RESUMEN
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.
Asunto(s)
Lesión Renal Aguda/patología , Asma/patología , Lesiones Traumáticas del Encéfalo/patología , Muerte Celular , Proteínas de Unión a Fosfatidiletanolamina/metabolismo , Lesión Renal Aguda/metabolismo , Animales , Apoptosis , Asma/metabolismo , Lesiones Traumáticas del Encéfalo/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular , Humanos , Isoenzimas/metabolismo , Lipooxigenasa/química , Lipooxigenasa/metabolismo , Ratones , Modelos Moleculares , Oxazolidinonas/farmacología , Oxidación-Reducción , Proteínas de Unión a Fosfatidiletanolamina/químicaRESUMEN
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.
Asunto(s)
Ferroptosis/fisiología , Fosfolipasas A2 Grupo VI/metabolismo , Animales , Araquidonato 15-Lipooxigenasa/metabolismo , Modelos Animales de Enfermedad , Femenino , Fosfolipasas A2 Grupo VI/fisiología , Humanos , Hierro/metabolismo , Leucotrienos/metabolismo , Metabolismo de los Lípidos/fisiología , Peróxidos Lipídicos/metabolismo , Lípidos/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Oxidación-Reducción , Enfermedad de Parkinson/metabolismo , Proteínas de Unión a Fosfatidiletanolamina/metabolismo , Fosfolipasas/metabolismo , Fosfolípidos/metabolismo , Ratas , Ratas Endogámicas LewRESUMEN
Ferroptotic death is the penalty for losing control over three processes-iron metabolism, lipid peroxidation and thiol regulation-that are common in the pro-inflammatory environment where professional phagocytes fulfill their functions and yet survive. We hypothesized that redox reprogramming of 15-lipoxygenase (15-LOX) during the generation of pro-ferroptotic signal 15-hydroperoxy-eicosa-tetra-enoyl-phosphatidylethanolamine (15-HpETE-PE) modulates ferroptotic endurance. Here, we have discovered that inducible nitric oxide synthase (iNOS)/NOâ¢-enrichment of activated M1 (but not alternatively activated M2) macrophages/microglia modulates susceptibility to ferroptosis. Genetic or pharmacologic depletion/inactivation of iNOS confers sensitivity on M1 cells, whereas NO⢠donors empower resistance of M2 cells to ferroptosis. In vivo, M1 phagocytes, in comparison to M2 phagocytes, exert higher resistance to pharmacologically induced ferroptosis. This resistance is diminished in iNOS-deficient cells in the pro-inflammatory conditions of brain trauma or the tumour microenvironment. The nitroxygenation of eicosatetraenoyl (ETE)-PE intermediates and oxidatively truncated species by NO⢠donors and/or suppression of NO⢠production by iNOS inhibitors represent a novel redox mechanism of regulation of ferroptosis in pro-inflammatory conditions.
Asunto(s)
Ferroptosis/fisiología , Macrófagos/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Animales , Araquidonato 15-Lipooxigenasa/metabolismo , Araquidonato 15-Lipooxigenasa/fisiología , Muerte Celular , Femenino , Hierro/metabolismo , Hierro/fisiología , Leucotrienos/metabolismo , Peroxidación de Lípido/fisiología , Peróxidos Lipídicos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Microglía/metabolismo , Óxido Nítrico Sintasa de Tipo II/fisiología , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismoRESUMEN
We recently discovered an anti-ferroptotic mechanism inherent to M1 macrophages whereby high levels of NOâ suppressed ferroptosis via inhibition of hydroperoxy-eicosatetraenoyl-phosphatidylethanolamine (HpETE-PE) production by 15-lipoxygenase (15LOX) complexed with PE-binding protein 1 (PEBP1). However, the mechanism of NOâ interference with 15LOX/PEBP1 activity remained unclear. Here, we use a biochemical model of recombinant 15LOX-2 complexed with PEBP1, LC-MS redox lipidomics, and structure-based modeling and simulations to uncover the mechanism through which NOâ suppresses ETE-PE oxidation. Our study reveals that O2 and NOâ use the same entry pores and channels connecting to 15LOX-2 catalytic site, resulting in a competition for the catalytic site. We identified residues that direct O2 and NOâ to the catalytic site, as well as those stabilizing the esterified ETE-PE phospholipid tail. The functional significance of these residues is supported by in silico saturation mutagenesis. We detected nitrosylated PE species in a biochemical system consisting of 15LOX-2/PEBP1 and NOâ donor and in RAW264.7 M2 macrophages treated with ferroptosis-inducer RSL3 in the presence of NOâ, in further support of the ability of NOâ to diffuse to, and react at, the 15LOX-2 catalytic site. The results provide first insights into the molecular mechanism of repression of the ferroptotic Hp-ETE-PE production by NOâ.
Asunto(s)
Ferroptosis/fisiología , Óxido Nítrico/metabolismo , Proteínas de Unión a Fosfatidiletanolamina/metabolismo , Araquidonato 15-Lipooxigenasa/metabolismo , Muerte Celular/fisiología , Humanos , Lipidómica , Macrófagos/metabolismo , Simulación de Dinámica Molecular , Oxidación-Reducción , Fosfatidiletanolaminas , Fosfolípidos/metabolismoRESUMEN
Myeloperoxidase (MPO), a key enzyme released by neutrophils during inflammation, has been shown to catalyze the biodegradation of carbon nanomaterials. In this work, we perform photoluminescence studies on the MPO-catalyzed oxidation of graphene oxide (GO) and surfactant-coated pristine (6,5) single-walled carbon nanotubes (SWCNTs). The enzymatic degradation mechanism involves the introduction of defects, which promotes further degradation. Interestingly, the photoluminescence responses of GO and SWCNTs to enzymatic degradation are counterposed. Although the near-infrared (NIR) fluorescence intensity of SWCNTs at 998 nm is either unchanged or decreases depending on the surfactant identity, the blue fluorescence intensity of GO at 440 nm increases with the progression of oxidation by MPO/H2O2/Cl- due to the formation of graphene quantum dots (GQDs). Turn-on GO fluorescence is also observed with neutrophil-like HL-60 cells, indicative of potential applications of GO for imaging MPO activity in live cells. Based on these results, we further construct two ratiometric sensors using SWCNT/GO nanoscrolls by incorporating surfactant-wrapped pristine SWCNTs as the internal either turn-off (with sodium cholate (SC)) or reference (with carboxymethylcellulose (CMC)) sensor. The ratiometric approach enables the sensors to be more stable to external noise by providing response invariant to the absolute intensity emitted from the sensors. Our sensors show linear response to MPO oxidative machinery and hold the promise to be used as self-calibrating carbon nanomaterial-based MPO activity indicators.
Asunto(s)
Grafito/metabolismo , Luminiscencia , Nanotubos de Carbono/química , Peroxidasa/metabolismo , Biocatálisis , Grafito/química , Células HL-60 , Humanos , Peroxidasa/química , Procesos FotoquímicosRESUMEN
Cytochrome c (Cytc) is a multifunctional protein that operates as an electron carrier in the mitochondrial electron transport chain and plays a key role in apoptosis. We have previously shown that tissue-specific phosphorylations of Cytc in the heart, liver, and kidney play an important role in the regulation of cellular respiration and cell death. Here, we report that Cytc purified from mammalian brain is phosphorylated on S47 and that this phosphorylation is lost during ischemia. We have characterized the functional effects in vitro using phosphorylated Cytc purified from pig brain tissue and a recombinant phosphomimetic mutant (S47E). We crystallized S47E phosphomimetic Cytc at 1.55 Å and suggest that it spatially matches S47-phosphorylated Cytc, making it a good model system. Both S47-phosphorylated and phosphomimetic Cytc showed a lower oxygen consumption rate in reaction with isolated Cytc oxidase, which we propose maintains intermediate mitochondrial membrane potentials under physiologic conditions, thus minimizing production of reactive oxygen species. S47-phosphorylated and phosphomimetic Cytc showed lower caspase-3 activity. Furthermore, phosphomimetic Cytc had decreased cardiolipin peroxidase activity and is more stable in the presence of H2O2. Our data suggest that S47 phosphorylation of Cytc is tissue protective and promotes cell survival in the brain.-Kalpage, H. A., Vaishnav, A., Liu, J., Varughese, A., Wan, J., Turner, A. A., Ji, Q., Zurek, M. P., Kapralov, A. A., Kagan, V. E., Brunzelle, J. S., Recanati, M.-A., Grossman, L. I., Sanderson, T. H., Lee, I., Salomon, A. R., Edwards, B. F. P, Hüttemann, M. Serine-47 phosphorylation of cytochrome c in the mammalian brain regulates cytochrome c oxidase and caspase-3 activity.
Asunto(s)
Encéfalo/metabolismo , Caspasa 3/metabolismo , Citocromos c/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Mitocondrias/metabolismo , Daño por Reperfusión/metabolismo , Serina/metabolismo , Animales , Apoptosis , Caspasa 3/genética , Respiración de la Célula , Cristalografía por Rayos X , Citocromos c/química , Citocromos c/genética , Complejo IV de Transporte de Electrones/genética , Potencial de la Membrana Mitocondrial , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Mutación , Oxidación-Reducción , Fosforilación , Conformación Proteica , Especies Reactivas de Oxígeno/metabolismo , Daño por Reperfusión/patología , Serina/química , Serina/genética , PorcinosRESUMEN
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.
Asunto(s)
Ácido Araquidónico/metabolismo , Ácidos Grasos Insaturados/metabolismo , Fosfolípidos/metabolismo , Animales , Ácido Araquidónico/antagonistas & inhibidores , Muerte Celular/efectos de los fármacos , Línea Celular , Coenzima A Ligasas/antagonistas & inhibidores , Coenzima A Ligasas/deficiencia , Coenzima A Ligasas/metabolismo , Ácidos Grasos Insaturados/antagonistas & inhibidores , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Mammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc isolated from kidneys is phosphorylated on Thr28, leading to a partial inhibition of respiration in the reaction with cytochrome c oxidase. To further study the effect of Cytc phosphorylation in vitro, we generated T28E phosphomimetic Cytc, revealing superior behavior regarding protein stability and its ability to degrade reactive oxygen species compared with wild-type unphosphorylated Cytc Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (ΔΨm), and ROS levels are reduced compared with wild type. As we show by high resolution crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr28 is located at a central position near the heme crevice, the most flexible epitope of the protein apart from the N and C termini. Finally, in silico prediction and our experimental data suggest that AMP kinase, which phosphorylates Cytc on Thr28 in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kidney, is the most likely candidate to phosphorylate Thr28 in vivo We conclude that Cytc phosphorylation is mediated in a tissue-specific manner and leads to regulation of electron transport chain flux via "controlled respiration," preventing ΔΨm hyperpolarization, a known cause of ROS and trigger of apoptosis.
Asunto(s)
Adenilato Quinasa/metabolismo , Respiración de la Célula/fisiología , Citocromos c/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Riñón/metabolismo , Treonina/metabolismo , Adenilato Quinasa/química , Animales , Apoptosis , Cristalografía por Rayos X , Citocromos c/química , Transporte de Electrón , Complejo IV de Transporte de Electrones/química , Riñón/citología , Potencial de la Membrana Mitocondrial , Ratones , Mitocondrias/metabolismo , Oxidación-Reducción , Fosforilación , Conformación Proteica , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Cytoglobin (Cygb) is a hexa-coordinated hemoprotein with yet to be defined physiological functions. The iron coordination and spin state of the Cygb heme group are sensitive to oxidation of two cysteine residues (Cys38/Cys83) and/or the binding of free fatty acids. However, the roles of redox vs lipid regulators of Cygb's structural rearrangements in the context of the protein peroxidase competence are not known. Searching for physiologically relevant lipid regulators of Cygb, here we report that anionic phospholipids, particularly phosphatidylinositolphosphates, affect structural organization of the protein and modulate its iron state and peroxidase activity both conjointly and/or independently of cysteine oxidation. Thus, different anionic lipids can operate in cysteine-dependent and cysteine-independent ways as inducers of the peroxidase activity. We establish that Cygb's peroxidase activity can be utilized for the catalysis of peroxidation of anionic phospholipids (including phosphatidylinositolphosphates) yielding mono-oxygenated molecular species. Combined with the computational simulations we propose a bipartite lipid binding model that rationalizes the modes of interactions with phospholipids, the effects on structural re-arrangements and the peroxidase activity of the hemoprotein.
Asunto(s)
Globinas/metabolismo , Peroxidación de Lípido , Peroxidasas/metabolismo , Fosfolípidos/metabolismo , Aniones , Catálisis , Cisteína/metabolismo , Citoglobina , Activación Enzimática , Globinas/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Hierro/metabolismo , Modelos Biológicos , Simulación de Dinámica Molecular , Oxidación-Reducción , Peroxidasas/química , Fosfolípidos/química , Conformación Proteica , Proteínas Recombinantes/metabolismo , Relación Estructura-ActividadRESUMEN
Biopersistence of carbon nanotubes, graphene oxide (GO) and several other types of carbonaceous nanomaterials is an essential determinant of their health effects. Successful biodegradation is one of the major factors defining the life span and biological responses to nanoparticles. Here, we review the role and contribution of different oxidative enzymes of inflammatory cells - myeloperoxidase, eosinophil peroxidase, lactoperoxidase, hemoglobin, and xanthine oxidase - to the reactions of nanoparticle biodegradation. We further focus on interactions of nanomaterials with hemoproteins dependent on the specific features of their physico-chemical and structural characteristics. Mechanistically, we highlight the significance of immobilized peroxidase reactive intermediates vs diffusible small molecule oxidants (hypochlorous and hypobromous acids) for the overall oxidative biodegradation process in neutrophils and eosinophils. We also accentuate the importance of peroxynitrite-driven pathways realized in macrophages via the engagement of NADPH oxidase- and NO synthase-triggered oxidative mechanisms. We consider possible involvement of oxidative machinery of other professional phagocytes such as microglial cells, myeloid-derived suppressor cells, in the context of biodegradation relevant to targeted drug delivery. We evaluate the importance of genetic factors and their manipulations for the enzymatic biodegradation in vivo. Finally, we emphasize a novel type of biodegradation realized via the activation of the "dormant" peroxidase activity of hemoproteins by the nano-surface. This is exemplified by the binding of GO to cyt c causing the unfolding and 'unmasking' of the peroxidase activity of the latter. We conclude with the strategies leading to safe by design carbonaceous nanoparticles with optimized characteristics for mechanism-based targeted delivery and regulatable life-span of drugs in circulation.
Asunto(s)
Nanopartículas/metabolismo , Estrés Oxidativo/fisiología , Peroxidasas/metabolismo , Animales , Humanos , Nanopartículas/química , Nanotubos de Carbono/química , Neutrófilos/metabolismo , Oxidación-Reducción , Peroxidasas/químicaRESUMEN
Cytochrome c (cyt c) release from mitochondria is accepted to be the point of no return for eliciting a cascade of interactions that lead to apoptosis. A strategy for containing sustained apoptosis is to reduce the mitochondrial permeability pore opening. Pore opening is enhanced by peroxidase activity of cyt c gained upon its complexation with cardiolipin in the presence of reactive oxygen species. Blocking access to the heme group has been proposed as an effective intervention method for reducing, if not eliminating, the peroxidase activity of cyt c. In the present study, using a combination of druggability simulations, pharmacophore modeling, virtual screening, and in vitro fluorescence measurements to probe peroxidase activity, we identified three repurposable drugs and seven compounds that are validated to effectively inhibit the peroxidase activity of cyt c.
Asunto(s)
Dominio Catalítico , Complejo IV de Transporte de Electrones/química , Inhibidores Enzimáticos/química , Peroxidasas/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/química , Secuencia de Aminoácidos , Complejo IV de Transporte de Electrones/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Humanos , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Peroxidasas/química , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Because of their unique stacked, cup-shaped, hollow compartments, nitrogen-doped carbon nanotube cups (NCNCs) have promising potential as nanoscale containers. Individual NCNCs are isolated from their stacked structure through acid oxidation and subsequent probe-tip sonication. The NCNCs are then effectively corked with gold nanoparticles (GNPs) by sodium citrate reduction with chloroauric acid, forming graphitic nanocapsules with significant surface-enhanced Raman signature. Mechanistically, the growth of the GNP corks starts from the nucleation and welding of gold seeds on the open rims of NCNCs enriched with nitrogen functionalities, as confirmed by density functional theory calculations. A potent oxidizing enzyme of neutrophils, myeloperoxidase (MPO), can effectively open the corked NCNCs through GNP detachment, with subsequent complete enzymatic degradation of the graphitic shells. This controlled opening and degradation was further carried out in vitro with human neutrophils. Furthermore, the GNP-corked NCNCs were demonstrated to function as novel drug delivery carriers, capable of effective (i) delivery of paclitaxel to tumor-associated myeloid-derived suppressor cells (MDSC), (ii) MPO-regulated release, and (iii) blockade of MDSC immunosuppressive potential.
Asunto(s)
Oro/química , Conformación Molecular , Nanotubos de Carbono/química , Peroxidasa/metabolismo , Animales , Línea Celular Tumoral , Citratos/química , Humanos , Peróxido de Hidrógeno/química , Nanopartículas del Metal/química , Ratones , Modelos Moleculares , Neutrófilos/metabolismo , Oxidación-Reducción , Cloruro de Sodio/química , Citrato de SodioRESUMEN
We have investigated whether the pro-apoptotic properties of the G41S mutant of human cytochrome c can be explained by a higher than wild-type peroxidase activity triggered by phospholipid binding. A key complex in mitochondrial apoptosis involves cytochrome c and the phospholipid cardiolipin. In this complex cytochrome c has its native axial Met(80) ligand dissociated from the haem-iron, considerably augmenting the peroxidase capability of the haem group upon H2O2 binding. By EPR spectroscopy we reveal that the magnitude of changes in the paramagnetic haem states, as well as the yield of protein-bound free radical, is dependent on the phospholipid used and is considerably greater in the G41S mutant. A high-resolution X-ray crystal structure of human cytochrome c was determined and, in combination with the radical EPR signal analysis, two tyrosine residues, Tyr(46) and Tyr(48), have been rationalized to be putative radical sites. Subsequent single and double tyrosine-to-phenylalanine mutations revealed that the EPR signal of the radical, found to be similar in all variants, including G41S and wild-type, originates not from a single tyrosine residue, but is instead a superimposition of multiple EPR signals from different radical sites. We propose a mechanism of multiple radical formations in the cytochrome c-phospholipid complexes under H2O2 treatment, consistent with the stabilization of the radical in the G41S mutant, which elicits a greater peroxidase activity from cytochrome c and thus has implications in mitochondrial apoptosis.
Asunto(s)
Apoptosis , Cardiolipinas/química , Citocromos c/química , Citocromos c/genética , Peróxido de Hidrógeno/química , Mutación Missense , Sustitución de Aminoácidos , Cardiolipinas/metabolismo , Citocromos c/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Humanos , Mitocondrias/enzimología , Mitocondrias/genética , Peroxidasa/química , Peroxidasa/genética , Peroxidasa/metabolismoRESUMEN
Significance: Lipid peroxidation and its products, oxygenated polyunsaturated lipids, act as essential signals coordinating metabolism and physiology and can be deleterious to membranes when they accumulate in excessive amounts. Recent Advances: There is an emerging understanding that regulation of polyunsaturated fatty acid (PUFA) phospholipid peroxidation, particularly of PUFA-phosphatidylethanolamine, is important in a newly discovered type of regulated cell death, ferroptosis. Among the most recently described regulatory mechanisms is the ferroptosis suppressor protein, which controls the peroxidation process due to its ability to reduce coenzyme Q (CoQ). Critical Issues: In this study, we reviewed the most recent data in the context of the concept of free radical reductases formulated in the 1980-1990s and focused on enzymatic mechanisms of CoQ reduction in different membranes (e.g., mitochondrial, endoplasmic reticulum, and plasma membrane electron transporters) as well as TCA cycle components and cytosolic reductases capable of recycling the high antioxidant efficiency of the CoQ/vitamin E system. Future Directions: We highlight the importance of individual components of the free radical reductase network in regulating the ferroptotic program and defining the sensitivity/tolerance of cells to ferroptotic death. Complete deciphering of the interactive complexity of this system may be important for designing effective antiferroptotic modalities. Antioxid. Redox Signal. 40, 317-328.
Asunto(s)
Ferroptosis , Ubiquinona , Vitamina E , Oxidación-Reducción , Oxidorreductasas/metabolismo , Peroxidación de Lípido , Radicales Libres/metabolismo , Complejo I de Transporte de Electrón/metabolismoRESUMEN
Eosinophil peroxidase (EPO) is one of the major oxidant-producing enzymes during inflammatory states in the human lung. The degradation of single-walled carbon nanotubes (SWCNTs) upon incubation with human EPO and H2O2 is reported. Biodegradation of SWCNTs is higher in the presence of NaBr, but neither EPO alone nor H2O2 alone caused the degradation of nanotubes. Molecular modeling reveals two binding sites for SWCNTs on EPO, one located at the proximal side (same side as the catalytic site) and the other on the distal side of EPO. The oxidized groups on SWCNTs in both cases are stabilized by electrostatic interactions with positively charged residues. Biodegradation of SWCNTs can also be executed in an ex vivo culture system using primary murine eosinophils stimulated to undergo degranulation. Biodegradation is proven by a range of methods including transmission electron microscopy, UV-visible-NIR spectroscopy, Raman spectroscopy, and confocal Raman imaging. Thus, human EPO (in vitro) and ex vivo activated eosinophils mediate biodegradation of SWCNTs: an observation that is relevant to pulmonary responses to these materials.
Asunto(s)
Nanotubos de Carbono/química , Animales , Biodegradación Ambiental , Peroxidasa del Eosinófilo/metabolismo , Eosinófilos/metabolismo , Humanos , RatonesRESUMEN
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.
Asunto(s)
Melanoma , Espectrometría de Masas en Tándem , Animales , Ratones , Humanos , Peroxidación de Lípido , Muerte Celular , Ratones Desnudos , Cromatografía Liquida , Oxidación-ReducciónRESUMEN
Formation of cytochrome c (cyt c)/cardiolipin (CL) peroxidase complex selective toward peroxidation of polyunsaturated CLs is a pre-requisite for mitochondrial membrane permeabilization. Tyrosine residues - via the generation of tyrosyl radicals (Tyr) - are likely reactive intermediates of the peroxidase cycle leading to CL peroxidation. We used mutants of horse heart cyt c in which each of the four Tyr residues was substituted for Phe and assessed their contribution to the peroxidase catalysis. Tyr67Phe mutation was associated with a partial loss of the oxygenase function of the cyt c/CL complex and the lowest concentration of H(2)O(2)-induced Tyr radicals in electron paramagnetic resonance (EPR) spectra. Our MS experiments directly demonstrated decreased production of CL-hydroperoxides (CL-OOH) by Tyr67Phe mutant. Similarly, oxidation of a phenolic substrate, Amplex Red, was affected to a greater extent in Tyr67Phe than in three other mutants. Tyr67Phe mutant exerted high resistance to H(2)O(2)-induced oligomerization. Measurements of Tyr fluorescence, hetero-nuclear magnetic resonance (NMR) and computer simulations position Tyr67 in close proximity to the porphyrin ring heme iron and one of the two axial heme-iron ligand residues, Met80. Thus, the highly conserved Tyr67 is a likely electron-donor (radical acceptor) in the oxygenase half-reaction of the cyt c/CL peroxidase complex.
Asunto(s)
Cardiolipinas/química , Citocromos c/química , Peroxidasas/química , Tirosina/química , Animales , Simulación por Computador , Espectroscopía de Resonancia por Spin del Electrón , Hemo/química , Caballos , Peróxido de Hidrógeno/química , Hierro/química , Espectroscopía de Resonancia Magnética/métodos , Membranas Mitocondriales/metabolismo , Mutación , Miocardio/metabolismo , Oxígeno/química , Oxigenasas/química , Peroxidasa/química , Fenilalanina/químicaRESUMEN
Ferroptosis is a programmed iron-dependent cell death associated with peroxidation of lipids particularly, phospholipids. Several studies suggested a possible contribution of mitochondria to ferroptosis although the mechanisms underlying mitochondria-mediated ferroptotic pathways remain elusive. Reduced glutathione (GSH) is a central player in ferroptosis that is required for glutathione peroxidase 4 to eliminate oxidized phospholipids. Mitochondria do not produce GSH, and although the transport of GSH to mitochondria is not fully understood, two carrier proteins, the dicarboxylate carrier (DIC, SLC25A10) and the oxoglutarate carrier (OGC, SLC25A11) have been suggested to participate in GSH transport. Here, we elucidated the role of DIC and OGC as well as mitochondrial bioenergetics in ferroptosis in H9c2 cardioblasts. Results showed that mitochondria are highly sensitive to ferroptotic stimuli displaying fragmentation, and lipid peroxidation shortly after the onset of ferroptotic stimulus. Inhibition of electron transport chain complexes and oxidative phosphorylation worsened RSL3-induced ferroptosis. LC-MS/MS analysis revealed a dramatic increase in the levels of pro-ferroptotic oxygenated phosphatidylethanolamine species in mitochondria in response to RSL3 (ferroptosis inducer) and cardiac ischemia-reperfusion. Inhibition of DIC and OGC aggravated ferroptosis and increased mitochondrial ROS, membrane depolarization, and GSH depletion. Dihydrolipoic acid, an essential cofactor for several mitochondrial multienzyme complexes, attenuated ferroptosis and induced direct reduction of pro-ferroptotic peroxidized phospholipids to hydroxy-phospholipids in vitro. In conclusion, we suggest that ferroptotic stimuli diminishes mitochondrial bioenergetics and stimulates GSH depletion and glutathione peroxidase 4 inactivation leading to ferroptosis.
Asunto(s)
Ferroptosis , Cromatografía Liquida , Glutatión , Mitocondrias , Miocitos Cardíacos , Espectrometría de Masas en TándemRESUMEN
In addition to the known prominent role of polyunsaturated (phospho)lipids as structural blocks of biomembranes, there is an emerging understanding of another important function of these molecules as a highly diversified signaling language utilized for intra- and extracellular communications. Technological developments in high-resolution mass spectrometry facilitated the development of a new branch of metabolomics, redox lipidomics. Analysis of lipid peroxidation reactions has already identified specific enzymatic mechanisms responsible for the biosynthesis of several unique signals in response to inflammation and regulated cell death programs. Obtaining comprehensive information about millions of signals encoded by oxidized phospholipids, represented by thousands of interactive reactions and pleiotropic (patho)physiological effects, is a daunting task. However, there is still reasonable hope that significant discoveries, of at least some of the important contributors to the overall overwhelmingly complex network of interactions triggered by inflammation, will lead to the discovery of new small molecule regulators and therapeutic modalities. For example, suppression of the production of AA-derived pro-inflammatory mediators, HXA3 and LTB4, by an iPLA2 γ inhibitor, R-BEL, mitigated injury associated with the activation of pro-inflammatory processes in animals exposed to whole-body irradiation. Further, technological developments promise to make redox lipidomics a powerful approach in the arsenal of diagnostic and therapeutic instruments for personalized medicine of inflammatory diseases and conditions.
Asunto(s)
Apoptosis , Inflamación/metabolismo , Lipidómica , Transducción de Señal/fisiología , Animales , Ácidos Grasos Insaturados/metabolismo , Humanos , Inflamación/etiología , Hierro/metabolismo , Peroxidación de Lípido , Oxidación-Reducción , Irradiación Corporal TotalRESUMEN
Tumor microenvironment is characterized by immunosuppressive mechanisms associated with the accumulation of immune regulatory cells - myeloid-derived suppressor cells (MDSC). Therapeutic depletion of MDSC has been associated with inhibition of tumor growth and therefore represents an attractive approach to cancer immunotherapy. MDSC in cancer are characterized by enhanced enzymatic capacity to generate reactive oxygen and nitrogen species (RONS) which have been shown to effectively degrade carbonaceous materials. We prepared enzymatically openable nitrogen-doped carbon nanotube cups (NCNC) corked with gold nanoparticles and loaded with paclitaxel as a therapeutic cargo. Loading and release of paclitaxel was confirmed through electron microscopy, Raman spectroscopy and LC-MS analysis. Under the assumption that RONS generated by MDSCs can be utilized as a dual targeting and oxidative degradation mechanism for NCNC, here we report that systemic administration of paclitaxel loaded NCNC delivers paclitaxel to circulating and lymphoid tissue MDSC resulting in the inhibition of growth of tumors (B16 melanoma cells inoculated into C57BL/6 mice) in vivo. Tumor growth inhibition was associated with decreased MDSC accumulation quantified by flow cytometry that correlated with bio-distribution of gold-corked NCNC resolved by ICP-MS detection of residual gold in mouse tissue. Thus, we developed a novel immunotherapeutic approach based on unique nanodelivery vehicles, which can be loaded with therapeutic agents that are released specifically in MDSC via NCNC selective enzymatic "opening" affecting change in the tumor microenvironment.