PEBP1 Wardens Ferroptosis by Enabling Lipoxygenase Generation of Lipid Death Signals.

Ferroptosis is a form of programmed cell death that is pathogenic to several acute and chronic diseases and executed via oxygenation of polyunsaturated phosphatidylethanolamines (PE) by 15-lipoxygenases (15-LO) that normally use free polyunsaturated fatty acids as substrates. Mechanisms of the altered 15-LO substrate specificity are enigmatic. We sought a common ferroptosis regulator for 15LO. We discovered that PEBP1, a scaffold protein inhibitor of protein kinase cascades, complexes with two 15LO isoforms, 15LO1 and 15LO2, and changes their substrate competence to generate hydroperoxy-PE. Inadequate reduction of hydroperoxy-PE due to insufficiency or dysfunction of a selenoperoxidase, GPX4, leads to ferroptosis. We demonstrated the importance of PEBP1-dependent regulatory mechanisms of ferroptotic death in airway epithelial cells in asthma, kidney epithelial cells in renal failure, and cortical and hippocampal neurons in brain trauma. As master regulators of ferroptotic cell death with profound implications for human disease, PEBP1/15LO complexes represent a new target for drug discovery.

Discovering selective antiferroptotic inhibitors of the 15LOX/PEBP1 complex noninterfering with biosynthesis of lipid mediators.

Programmed ferroptotic death eliminates cells in all major organs and tissues with imbalanced redox metabolism due to overwhelming iron-catalyzed lipid peroxidation under insufficient control by thiols (Glutathione (GSH)). Ferroptosis has been associated with the pathogenesis of major chronic degenerative diseases and acute injuries of the brain, cardiovascular system, liver, kidneys, and other organs, and its manipulation offers a promising new strategy for anticancer therapy. This explains the high interest in designing new small-molecule-specific inhibitors against ferroptosis. Given the role of 15-lipoxygenase (15LOX) association with phosphatidylethanolamine (PE)-binding protein 1 (PEBP1) in initiating ferroptosis-specific peroxidation of polyunsaturated PE, we propose a strategy of discovering antiferroptotic agents as inhibitors of the 15LOX/PEBP1 catalytic complex rather than 15LOX alone. Here we designed, synthesized, and tested a customized library of 26 compounds using biochemical, molecular, and cell biology models along with redox lipidomic and computational analyses. We selected two lead compounds, FerroLOXIN-1 and 2, which effectively suppressed ferroptosis in vitro and in vivo without affecting the biosynthesis of pro-/anti-inflammatory lipid mediators in vivo. The effectiveness of these lead compounds is not due to radical scavenging or iron-chelation but results from their specific mechanisms of interaction with the 15LOX-2/PEBP1 complex, which either alters the binding pose of the substrate [eicosatetraenoyl-PE (ETE-PE)] in a nonproductive way or blocks the predominant oxygen channel thus preventing the catalysis of ETE-PE peroxidation. Our successful strategy may be adapted to the design of additional chemical libraries to reveal new ferroptosis-targeting therapeutic modalities.

Strikingly High Activity of 15-Lipoxygenase Towards Di-Polyunsaturated Arachidonoyl/Adrenoyl-Phosphatidylethanolamines Generates Peroxidation Signals of Ferroptotic Cell Death.

The vast majority of membrane phospholipids (PLs) include two asymmetrically positioned fatty acyls: oxidizable polyunsaturated fatty acids (PUFA) attached predominantly at the sn2 position, and non-oxidizable saturated/monounsaturated acids (SFA/MUFA) localized at the sn1 position. The peroxidation of PUFA-PLs, particularly sn2-arachidonoyl(AA)- and sn2-adrenoyl(AdA)-containing phosphatidylethanolamines (PE), has been associated with the execution of ferroptosis, a program of regulated cell death. There is a minor subpopulation (≈1-2 mol %) of doubly PUFA-acylated phospholipids (di-PUFA-PLs) whose role in ferroptosis remains enigmatic. Here we report that 15-lipoxygenase (15LOX) exhibits unexpectedly high pro-ferroptotic peroxidation activity towards di-PUFA-PEs. We revealed that peroxidation of several molecular species of di-PUFA-PEs occurred early in ferroptosis. Ferrostatin-1, a typical ferroptosis inhibitor, effectively prevented peroxidation of di-PUFA-PEs. Furthermore, co-incubation of cells with di-AA-PE and 15LOX produced PUFA-PE peroxidation and induced ferroptotic death. The decreased contents of di-PUFA-PEs in ACSL4 KO A375 cells was associated with lower levels of di-PUFA-PE peroxidation and enhanced resistance to ferroptosis. Thus, di-PUFA-PE species are newly identified phospholipid peroxidation substrates and regulators of ferroptosis, representing a promising therapeutic target for many diseases related to ferroptotic death.

Phospholipase iPLA2ß averts ferroptosis by eliminating a redox lipid death signal.

Ferroptosis, triggered by discoordination of iron, thiols and lipids, leads to the accumulation of 15-hydroperoxy (Hp)-arachidonoyl-phosphatidylethanolamine (15-HpETE-PE), generated by complexes of 15-lipoxygenase (15-LOX) and a scaffold protein, phosphatidylethanolamine (PE)-binding protein (PEBP)1. As the Ca2+-independent phospholipase A2ß (iPLA2ß, PLA2G6 or PNPLA9 gene) can preferentially hydrolyze peroxidized phospholipids, it may eliminate the ferroptotic 15-HpETE-PE death signal. Here, we demonstrate that by hydrolyzing 15-HpETE-PE, iPLA2ß averts ferroptosis, whereas its genetic or pharmacological inactivation sensitizes cells to ferroptosis. Given that PLA2G6 mutations relate to neurodegeneration, we examined fibroblasts from a patient with a Parkinson's disease (PD)-associated mutation (fPDR747W) and found selectively decreased 15-HpETE-PE-hydrolyzing activity, 15-HpETE-PE accumulation and elevated sensitivity to ferroptosis. CRISPR-Cas9-engineered Pnpla9R748W/R748W mice exhibited progressive parkinsonian motor deficits and 15-HpETE-PE accumulation. Elevated 15-HpETE-PE levels were also detected in midbrains of rotenone-infused parkinsonian rats and α-synuclein-mutant SncaA53T mice, with decreased iPLA2ß expression and a PD-relevant phenotype. Thus, iPLA2ß is a new ferroptosis regulator, and its mutations may be implicated in PD pathogenesis.

Successive High-Resolution (H2O)n-GCIB and C60-SIMS Imaging Integrates Multi-Omics in Different Cell Types in Breast Cancer Tissue.

The temporo-spatial organization of different cells in the tumor microenvironment (TME) is the key to understanding their complex communication networks and the immune landscape that exists within compromised tissues. Multi-omics profiling of single-interacting cells in the native TME is critical for providing further information regarding the reprograming mechanisms leading to immunosuppression and tumor progression. This requires new technologies for biomolecular profiling of phenotypically heterogeneous cells on the same tissue sample. Here, we developed a new methodology for comprehensive lipidomic and metabolomic profiling of individual cells on frozen-hydrated tissue sections using water gas cluster ion beam secondary ion mass spectrometry ((H2O)n-GCIB-SIMS) (at 1.6 µm beam spot size), followed by profiling cell-type specific lanthanide antibodies on the same tissue section using C60-SIMS (at 1.1 µm beam spot size). We revealed distinct variations of distribution and intensities of >150 key ions (e.g., lipids and important metabolites) in different types of the TME individual cells, such as actively proliferating tumor cells as well as infiltrating immune cells. The demonstrated feasibility of SIMS imaging to integrate the multi-omics profiling in the same tissue section at the single-cell level will lead to new insights into the role of lipid reprogramming and metabolic response in normal regulation or pathogenic discoordination of cell-cell interactions in a variety of tissue microenvironments.

Direct Mapping of Phospholipid Ferroptotic Death Signals in Cells and Tissues by Gas Cluster Ion Beam Secondary Ion Mass Spectrometry (GCIB-SIMS).

Peroxidized phosphatidylethanolamine (PEox) species have been identified by liquid chromatography mass spectrometry (LC-MS) as predictive biomarkers of ferroptosis, a new program of regulated cell death. However, the presence and subcellular distribution of PEox in specific cell types and tissues have not been directly detected by imaging protocols. By applying gas cluster ion beam secondary ion mass spectrometry (GCIB-SIMS) imaging with a 70 keV (H2 O)n+ (n>28 000) cluster ion beam, we were able to map PEox with 1.2 µm spatial resolution at the single cell/subcellular level in ferroptotic H9c2 cardiomyocytes and cortical/hippocampal neurons after traumatic brain injury. Application of this protocol affords visualization of physiologically relevant levels of very low abundance (20 pmol µmol-1 lipid) peroxidized lipids in subcellular compartments and their accumulation in disease conditions.

Lipidomics Detection of Brain Cardiolipins in Plasma Is Associated With Outcome After Cardiac Arrest.

OBJECTIVES: Brain mitochondrial dysfunction limits neurologic recovery after cardiac arrest. Brain polyunsaturated cardiolipins, mitochondria-unique and functionally essential phospholipids, have unprecedented diversification. Since brain cardiolipins are not present in plasma normally, we hypothesized their appearance would correlate with brain injury severity early after cardiac arrest and return of spontaneous circulation. DESIGN: Observational case-control study. SETTING: Two medical centers within one city. PARTICIPANTS (SUBJECTS): We enrolled 41 adult cardiac arrest patients in whom blood could be obtained within 6 hours of resuscitation. Two subjects were excluded following outlier analysis. Ten healthy subjects were controls. Sprague-Dawley rats were used in asphyxial cardiac arrest studies. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We developed a high-resolution liquid chromatography/mass spectrometry method and determined cardiolipins speciation in human brain, heart, and plasma within 6 hours of (return of spontaneous circulation) from 39 patients with cardiac arrest, 5 with myocardial infarction, and 10 healthy controls. Cerebral score was derived from brain-specific cardiolipins identified in plasma of patients with varying neurologic injury and outcome. Using a rat model of cardiac arrest, cardiolipins were quantified in plasma, brain, and heart. Human brain exhibited a highly diverse cardiolipinome compared with heart that allowed the identification of brain-specific cardiolipins. Nine of 26 brain-specific cardiolipins were detected in plasma and correlated with brain injury. The cerebral score correlated with early neurologic injury and predicted discharge neurologic/functional outcome. Cardiolipin (70:5) emerged as a potential point-of-care marker predicting injury severity and outcome. In rat cardiac arrest, a significant reduction in hippocampal cardiolipins corresponded to their release from the brain into systemic circulation. Cerebral score was significantly increased in 10 minutes versus 5 minutes no-flow cardiac arrest and naïve controls. CONCLUSIONS: Brain-specific cardiolipins accumulate in plasma early after return of spontaneous circulation and proportional to neurologic injury representing a promising novel biomarker.

Oxidized arachidonic and adrenic PEs navigate cells to ferroptosis.

Enigmatic lipid peroxidation products have been claimed as the proximate executioners of ferroptosis-a specialized death program triggered by insufficiency of glutathione peroxidase 4 (GPX4). Using quantitative redox lipidomics, reverse genetics, bioinformatics and systems biology, we discovered that ferroptosis involves a highly organized oxygenation center, wherein oxidation in endoplasmic-reticulum-associated compartments occurs on only one class of phospholipids (phosphatidylethanolamines (PEs)) and is specific toward two fatty acyls-arachidonoyl (AA) and adrenoyl (AdA). Suppression of AA or AdA esterification into PE by genetic or pharmacological inhibition of acyl-CoA synthase 4 (ACSL4) acts as a specific antiferroptotic rescue pathway. Lipoxygenase (LOX) generates doubly and triply-oxygenated (15-hydroperoxy)-diacylated PE species, which act as death signals, and tocopherols and tocotrienols (vitamin E) suppress LOX and protect against ferroptosis, suggesting a homeostatic physiological role for vitamin E. This oxidative PE death pathway may also represent a target for drug discovery.

Secondary-Ion Mass Spectrometry Images Cardiolipins and Phosphatidylethanolamines at the Subcellular Level.

Millions of diverse molecules constituting the lipidome act as important signals within cells. Of these, cardiolipin (CL) and phosphatidylethanolamine (PE) participate in apoptosis and ferroptosis, respectively. Their subcellular distribution is largely unknown. Imaging mass spectrometry is capable of deciphering the spatial distribution of multiple lipids at subcellular levels. Here we report the development of a unique 70 keV gas-cluster ion beam that consists of (CO2 )n+ (n>10 000) projectiles. Coupled with direct current beam buncher-time-of-flight secondary-ion mass spectrometry, it is optimized for sensitivity towards high-mass species (up to m/z 3000) at high spatial resolution (1 µm). In combination with immunohistochemistry, phospholipids, including PE and CL, have been assessed in subcellular compartments of mouse hippocampal neuronal cells and rat brain tissue.

Empowerment of 15-Lipoxygenase Catalytic Competence in Selective Oxidation of Membrane ETE-PE to Ferroptotic Death Signals, HpETE-PE.

sn2-15-Hydroperoxy-eicasotetraenoyl-phosphatidylethanolamines ( sn2-15-HpETE-PE) generated by mammalian 15-lipoxygenase/phosphatidylethanolamine binding protein-1 (15-LO/PEBP1) complex is a death signal in a recently identified type of programmed cell demise, ferroptosis. How the enzymatic complex selects sn2-ETE-PE as the substrate among 1 of ∼100 total oxidizable membrane PUFA phospholipids is a central, yet unresolved question. To unearth the highly selective and specific mechanisms of catalytic competence, we used a combination of redox lipidomics, mutational and computational structural analysis to show they stem from (i) reactivity toward readily accessible hexagonally organized membrane sn2-ETE-PEs, (ii) relative preponderance of sn2-ETE-PE species vs other sn2-ETE-PLs, and (iii) allosteric modification of the enzyme in the complex with PEBP1. This emphasizes the role of enzymatic vs random stochastic free radical reactions in ferroptotic death signaling.

Gas Cluster Ion Beam Time-of-Flight Secondary Ion Mass Spectrometry High-Resolution Imaging of Cardiolipin Speciation in the Brain: Identification of Molecular Losses after Traumatic Injury.

Gas cluster ion beam-secondary ion mass spectrometry (GCIB-SIMS) has shown the full potential of mapping intact lipids in biological systems with better than 10 µm lateral resolution. This study investigated further the capability of GCIB-SIMS in imaging high-mass signals from intact cardiolipin (CL) and gangliosides in normal brain and the effect of a controlled cortical impact model (CCI) of traumatic brain injury (TBI) on their distribution. A combination of enzymatic and chemical treatments was employed to suppress the signals from the most abundant phospholipids (phosphatidylcholine (PC) and phosphatidylethanolamine (PE)) and enhance the signals from the low-abundance CLs and gangliosides to allow their GCIB-SIMS detection at 8 and 16 µm spatial resolution. Brain CLs have not been observed previously using other contemporary imaging mass spectrometry techniques at better than 50 µm spatial resolution. High-resolution images of naive and injured brain tissue facilitated the comparison of CL species across three multicell layers in the CA1, CA3, and DG regions of the hippocampus. GCIB-SIMS also reliably mapped losses of oxidizable polyunsaturated CL species (but not the oxidation-resistant saturated and monounsaturated gangliosides) to regions including the CA1 and CA3 of the hippocampus after CCI. This work extends the detection range for SIMS measurements of intact lipids to above m/z 2000, bridging the mass range gap compared with MALDI. Further advances in high-resolution SIMS of CLs, with the potential for single cell or supra-cellular imaging, will be essential for the understanding of CL's functional and structural organization in normal and injured brain.

Global assessment of oxidized free fatty acids in brain reveals an enzymatic predominance to oxidative signaling after trauma.

Traumatic brain injury (TBI) is a major health problem associated with significant morbidity and mortality. The pathophysiology of TBI is complex involving signaling through multiple cascades, including lipid peroxidation. Oxidized free fatty acids, a prominent product of lipid peroxidation, are potent cellular mediators involved in induction and resolution of inflammation and modulation of vasomotor tone. While previous studies have assessed lipid peroxidation after TBI, to our knowledge no studies have used a systematic approach to quantify the global oxidative changes in free fatty acids. In this study, we identified and quantified 244 free fatty acid oxidation products using a newly developed global liquid chromatography tandem-mass spectrometry (LC-MS/MS) method. This methodology was used to follow the time course of these lipid species in the contusional cortex of our pediatric rat model of TBI. We show that oxidation peaked at 1h after controlled cortical impact and was progressively attenuated at 4 and 24h time points. While enzymatic and non-enzymatic pathways were activated at 1h post-TBI, enzymatic lipid peroxidation was the predominant mechanism with 15-lipoxygenase (LOX) contributing to the majority of total oxidized fatty acid content. Pro-inflammatory lipid mediators were significantly increased at 1 and 4h after TBI with return to basal levels by 24h. Anti-inflammatory lipid mediators remained significantly increased across all three time points, indicating an elevated and sustained anti-inflammatory response following TBI.

Known unknowns of cardiolipin signaling: The best is yet to come.

Since its discovery 75years ago, a wealth of knowledge has accumulated on the role of cardiolipin, the hallmark phospholipid of mitochondria, in bioenergetics and particularly on the structural organization of the inner mitochondrial membrane. A surge of interest in this anionic doubly-charged tetra-acylated lipid found in both prokaryotes and mitochondria has emerged based on its newly discovered signaling functions. Cardiolipin displays organ, tissue, cellular and transmembrane distribution asymmetries. A collapse of the membrane asymmetry represents a pro-mitophageal mechanism whereby externalized cardiolipin acts as an "eat-me" signal. Oxidation of cardiolipin's polyunsaturated acyl chains - catalyzed by cardiolipin complexes with cytochrome c. - is a pro-apoptotic signal. The messaging functions of myriads of cardiolipin species and their oxidation products are now being recognized as important intracellular and extracellular signals for innate and adaptive immune systems. This newly developing field of research exploring cardiolipin signaling is the main subject of this review. This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum.

Defects of Lipid Synthesis Are Linked to the Age-Dependent Demyelination Caused by Lamin B1 Overexpression.

Lamin B1 is a component of the nuclear lamina and plays a critical role in maintaining nuclear architecture, regulating gene expression and modulating chromatin positioning. We have previously shown that LMNB1 gene duplications cause autosomal dominant leukodystrophy (ADLD), a fatal adult onset demyelinating disease. The mechanisms by which increased LMNB1 levels cause ADLD are unclear. To address this, we used a transgenic mouse model where Lamin B1 overexpression is targeted to oligodendrocytes. These mice showed severe vacuolar degeneration of the spinal cord white matter together with marked astrogliosis, microglial infiltration, and secondary axonal damage. Oligodendrocytes in the transgenic mice revealed alterations in histone modifications favoring a transcriptionally repressed state. Chromatin changes were accompanied by reduced expression of genes involved in lipid synthesis pathways, many of which are known to play important roles in myelin regulation and are preferentially expressed in oligodendrocytes. Decreased lipogenic gene expression resulted in a significant reduction in multiple classes of lipids involved in myelin formation. Many of these gene expression changes and lipid alterations were observed even before the onset of the phenotype, suggesting a causal role. Our findings establish, for the first time, a link between LMNB1 and lipid synthesis in oligodendrocytes, and provide a mechanistic framework to explain the age dependence and white matter involvement of the disease phenotype. These results have implications for disease pathogenesis and may also shed light on the regulation of lipid synthesis pathways in myelin maintenance and turnover. SIGNIFICANCE STATEMENT: Autosomal dominant leukodystrophy (ADLD) is fatal neurological disorder caused by increased levels of the nuclear protein, Lamin B1. The disease is characterized by an age-dependent loss of myelin, the fatty sheath that covers nerve fibers. We have studied a mouse model where Lamin B1 level are increased in oligodendrocytes, the cell type that produces myelin in the CNS. We demonstrate that destruction of myelin in the spinal cord is responsible for the degenerative phenotype in our mouse model. We show that this degeneration is mediated by reduced expression of lipid synthesis genes and the subsequent reduction in myelin enriched lipids. These findings provide a mechanistic framework to explain the age dependence and tissue specificity of the ADLD disease phenotype.

Biosynthesis of oxidized lipid mediators via lipoprotein-associated phospholipase A2 hydrolysis of extracellular cardiolipin induces endothelial toxicity.

We (66) have previously described an NSAID-insensitive intramitochondrial biosynthetic pathway involving oxidation of the polyunsaturated mitochondrial phospholipid, cardiolipin (CL), followed by hydrolysis [by calcium-independent mitochondrial calcium-independent phospholipase A2-γ (iPLA2γ)] of oxidized CL (CLox), leading to the formation of lysoCL and oxygenated octadecadienoic metabolites. We now describe a model system utilizing oxidative lipidomics/mass spectrometry and bioassays on cultured bovine pulmonary artery endothelial cells (BPAECs) to assess the impact of CLox that we show, in vivo, can be released to the extracellular space and may be hydrolyzed by lipoprotein-associated PLA2 (Lp-PLA2). Chemically oxidized liposomes containing bovine heart CL produced multiple oxygenated species. Addition of Lp-PLA2 hydrolyzed CLox and produced (oxygenated) monolysoCL and dilysoCL and oxidized octadecadienoic metabolites including 9- and 13-hydroxyoctadecadienoic (HODE) acids. CLox caused BPAEC necrosis that was exacerbated by Lp-PLA2 Lower doses of nonlethal CLox increased permeability of BPAEC monolayers. This effect was exacerbated by Lp-PLA2 and partially mimicked by authentic monolysoCL or 9- or 13-HODE. Control mice plasma contained virtually no detectable CLox; in contrast, 4 h after Pseudomonas aeruginosa (P. aeruginosa) infection, 34 ± 8 mol% (n = 6; P < 0.02) of circulating CL was oxidized. In addition, molar percentage of monolysoCL increased twofold after P. aeruginosa in a subgroup analyzed for these changes. Collectively, these studies suggest an important role for 1) oxidation of CL in proinflammatory environments and 2) possible hydrolysis of CLox in extracellular spaces producing lysoCL and oxidized octadecadienoic acid metabolites that may lead to impairment of pulmonary endothelial barrier function and necrosis.

Imaging mass spectrometry reveals loss of polyunsaturated cardiolipins in the cortical contusion, hippocampus, and thalamus after traumatic brain injury.

Traumatic brain injury (TBI) leads to changes in ion fluxes, alterations in mitochondrial function, and increased generation of reactive oxygen species, resulting in secondary tissue damage. Mitochondria play important signaling roles in coordination of multiple metabolic platforms in addition to their well-known role in bioenergetics. Mitochondrial signaling strongly depends on cardiolipin (CL), a mitochondria-specific structurally unusual anionic phospholipid containing four fatty acyl chains. While our previous reports indicated that CL is selectively oxidized and presents itself as a target for the redox therapy following TBI, the topography of changes of CL in the injured brain remained to be defined. Here, we present a matrix-assisted laser desorption/ionization imaging study which reports regio-specific changes in CL, in a controlled cortical impact model of TBI in rats. Matrix-assisted laser desorption/ionization imaging revealed that TBI caused early decreases in CL in the contusional cortex, ipsilateral hippocampus, and thalamus with the most highly unsaturated CL species being most susceptible to loss. Phosphatidylinositol was the only other lipid species that exhibited a significant decrease, albeit to a lesser extent than CL. Signals for other lipids remained unchanged. This is the first study evaluating the spatial distribution of CL loss after acute brain injury. We propose that the CL loss may constitute an upstream mechanism for CL-driven signaling in different brain regions as an early response mechanism and may also underlie the bioenergetic changes that occur in hippocampal, cortical, and thalamic mitochondria after TBI.

Dual function of mitochondrial Nm23-H4 protein in phosphotransfer and intermembrane lipid transfer: a cardiolipin-dependent switch.

The nucleoside diphosphate kinase Nm23-H4/NDPK-D forms symmetrical hexameric complexes in the mitochondrial intermembrane space with phosphotransfer activity using mitochondrial ATP to regenerate nucleoside triphosphates. We demonstrate the complex formation between Nm23-H4 and mitochondrial GTPase OPA1 in rat liver, suggesting its involvement in local and direct GTP delivery. Similar to OPA1, Nm23-H4 is further known to strongly bind in vitro to anionic phospholipids, mainly cardiolipin, and in vivo to the inner mitochondrial membrane. We show here that such protein-lipid complexes inhibit nucleoside diphosphate kinase activity but are necessary for another function of Nm23-H4, selective intermembrane lipid transfer. Mitochondrial lipid distribution was analyzed by liquid chromatography-mass spectrometry using HeLa cells expressing either wild-type Nm23-H4 or a membrane binding-deficient mutant at a site predicted based on molecular modeling to be crucial for cardiolipin binding and transfer mechanism. We found that wild type, but not the mutant enzyme, selectively increased the content of cardiolipin in the outer mitochondrial membrane, but the distribution of other more abundant phospholipids (e.g. phosphatidylcholine) remained unchanged. HeLa cells expressing the wild-type enzyme showed increased accumulation of Bax in mitochondria and were sensitized to rotenone-induced apoptosis as revealed by stimulated release of cytochrome c into the cytosol, elevated caspase 3/7 activity, and increased annexin V binding. Based on these data and molecular modeling, we propose that Nm23-H4 acts as a lipid-dependent mitochondrial switch with dual function in phosphotransfer serving local GTP supply and cardiolipin transfer for apoptotic signaling and putative other functions.

PHLDA2-mediated phosphatidic acid peroxidation triggers a distinct ferroptotic response during tumor suppression.

Although the role of ferroptosis in killing tumor cells is well established, recent studies indicate that ferroptosis inducers also sabotage anti-tumor immunity by killing neutrophils and thus unexpectedly stimulate tumor growth, raising a serious issue about whether ferroptosis effectively suppresses tumor development in vivo. Through genome-wide CRISPR-Cas9 screenings, we discover a pleckstrin homology-like domain family A member 2 (PHLDA2)-mediated ferroptosis pathway that is neither ACSL4-dependent nor requires common ferroptosis inducers. PHLDA2-mediated ferroptosis acts through the peroxidation of phosphatidic acid (PA) upon high levels of reactive oxygen species (ROS). ROS-induced ferroptosis is critical for tumor growth in the absence of common ferroptosis inducers; strikingly, loss of PHLDA2 abrogates ROS-induced ferroptosis and promotes tumor growth but has no obvious effect in normal tissues in both immunodeficient and immunocompetent mouse tumor models. These data demonstrate that PHLDA2-mediated PA peroxidation triggers a distinct ferroptosis response critical for tumor suppression and reveal that PHLDA2-mediated ferroptosis occurs naturally in vivo without any treatment from ferroptosis inducers.

Oxidized phospholipids as biomarkers of tissue and cell damage with a focus on cardiolipin.

Oxidized phospholipid species are important, biologically relevant, lipid signaling molecules that usually exist in low abundance in biological tissues. Along with their inherent stability issues, these oxidized lipids present themselves as a challenge in their detection and identification. Often times, oxidized lipid species can co-chromatograph with non-oxidized species making the detection of the former extremely difficult, even with the use of mass spectrometry. In this study, a normal-phase and reverse-phase two dimensional high performance liquid chromatography (HPLC)-mass spectrometric system was applied to separate oxidized phospholipids from their non-oxidized counterparts, allowing unambiguous detection in a total lipid extract. We have utilized bovine heart cardiolipin as well as commercially available tetralinoleoyl cardiolipin oxidized with cytochrome c (cyt c) and hydrogen peroxide as well as with lipoxygenase to test the separation power of the system. Our findings indicate that oxidized species of not only cardiolipin, but other phospholipid species, can be effectively separated from their non-oxidized counterparts in this two dimensional system. We utilized three types of biological tissues and oxidative insults, namely rotenone treatment of lymphocytes to induce mitochondrial damage and cell death, pulmonary inhalation exposure to single walled carbon nanotubes, as well as total body irradiation, in order to identify cardiolipin oxidation products, critical to the cell damage/cell death pathways in these tissues following cellular stress/injury. Our results indicate that selective cardiolipin (CL) oxidation is a result of a non-random free radical process. In addition, we assessed the ability of the system to identify CL oxidation products in the brain, a tissue known for its extreme complexity and diversity of CL species. The ability of the two dimensional HPLC-mass spectrometric system to detect and characterize oxidized lipid products will allow new studies to be formulated to probe the answers to biologically important questions with regard to oxidative lipidomics and cellular insult. This article is part of a Special Issue entitled: Oxidized phospholipids - their properties and interactions with proteins.

Redox phospholipidomics discovers pro-ferroptotic death signals in A375 melanoma cells in vitro and in vivo.

Growing cancer cells effectively evade most programs of regulated cell death, particularly apoptosis. This necessitates a search for alternative therapeutic modalities to cause cancer cell's demise, among them - ferroptosis. One of the obstacles to using pro-ferroptotic agents to treat cancer is the lack of adequate biomarkers of ferroptosis. Ferroptosis is accompanied by peroxidation of polyunsaturated species of phosphatidylethanolamine (PE) to hydroperoxy- (-OOH) derivatives, which act as death signals. We demonstrate that RSL3-induced death of A375 melanoma cells in vitro was fully preventable by ferrostatin-1, suggesting their high susceptibility to ferroptosis. Treatment of A375 cells with RSL3 caused a significant accumulation of PE-(18:0/20:4-OOH) and PE-(18:0/22:4-OOH), the biomarkers of ferroptosis, as well as oxidatively truncated products - PE-(18:0/hydroxy-8-oxo-oct-6-enoic acid (HOOA) and PC-(18:0/HOOA). A significant suppressive effect of RSL3 on melanoma growth was observed in vivo (utilizing a xenograft model of inoculation of GFP-labeled A375 cells into immune-deficient athymic nude mice). Redox phospholipidomics revealed elevated levels of 18:0/20:4-OOH in RSL3-treated group vs controls. In addition, PE-(18:0/20:4-OOH) species were identified as major contributors to the separation of control and RSL3-treated groups, with the highest variable importance in projection predictive score. Pearson correlation analysis revealed an association between tumor weight and contents of PE-(18:0/20:4-OOH) (r = -0.505), PE-18:0/HOOA (r = -0.547) and PE 16:0-HOOA (r = -0.503). Thus, LC-MS/MS based redox lipidomics is a sensitive and precise approach for the detection and characterization of phospholipid biomarkers of ferroptosis induced in cancer cells by radio- and chemotherapy.