

Fig. 5.. A schema explaining the major stages of LC-MS based non-targeted Global (Redox) Lipidomics Analysis (GRLA).

GRLA includes analysis of phospholipids and neutral lipids including their oxidized and oxidatively truncated species and consists of several steps. The first step is separation of lipids by liquid chromatography and their detection by mass spectrometry. Normal and reverse phase chromatographic protocols are used for separation of lipids based on their polarity and hydrophobicity. Electrospray ionization (ESI) is widely used for the detection of lipids and their oxidatively modified species. Second step is the identification of lipids and their oxidation products. This is achieved by using ion trap or high resolution mass spectrometry (MS) orbitrap instrumentations with unlimited fragmentation capacity. Step three includes quantitative analysis and normalization of results. LC-MS and LC-MS/MS can be set up for quantitative analysis using internal standards and calibration curves established with reference standards.

Fig. 6.. Schematic representation of the major stages of lipid redox metabolism leading to the formation of HOO-AA-PE as a pro-ferroptotic signal.

Esterification of arachidonic acid (AA) into phosphataidylethanolamine (PE) requires acyl-CoA synthase 4 (ACSL4) catalyzed formation of AA-CoA which is used for the AA esterification into lyso-PE driven by lysophosphatidyl-choline acyltransferase 3 (LPCAT3) to yield AA-PE. Assembly and engagement of 15-LOX/PEBP1 enzymatic complexes changes 15-LOX substrate selectivity from AA to AA-PE thus facilitating the generation of specific product (15-HOO-AA-PE). Under ferroptotic conditions, the activity of GPX4 is inhibited, thus HOO-AA-PE cannot be reduced to HO-AA-PE. In contrast oxidatively-truncated electrophilic products of HOO-AA-PE with shortened side chains and electrophilic oxygen functionalities are formed. These oxidatively-truncated PE products form adducts by attacking nucleophilic sites in target proteins thus leading to the yet to be identified gateway ferroptotic complexes.

Figure 7.

Metabolism of GSH. Cystine is imported into the cells with an expense of glutamate by system Xc-. Glutamate levels in the cytoplasm dictates the cystine transport. Cytosolic glutamate level is controlled by its transport to mitochondria through two transporters aspartate glutamate exchanger and glutamate carrier. Mitochondrial glutamate is used in the TCA cycle after its conversion to α-ketoglutarate. Transported cystine is converted to cysteine by thioredoxin (TRX) or by GSH. Cysteine is then converted to γ-glutamylcysteine (γ-GluCys) by glutamate cysteine ligase (GCS). GSH is synthesized by the addition of a glycine group to γ-GluCys by glutathione synthetase (GS). 10–15% of the GSH are transported to the mitochondria by dicarboxylate carrier (DIC) and the 2-oxoglutarate carrier (OGC). GSH is also transported across various transporters. PE-OOH produced from the organelle membrane is reduced to PE-OH by glutathione peroxidase4 (GPX4) using GSH. During the reduction process of PE-OOH to PE-OH, GSH is oxidized to GSSG, which are then converted into GSH by glutathione reductase (GRX).