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1.
Nature ; 619(7969): 371-377, 2023 Jul.
Article in English | MEDLINE | ID: mdl-37380771

ABSTRACT

Ferroptosis is evolving as a highly promising approach to combat difficult-to-treat tumour entities including therapy-refractory and dedifferentiating cancers1-3. Recently, ferroptosis suppressor protein-1 (FSP1), along with extramitochondrial ubiquinone or exogenous vitamin K and NAD(P)H/H+ as an electron donor, has been identified as the second ferroptosis-suppressing system, which efficiently prevents lipid peroxidation independently of the cyst(e)ine-glutathione (GSH)-glutathione peroxidase 4 (GPX4) axis4-6. To develop FSP1 inhibitors as next-generation therapeutic ferroptosis inducers, here we performed a small molecule library screen and identified the compound class of 3-phenylquinazolinones (represented by icFSP1) as potent FSP1 inhibitors. We show that icFSP1, unlike iFSP1, the first described on-target FSP1 inhibitor5, does not competitively inhibit FSP1 enzyme activity, but instead triggers subcellular relocalization of FSP1 from the membrane and FSP1 condensation before ferroptosis induction, in synergism with GPX4 inhibition. icFSP1-induced FSP1 condensates show droplet-like properties consistent with phase separation, an emerging and widespread mechanism to modulate biological activity7. N-terminal myristoylation, distinct amino acid residues and intrinsically disordered, low-complexity regions in FSP1 were identified to be essential for FSP1-dependent phase separation in cells and in vitro. We further demonstrate that icFSP1 impairs tumour growth and induces FSP1 condensates in tumours in vivo. Hence, our results suggest that icFSP1 exhibits a unique mechanism of action and synergizes with ferroptosis-inducing agents to potentiate the ferroptotic cell death response, thus providing a rationale for targeting FSP1-dependent phase separation as an efficient anti-cancer therapy.


Subject(s)
Apoptosis Regulatory Proteins , Ferroptosis , Mitochondrial Proteins , Humans , Amino Acids/metabolism , Cysteine/metabolism , Ferroptosis/drug effects , Glutathione/metabolism , NAD/metabolism , NADP/metabolism , Neoplasms/drug therapy , Quinazolines/pharmacology , Small Molecule Libraries , Ubiquinone/metabolism , Vitamin K/metabolism , Apoptosis Regulatory Proteins/antagonists & inhibitors , Apoptosis Regulatory Proteins/metabolism , Mitochondrial Proteins/antagonists & inhibitors , Mitochondrial Proteins/metabolism
2.
Nature ; 608(7924): 778-783, 2022 08.
Article in English | MEDLINE | ID: mdl-35922516

ABSTRACT

Ferroptosis, a non-apoptotic form of cell death marked by iron-dependent lipid peroxidation1, has a key role in organ injury, degenerative disease and vulnerability of therapy-resistant cancers2. Although substantial progress has been made in understanding the molecular processes relevant to ferroptosis, additional cell-extrinsic and cell-intrinsic processes that determine cell sensitivity toward ferroptosis remain unknown. Here we show that the fully reduced forms of vitamin K-a group of naphthoquinones that includes menaquinone and phylloquinone3-confer a strong anti-ferroptotic function, in addition to the conventional function linked to blood clotting by acting as a cofactor for γ-glutamyl carboxylase. Ferroptosis suppressor protein 1 (FSP1), a NAD(P)H-ubiquinone reductase and the second mainstay of ferroptosis control after glutathione peroxidase-44,5, was found to efficiently reduce vitamin K to its hydroquinone, a potent radical-trapping antioxidant and inhibitor of (phospho)lipid peroxidation. The FSP1-mediated reduction of vitamin K was also responsible for the antidotal effect of vitamin K against warfarin poisoning. It follows that FSP1 is the enzyme mediating warfarin-resistant vitamin K reduction in the canonical vitamin K cycle6. The FSP1-dependent non-canonical vitamin K cycle can act to protect cells against detrimental lipid peroxidation and ferroptosis.


Subject(s)
Ferroptosis , Vitamin K , Antidotes/pharmacology , Antioxidants/metabolism , Antioxidants/pharmacology , Carbon-Carbon Ligases/metabolism , Coenzymes/metabolism , Ferroptosis/drug effects , Hydroquinones/metabolism , Hydroquinones/pharmacology , Lipid Peroxidation/drug effects , Oxidation-Reduction , S100 Calcium-Binding Protein A4/metabolism , Vitamin K/metabolism , Vitamin K/pharmacology , Warfarin/adverse effects
3.
Nature ; 575(7784): 693-698, 2019 11.
Article in English | MEDLINE | ID: mdl-31634899

ABSTRACT

Ferroptosis is an iron-dependent form of necrotic cell death marked by oxidative damage to phospholipids1,2. To date, ferroptosis has been thought to be controlled only by the phospholipid hydroperoxide-reducing enzyme glutathione peroxidase 4 (GPX4)3,4 and radical-trapping antioxidants5,6. However, elucidation of the factors that underlie the sensitivity of a given cell type to ferroptosis7 is crucial to understand the pathophysiological role of ferroptosis and how it may be exploited for the treatment of cancer. Although metabolic constraints8 and phospholipid composition9,10 contribute to ferroptosis sensitivity, no cell-autonomous mechanisms have been identified that account for the resistance of cells to ferroptosis. Here we used an expression cloning approach to identify genes in human cancer cells that are able to complement the loss of GPX4. We found that the flavoprotein apoptosis-inducing factor mitochondria-associated 2 (AIFM2) is a previously unrecognized anti-ferroptotic gene. AIFM2, which we renamed ferroptosis suppressor protein 1 (FSP1) and which was initially described as a pro-apoptotic gene11, confers protection against ferroptosis elicited by GPX4 deletion. We further demonstrate that the suppression of ferroptosis by FSP1 is mediated by ubiquinone (also known as coenzyme Q10, CoQ10): the reduced form, ubiquinol, traps lipid peroxyl radicals that mediate lipid peroxidation, whereas FSP1 catalyses the regeneration of CoQ10 using NAD(P)H. Pharmacological targeting of FSP1 strongly synergizes with GPX4 inhibitors to trigger ferroptosis in a number of cancer entities. In conclusion, the FSP1-CoQ10-NAD(P)H pathway exists as a stand-alone parallel system, which co-operates with GPX4 and glutathione to suppress phospholipid peroxidation and ferroptosis.


Subject(s)
Apoptosis Regulatory Proteins/metabolism , Ferroptosis/genetics , Glutathione/metabolism , Mitochondrial Proteins/metabolism , Animals , Apoptosis Regulatory Proteins/genetics , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Gene Knockout Techniques , Humans , Lipid Peroxidation/genetics , Mice , Mitochondrial Proteins/genetics , Ubiquinone/analogs & derivatives , Ubiquinone/metabolism
5.
Proc Natl Acad Sci U S A ; 116(16): 8038-8047, 2019 04 16.
Article in English | MEDLINE | ID: mdl-30944221

ABSTRACT

Abdominal aortic aneurysm (AAA) is an inflammatory vascular disease with high mortality and limited treatment options. How blood lipids regulate AAA development is unknown. Here lipidomics and genetic models demonstrate a central role for procoagulant enzymatically oxidized phospholipids (eoxPL) in regulating AAA. Specifically, through activating coagulation, eoxPL either promoted or inhibited AAA depending on tissue localization. Ang II administration to ApoE-/- mice increased intravascular coagulation during AAA development. Lipidomics revealed large numbers of eoxPL formed within mouse and human AAA lesions. Deletion of eoxPL-generating enzymes (Alox12 or Alox15) or administration of the factor Xa inhibitor rivaroxaban significantly reduced AAA. Alox-deficient mice displayed constitutively dysregulated hemostasis, including a consumptive coagulopathy, characterized by compensatory increase in prothrombotic aminophospholipids (aPL) in circulating cell membranes. Intravenously administered procoagulant PL caused clotting factor activation and depletion, induced a bleeding defect, and significantly reduced AAA development. These data suggest that Alox deletion reduces AAA through diverting coagulation away from the vessel wall due to eoxPL deficiency, instead activating clotting factor consumption and depletion in the circulation. In mouse whole blood, ∼44 eoxPL molecular species formed within minutes of clot initiation. These were significantly elevated with ApoE-/- deletion, and many were absent in Alox-/- mice, identifying specific eoxPL that modulate AAA. Correlation networks demonstrated eoxPL belonged to subfamilies defined by oxylipin composition. Thus, procoagulant PL regulate AAA development through complex interactions with clotting factors. Modulation of the delicate balance between bleeding and thrombosis within either the vessel wall or circulation was revealed that can either drive or prevent disease development.


Subject(s)
Aorta, Abdominal/physiopathology , Aortic Aneurysm, Abdominal , Phospholipids , Angiotensins/metabolism , Animals , Aortic Aneurysm, Abdominal/genetics , Aortic Aneurysm, Abdominal/metabolism , Aortic Aneurysm, Abdominal/physiopathology , Blood Coagulation Factors/genetics , Blood Coagulation Factors/metabolism , Disease Models, Animal , Female , Lipoxygenase/genetics , Lipoxygenase/metabolism , Male , Mice , Mice, Knockout, ApoE , Phospholipids/genetics , Phospholipids/metabolism
6.
Int J Mol Sci ; 23(17)2022 Aug 31.
Article in English | MEDLINE | ID: mdl-36077303

ABSTRACT

Glutathione peroxidase 4 (Gpx4) and arachidonic acid 15 lipoxygenase (Alox15) are counterplayers in oxidative lipid metabolism and both enzymes have been implicated in spermatogenesis. However, the roles of the two proteins in acrosomal exocytosis have not been explored in detail. Here we characterized Gpx4 distribution in mouse sperm and detected the enzyme not only in the midpiece of the resting sperm but also at the anterior region of the head, where the acrosome is localized. During sperm capacitation, Gpx4 translocated to the post-acrosomal compartment. Sperm from Gpx4+/Sec46Ala mice heterozygously expressing a catalytically silent enzyme displayed an increased expression of phosphotyrosyl proteins, impaired acrosomal exocytosis after in vitro capacitation and were not suitable for in vitro fertilization. Alox15-deficient sperm showed normal acrosome reactions but when crossed into a Gpx4-deficient background spontaneous acrosomal exocytosis was observed during capacitation and these cells were even less suitable for in vitro fertilization. Taken together, our data indicate that heterozygous expression of a catalytically silent Gpx4 variant impairs acrosomal exocytosis and in vitro fertilization. Alox15 deficiency hardly impacted the acrosome reaction but when crossed into the Gpx4-deficient background spontaneous acrosomal exocytosis was induced. The detailed molecular mechanisms for the observed effects may be related to the compromised redox homeostasis.


Subject(s)
Acrosome Reaction , Arachidonate 15-Lipoxygenase , Acrosome/metabolism , Animals , Arachidonate 15-Lipoxygenase/genetics , Arachidonate 15-Lipoxygenase/metabolism , Exocytosis , Fertilization in Vitro , Male , Mice , Phospholipid Hydroperoxide Glutathione Peroxidase , Semen , Spermatozoa/metabolism
7.
PLoS Pathog ; 15(3): e1007597, 2019 03.
Article in English | MEDLINE | ID: mdl-30921435

ABSTRACT

Cryptococcus neoformans is one of the leading causes of invasive fungal infection in humans worldwide. C. neoformans uses macrophages as a proliferative niche to increase infective burden and avoid immune surveillance. However, the specific mechanisms by which C. neoformans manipulates host immunity to promote its growth during infection remain ill-defined. Here we demonstrate that eicosanoid lipid mediators manipulated and/or produced by C. neoformans play a key role in regulating pathogenesis. C. neoformans is known to secrete several eicosanoids that are highly similar to those found in vertebrate hosts. Using eicosanoid deficient cryptococcal mutants Δplb1 and Δlac1, we demonstrate that prostaglandin E2 is required by C. neoformans for proliferation within macrophages and in vivo during infection. Genetic and pharmacological disruption of host PGE2 synthesis is not required for promotion of cryptococcal growth by eicosanoid production. We find that PGE2 must be dehydrogenated into 15-keto-PGE2 to promote fungal growth, a finding that implicated the host nuclear receptor PPAR-γ. C. neoformans infection of macrophages activates host PPAR-γ and its inhibition is sufficient to abrogate the effect of 15-keto-PGE2 in promoting fungal growth during infection. Thus, we describe the first mechanism of reliance on pathogen-derived eicosanoids in fungal pathogenesis and the specific role of 15-keto-PGE2 and host PPAR-γ in cryptococcosis.


Subject(s)
Cryptococcus neoformans/metabolism , Dinoprostone/analogs & derivatives , Eicosanoids/metabolism , Animals , Animals, Genetically Modified , Cell Culture Techniques , Cryptococcosis/metabolism , Cryptococcus neoformans/growth & development , Cryptococcus neoformans/pathogenicity , Dinoprostone/metabolism , Dinoprostone/physiology , Disease Models, Animal , Eicosanoids/immunology , Host-Pathogen Interactions/physiology , Humans , Macrophages/microbiology , PPAR gamma/metabolism , Virulence/physiology , Zebrafish/microbiology
8.
J Biol Chem ; 294(23): 9225-9238, 2019 06 07.
Article in English | MEDLINE | ID: mdl-31061099

ABSTRACT

Eicosanoids are critical mediators of fever, pain, and inflammation generated by immune and tissue cells. We recently described a new bioactive eicosanoid generated by cyclooxygenase-1 (COX-1) turnover during platelet activation that can stimulate human neutrophil integrin expression. On the basis of mass spectrometry (MS/MS and MS3), stable isotope labeling, and GC-MS analysis, we previously proposed a structure of 8-hydroxy-9,11-dioxolane eicosatetraenoic acid (DXA3). Here, we achieved enzymatic synthesis and 1H NMR characterization of this compound with results in conflict with the previously proposed structural assignment. Accordingly, by using LC-MS, we screened autoxidation reactions of 11-hydroperoxy-eicosatetraenoic acid (11-HpETE) and thereby identified a candidate sharing the precise reverse-phase chromatographic and MS characteristics of the platelet product. We optimized these methods to increase yield, allowing full structural analysis by 1H NMR. The revised assignment is presented here as 8,9-11,12-diepoxy-13-hydroxyeicosadienoic acid, abbreviated to 8,9-11,12-DiEp-13-HEDE or DiEpHEDE, substituted for the previous name DXA3 We found that in platelets, the lipid likely forms via dioxolane ring opening with rearrangement to the diepoxy moieties followed by oxygen insertion at C13. We present its enzymatic biosynthetic pathway and MS/MS fragmentation pattern and, using the synthetic compound, demonstrate that it has bioactivity. For the platelet lipid, we estimate 16 isomers based on our current knowledge (and four isomers for the synthetic lipid). Determining the exact isomeric structure of the platelet lipid remains to be undertaken.


Subject(s)
Blood Platelets/metabolism , Eicosanoids/chemistry , Hydroxyeicosatetraenoic Acids/chemistry , Chromatography, High Pressure Liquid , Cyclooxygenase 1/metabolism , Eicosanoids/analysis , Gas Chromatography-Mass Spectrometry , Humans , Hydroxyeicosatetraenoic Acids/analysis , Hydroxyeicosatetraenoic Acids/chemical synthesis , Isomerism , Magnetic Resonance Spectroscopy , Molecular Conformation , Tandem Mass Spectrometry
9.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1863(2): 152-164, 2018 Feb.
Article in English | MEDLINE | ID: mdl-29146531

ABSTRACT

Pseudomonas aeruginosa is a gram-negative pathogen, which causes life-threatening infections in immunocompromized patients. These bacteria express a secreted lipoxygenase (PA-LOX), which oxygenates free arachidonic acid to 15S-hydro(pero)xyeicosatetraenoic acid. It binds phospholipids at its active site and physically interacts with lipid vesicles. When incubated with red blood cells membrane lipids are oxidized and hemolysis is induced but the structures of the oxygenated membrane lipids have not been determined. Using a lipidomic approach, we analyzed the formation of oxidized phospholipids generated during the in vitro incubation of recombinant PA-LOX with human erythrocytes and cultured human lung epithelial cells. Precursor scanning of lipid extracts prepared from these cells followed by multiple reaction monitoring and MS/MS analysis revealed a complex mixture of oxidation products. For human red blood cells this mixture comprised forty different phosphatidylethanolamine and phosphatidylcholine species carrying oxidized fatty acid residues, such as hydroxy-octadecadienoic acids, hydroxy- and keto-eicosatetraenoic acid, hydroxy-docosahexaenoic acid as well as oxygenated derivatives of less frequently occurring polyenoic fatty acids. Similar oxygenation products were also detected when cultured lung epithelial cells were employed but here the amounts of oxygenated lipids were smaller and under identical experimental conditions we did not detect major signs of cell lysis. However, live imaging indicated an impaired capacity for trypan blue exclusion and an augmented mitosis rate. Taken together these data indicate that PA-LOX can oxidize the membrane lipids of eukaryotic cells and that the functional consequences of this reaction strongly depend on the cell type.


Subject(s)
Bacterial Proteins/metabolism , Erythrocyte Membrane/metabolism , Lipoxygenase/metabolism , Phospholipids/metabolism , Pseudomonas aeruginosa/enzymology , A549 Cells , Bacterial Proteins/chemistry , Erythrocyte Membrane/chemistry , Erythrocyte Membrane/pathology , Humans , Lipoxygenase/chemistry , Oxidation-Reduction , Phospholipids/chemistry
10.
Am J Respir Cell Mol Biol ; 57(6): 692-701, 2017 12.
Article in English | MEDLINE | ID: mdl-28723225

ABSTRACT

Type 2-associated goblet cell hyperplasia and mucus hypersecretion are well known features of asthma. 15-Lipoxygenase-1 (15LO1) is induced by the type 2 cytokine IL-13 in human airway epithelial cells (HAECs) in vitro and is increased in fresh asthmatic HAECs ex vivo. 15LO1 generates a variety of products, including 15-hydroxyeicosatetraenoic acid (15-HETE), 15-HETE-phosphatidylethanolamine (15-HETE-PE), and 13-hydroxyoctadecadienoic acid (13-HODE). In this study, we investigated the 15LO1 metabolite profile at baseline and after IL-13 treatment, as well as its influence on goblet cell differentiation in HAECs. Primary HAECs obtained from bronchial brushings of asthmatic and healthy subjects were cultured under air-liquid interface culture supplemented with arachidonic acid and linoleic acid (10 µM each) and exposed to IL-13 for 7 days. Short interfering RNA transfection and 15LO1 inhibition were applied to suppress 15LO1 expression and activity. IL-13 stimulation induced expression of 15LO1 and preferentially generated 15-HETE-PE in vitro, both of which persisted after removal of IL-13. 15LO1 inhibition (by short interfering RNA and chemical inhibitor) decreased IL-13-induced forkhead box protein A3 (FOXA3) expression and enhanced FOXA2 expression. These changes were associated with reductions in both mucin 5AC and periostin. Exogenous 15-HETE-PE stimulation (alone) recapitulated IL-13-induced FOXA3, mucin 5AC, and periostin expression. The results of this study confirm the central importance of 15LO1 and its primary product, 15-HETE-PE, for epithelial cell remodeling in HAECs.


Subject(s)
Cell Differentiation/drug effects , Epithelial Cells/metabolism , Goblet Cells/metabolism , Hydroxyeicosatetraenoic Acids/biosynthesis , Interleukin-13/pharmacology , Airway Remodeling/drug effects , Arachidonate 15-Lipoxygenase/metabolism , Gene Expression Regulation/drug effects , Hepatocyte Nuclear Factor 3-beta/biosynthesis , Hepatocyte Nuclear Factor 3-gamma/biosynthesis , Humans , Linoleic Acids/biosynthesis , Mucin 5AC/biosynthesis
11.
J Biol Chem ; 291(26): 13448-64, 2016 Jun 24.
Article in English | MEDLINE | ID: mdl-27129261

ABSTRACT

Eicosanoids are important mediators of fever, pain, and inflammation that modulate cell signaling during acute and chronic disease. We show by using lipidomics that thrombin-activated human platelets generate a new type of eicosanoid that both stimulates and primes human neutrophil integrin (Mac-1) expression, in response to formylmethionylleucylphenylalanine. Detailed characterization proposes a dioxolane structure, 8-hydroxy-9,11-dioxolane eicosatetraenoic acid (dioxolane A3, DXA3). The lipid is generated in nanogram amounts by platelets from endogenous arachidonate during physiological activation, with inhibition by aspirin in vitro or in vivo, implicating cyclooxygenase-1 (COX). Pharmacological and genetic studies on human/murine platelets revealed that DXA3 formation requires protease-activated receptors 1 and 4, cytosolic phospholipase A2 (cPLA2), Src tyrosine kinases, p38 MAPK, phospholipase C, and intracellular calcium. From data generated by purified COX isoforms and chemical oxidation, we propose that DXA3 is generated by release of an intermediate from the active site followed by oxygenation at C8. In summary, a new neutrophil-activating platelet-derived lipid generated by COX-1 is presented that can activate or prime human neutrophils, suggesting a role in innate immunity and acute inflammation.


Subject(s)
Blood Platelets/enzymology , Cyclooxygenase 1/metabolism , Dioxolanes/metabolism , MAP Kinase Signaling System/physiology , Neutrophil Activation/physiology , Neutrophils/metabolism , Animals , Aspirin/pharmacology , Blood Platelets/immunology , Cyclooxygenase 1/immunology , Dioxolanes/immunology , Immunity, Innate/drug effects , Immunity, Innate/physiology , MAP Kinase Signaling System/drug effects , Macrophage-1 Antigen/immunology , Macrophage-1 Antigen/metabolism , Male , Membrane Proteins/immunology , Membrane Proteins/metabolism , Mice , Neutrophil Activation/drug effects , Neutrophils/immunology , Platelet Activation/drug effects , Platelet Activation/physiology
12.
Proc Natl Acad Sci U S A ; 110(15): 5875-80, 2013 Apr 09.
Article in English | MEDLINE | ID: mdl-23530199

ABSTRACT

Aminophospholipid (APL) trafficking across the plasma membrane is a key event in cell activation, apoptosis, and aging and is required for clearance of dying cells and coagulation. Currently the phospholipid molecular species externalized are unknown. Using a lipidomic method, we show that thrombin, collagen, or ionophore-activated human platelets externalize two phosphatidylserines (PSs) and five phosphatidylethanolamines (PEs). Four percent of the total cellular PE/PS pool (∼300 ng/2 × 10(8) cells, thrombin), is externalized via calcium mobilization and protease-activated receptors-1 and -4, and 48% is contained in microparticles. Apoptosis and energy depletion (aging) externalized the same APLs in a calcium-dependent manner, and all stimuli externalized oxidized phospholipids, termed hydroxyeicosatetraenoic acid-PEs. Transmembrane protein-16F (TMEM-16F), the protein mutated in Scott syndrome, was required for PE/PS externalization during thrombin activation and energy depletion, but not apoptosis. Platelet-specific APLs optimally supported tissue factor-dependent coagulation in human plasma, vs. APL with longer or shorter fatty acyl chains. This finding demonstrates fatty acids as molecular determinants of APL that regulate hemostasis. Thus, the molecular species of externalized APL during platelet activation, apoptosis, and energy depletion were characterized, and their ability to support coagulation revealed. The findings have therapeutic implications for bleeding disorders and transfusion therapy. The assay could be applied to other cell events characterized by APL externalization, including cell division and vesiculation.


Subject(s)
Blood Coagulation , Blood Platelets/metabolism , Fatty Acids/chemistry , Gene Expression Regulation , Phospholipids/chemistry , Aging , Annexin A5/chemistry , Apoptosis , Biotinylation , Calcium/metabolism , Cell Membrane/metabolism , Dose-Response Relationship, Drug , Humans , Thrombin/chemistry , Thrombin/metabolism , Time Factors
13.
J Lipid Res ; 54(11): 3085-97, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23883581

ABSTRACT

Oxidized phospholipids (oxPLs) generated nonenzymatically display pleiotropic biological actions in inflammation. Their generation by cellular cyclooxygenases (COXs) is currently unknown. To determine whether platelets generate prostaglandin (PG)-containing oxPLs, then characterize their structures and mechanisms of formation, we applied precursor scanning-tandem mass spectrometry to lipid extracts of agonist-activated human platelets. Thrombin, collagen, or ionophore activation stimulated generation of families of PGs comprising PGE2 and D2 attached to four phosphatidylethanolamine (PE) phospholipids (16:0p/, 18:1p/, 18:0p/, and 18:0a/). They formed within 2 to 5 min of activation in a calcium, phospholipase C, p38 MAP kinases, MEK1, cPLA2, and src tyrosine kinase-dependent manner (28.1 ± 2.3 pg/2 × 108 platelets). Unlike free PGs, they remained cell associated, suggesting an autocrine mode of action. Their generation was inhibited by in vivo aspirin supplementation (75 mg/day) or in vitro COX-1 blockade. Inhibitors of fatty acyl reesterification blocked generation significantly, while purified COX-1 was unable to directly oxidize PE in vitro. This indicates that they form in platelets via rapid esterification of COX-1 derived PGE2/D2 into PE. In summary, COX-1 in human platelets acutely mediates membrane phospholipid oxidation via formation of PG-esterified PLs in response to pathophysiological agonists.


Subject(s)
Aspirin/pharmacology , Blood Platelets/drug effects , Blood Platelets/metabolism , Cyclooxygenase 1/metabolism , Cyclooxygenase Inhibitors/pharmacology , Phospholipids/metabolism , Prostaglandins/metabolism , Blood Platelets/physiology , Calcium/metabolism , Dinoprostone/metabolism , Dose-Response Relationship, Drug , Esterification/drug effects , Feedback, Physiological/drug effects , Humans , Intracellular Space/drug effects , Intracellular Space/metabolism , MAP Kinase Kinase 1/metabolism , Phosphatidylethanolamines/metabolism , Platelet Activation/drug effects , Prostaglandin D2/metabolism , Protein Kinase C/metabolism , Receptor, PAR-1/metabolism , Thrombin/metabolism , src-Family Kinases/metabolism
14.
Curr Atheroscler Rep ; 15(5): 323, 2013 May.
Article in English | MEDLINE | ID: mdl-23512607

ABSTRACT

Oxidized PLs (OxPLs) generated in health and disease are now recognized as important mediators of cellular signalling. There is an increasing body of evidence showing that PL peroxidation is not only increased in vascular disorders, but is also a physiological event of relevance to coagulation, innate immunity, and self-tolerance. Nonenzymatically formed OxPLs generated during chronic inflammation is an uncontrolled event, generating hundreds of diverse structures, and prone to more deleterious bioactivities. In contrast, enzymatic formation of OxPLs is tightly regulated, involving receptors and intracellular signaling, acting as part of the normal physiological response to injury in order to restore homeostasis. In the present review, the major nonenzymatic OxPLs structures found during vascular inflammation are summarized, along with a brief description of their known biological activities. Also, we review what is currently known about enzymatic formation of OxPLs by acutely activated immune cells and their signaling actions under homeostatic and pathological conditions.


Subject(s)
Atherosclerosis/metabolism , Oxidation-Reduction , Phospholipids/metabolism , Vasculitis/metabolism , Atherosclerosis/physiopathology , Humans , Lipoxygenases/physiology , Phospholipids/physiology , Prostaglandin-Endoperoxide Synthases/physiology , Reactive Oxygen Species , Signal Transduction/physiology , Vasculitis/physiopathology
15.
Nat Commun ; 13(1): 139, 2022 01 10.
Article in English | MEDLINE | ID: mdl-35013270

ABSTRACT

Oxylipins are potent biological mediators requiring strict control, but how they are removed en masse during infection and inflammation is unknown. Here we show that lipopolysaccharide (LPS) dynamically enhances oxylipin removal via mitochondrial ß-oxidation. Specifically, genetic or pharmacological targeting of carnitine palmitoyl transferase 1 (CPT1), a mitochondrial importer of fatty acids, reveal that many oxylipins are removed by this protein during inflammation in vitro and in vivo. Using stable isotope-tracing lipidomics, we find secretion-reuptake recycling for 12-HETE and its intermediate metabolites. Meanwhile, oxylipin ß-oxidation is uncoupled from oxidative phosphorylation, thus not contributing to energy generation. Testing for genetic control checkpoints, transcriptional interrogation of human neonatal sepsis finds upregulation of many genes involved in mitochondrial removal of long-chain fatty acyls, such as ACSL1,3,4, ACADVL, CPT1B, CPT2 and HADHB. Also, ACSL1/Acsl1 upregulation is consistently observed following the treatment of human/murine macrophages with LPS and IFN-γ. Last, dampening oxylipin levels by ß-oxidation is suggested to impact on their regulation of leukocyte functions. In summary, we propose mitochondrial ß-oxidation as a regulatory metabolic checkpoint for oxylipins during inflammation.


Subject(s)
12-Hydroxy-5,8,10,14-eicosatetraenoic Acid/metabolism , Lipid Metabolism/genetics , Mitochondria/drug effects , Oxylipins/metabolism , Peritonitis/genetics , Sepsis/genetics , Acyl-CoA Dehydrogenase, Long-Chain/blood , Acyl-CoA Dehydrogenase, Long-Chain/genetics , Animals , Carnitine O-Palmitoyltransferase/blood , Carnitine O-Palmitoyltransferase/genetics , Coenzyme A Ligases/blood , Coenzyme A Ligases/genetics , Female , Gene Expression Regulation , Humans , Infant, Newborn , Interferon-gamma/pharmacology , Lipidomics/methods , Lipopolysaccharides/pharmacology , Macrophages/drug effects , Macrophages/metabolism , Macrophages/pathology , Male , Mice , Mice, Inbred C57BL , Mitochondria/metabolism , Mitochondrial Trifunctional Protein, beta Subunit/blood , Mitochondrial Trifunctional Protein, beta Subunit/genetics , Oxidation-Reduction , Peritonitis/blood , Peritonitis/chemically induced , Peritonitis/pathology , RAW 264.7 Cells , Sepsis/blood , Sepsis/pathology
16.
Trends Endocrinol Metab ; 32(7): 463-473, 2021 07.
Article in English | MEDLINE | ID: mdl-33985897

ABSTRACT

Lipid peroxidation (LPO) is the molecular mechanism involved in oxidative damage of cellular membranes and the hallmark of a nonapoptotic form of cell death, known as ferroptosis. This iron-dependent cell death is an emerging strategy in cancer treatment and one of the central cell death mechanisms accounting for early cell loss and organ dysfunction in both neurodegenerative disease and ischemia-reperfusion injury. Although the biological roles of LPO products have attracted considerable attention, not only for their pathological mechanisms but also for their potential clinical application as biomarkers, the existence of a common lethal lipid death signal generated during ferroptosis remains poorly explored. A better understanding of the LPO process, however, may unleash unprecedented opportunities for therapeutic intervention of as-yet incurable diseases.


Subject(s)
Ferroptosis , Lipids , Neurodegenerative Diseases , Humans , Lipid Peroxidation
17.
PLoS One ; 15(10): e0240189, 2020.
Article in English | MEDLINE | ID: mdl-33031441

ABSTRACT

Tissue factor (TF) is critical for the activation of blood coagulation. TF function is regulated by the amount of externalised phosphatidylserine (PS) and phosphatidylethanolamine (PE) on the surface of the cell in which it is expressed. We investigated the role PS and PE in fibroblast TF function. Fibroblasts expressed 6-9 x 104 TF molecules/cell but had low specific activity for FXa generation. We confirmed that this was associated with minimal externalized PS and PE and characterised for the first time the molecular species of PS/PE demonstrating that these differed from those found in platelets. Mechanical damage of fibroblasts, used to simulate vascular injury, increased externalized PS/PE and led to a 7-fold increase in FXa generation that was inhibited by annexin V and an anti-TF antibody. Platelet-derived extracellular vesicles (EVs), that did not express TF, supported minimal FVIIa-dependent FXa generation but substantially increased fibroblast TF activity. This enhancement in fibroblast TF activity could also be achieved using synthetic liposomes comprising 10% PS without TF. In conclusion, despite high levels of surface TF expression, healthy fibroblasts express low levels of external-facing PS and PE limiting their ability to generate FXa. Addition of platelet-derived TF-negative EVs or artificial liposomes enhanced fibroblast TF activity in a PS dependent manner. These findings contribute information about the mechanisms that control TF function in the fibroblast membrane.


Subject(s)
Extracellular Vesicles/metabolism , Fibroblasts/metabolism , Thromboplastin/metabolism , Blood Coagulation , Blood Platelets/metabolism , Cell Line , Humans , Liposomes/metabolism , Phosphatidylethanolamines/metabolism , Phosphatidylserines/metabolism , Thromboplastin/genetics
18.
Circ Genom Precis Med ; 13(3): e002806, 2020 06.
Article in English | MEDLINE | ID: mdl-32396387

ABSTRACT

BACKGROUND: Common chromosome 9p21 single nucleotide polymorphisms (SNPs) increase coronary heart disease risk, independent of traditional lipid risk factors. However, lipids comprise large numbers of structurally related molecules not measured in traditional risk measurements, and many have inflammatory bioactivities. Here, we applied lipidomic and genomic approaches to 3 model systems to characterize lipid metabolic changes in common Chr9p21 SNPs, which confer ≈30% elevated coronary heart disease risk associated with altered expression of ANRIL, a long ncRNA. METHODS: Untargeted and targeted lipidomics was applied to plasma from NPHSII (Northwick Park Heart Study II) homozygotes for AA or GG in rs10757274, followed by correlation and network analysis. To identify candidate genes, transcriptomic data from shRNA downregulation of ANRIL in HEK-293 cells was mined. Transcriptional data from vascular smooth muscle cells differentiated from induced pluripotent stem cells of individuals with/without Chr9p21 risk, nonrisk alleles, and corresponding knockout isogenic lines were next examined. Last, an in-silico analysis of miRNAs was conducted to identify how ANRIL might control lysoPL (lysophosphospholipid)/lysoPA (lysophosphatidic acid) genes. RESULTS: Elevated risk GG correlated with reduced lysoPLs, lysoPA, and ATX (autotaxin). Five other risk SNPs did not show this phenotype. LysoPL-lysoPA interconversion was uncoupled from ATX in GG plasma, suggesting metabolic dysregulation. Significantly altered expression of several lysoPL/lysoPA metabolizing enzymes was found in HEK cells lacking ANRIL. In the vascular smooth muscle cells data set, the presence of risk alleles associated with altered expression of several lysoPL/lysoPA enzymes. Deletion of the risk locus reversed the expression of several lysoPL/lysoPA genes to nonrisk haplotype levels. Genes that were altered across both cell data sets were DGKA, MBOAT2, PLPP1, and LPL. The in-silico analysis identified 4 ANRIL-regulated miRNAs that control lysoPL genes as miR-186-3p, miR-34a-3p, miR-122-5p, and miR-34a-5p. CONCLUSIONS: A Chr9p21 risk SNP associates with complex alterations in immune-bioactive phospholipids and their metabolism. Lipid metabolites and genomic pathways associated with coronary heart disease pathogenesis in Chr9p21 and ANRIL-associated disease are demonstrated.


Subject(s)
Chromosomes, Human, Pair 9/genetics , Coronary Disease , Lysophospholipids , Phosphoric Diester Hydrolases , Polymorphism, Single Nucleotide , Chromosomes, Human, Pair 9/metabolism , Coronary Disease/genetics , Coronary Disease/metabolism , HEK293 Cells , Humans , Lysophospholipids/genetics , Lysophospholipids/metabolism , Male , Middle Aged , Phosphoric Diester Hydrolases/genetics , Phosphoric Diester Hydrolases/metabolism
19.
Sci Signal ; 12(574)2019 03 26.
Article in English | MEDLINE | ID: mdl-30914483

ABSTRACT

Enzymatically oxidized phospholipids (eoxPLs) are formed through regulated processes by which eicosanoids or prostaglandins are attached to phospholipids (PLs) in immune cells. These eoxPLs comprise structurally diverse families of biomolecules with potent bioactivities, and they have important immunoregulatory roles in both health and disease. The formation of oxPLs through enzymatic pathways and their signaling capabilities are emerging concepts. This paradigm is changing our understanding of eicosanoid, prostaglandin, and PL biology in health and disease. eoxPLs have roles in cellular events such as ferroptosis, apoptosis, and blood clotting and diseases such as arthritis, diabetes, and cardiovascular disease. They are increasingly recognized as endogenous bioactive mediators and potential targets for drug development. This review will describe recent evidence that places eoxPLs and their biosynthetic pathways center stage in immunoregulation.


Subject(s)
Ferroptosis/physiology , Immunity, Innate/physiology , Lipoxygenases/physiology , Phospholipids/physiology , Prostaglandin-Endoperoxide Synthases/physiology , Animals , Blood Platelets/metabolism , Eicosanoids/metabolism , Hemostasis/physiology , Humans , Immune Tolerance , Inflammation/immunology , Inflammation/metabolism , Lipid Peroxidation , Neutrophils/metabolism , Oxidation-Reduction , Phospholipids/chemistry , Prostaglandins/metabolism , Spectrometry, Mass, Electrospray Ionization , Stereoisomerism , Thrombosis/metabolism , Vascular Diseases/immunology , Vascular Diseases/metabolism
20.
JCI Insight ; 3(6)2018 03 22.
Article in English | MEDLINE | ID: mdl-29563336

ABSTRACT

Hemostatic defects are treated using coagulation factors; however, clot formation also requires a procoagulant phospholipid (PL) surface. Here, we show that innate immune cell-derived enzymatically oxidized phospholipids (eoxPL) termed hydroxyeicosatetraenoic acid-phospholipids (HETE-PLs) restore hemostasis in human and murine conditions of pathological bleeding. HETE-PLs abolished blood loss in murine hemophilia A and enhanced coagulation in factor VIII- (FVIII-), FIX-, and FX-deficient human plasma . HETE-PLs were decreased in platelets from patients after cardiopulmonary bypass (CPB). To explore molecular mechanisms, the ability of eoxPL to stimulate individual isolated coagulation factor/cofactor complexes was tested in vitro. Extrinsic tenase (FVIIa/tissue factor [TF]), intrinsic tenase (FVIIIa/FIXa), and prothrombinase (FVa/FXa) all were enhanced by both HETE-PEs and HETE-PCs, suggesting a common mechanism involving the fatty acid moiety. In plasma, 9-, 15-, and 12-HETE-PLs were more effective than 5-, 11-, or 8-HETE-PLs, indicating positional isomer specificity. Coagulation was enhanced at lower lipid/factor ratios, consistent with a more concentrated area for protein binding. Surface plasmon resonance confirmed binding of FII and FX to HETE-PEs. HETE-PEs increased membrane curvature and thickness, but not surface charge or homogeneity, possibly suggesting increased accessibility to cations/factors. In summary, innate immune-derived eoxPL enhance calcium-dependent coagulation factor function, and their potential utility in bleeding disorders is proposed.


Subject(s)
Blood Coagulation Factors/metabolism , Hemorrhage/enzymology , Hemorrhage/metabolism , Phospholipids/metabolism , Thrombin/metabolism , Adult , Aged , Aged, 80 and over , Animals , Blood Coagulation , Blood Coagulation Factors/genetics , Blood Platelets , Cardiopulmonary Bypass/adverse effects , Carrier Proteins , Cysteine Endopeptidases , Factor IX/genetics , Factor VIII/genetics , Factor VIIa/metabolism , Factor X/genetics , Hemophilia A , Hemorrhage/prevention & control , Hemostasis , Humans , Hydroxyeicosatetraenoic Acids , Lipoproteins/pharmacology , Male , Mice , Mice, Inbred C57BL , Middle Aged , Neoplasm Proteins , Surface Plasmon Resonance , Thromboplastin/antagonists & inhibitors , Thromboplastin/metabolism
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