RESUMO
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.
Assuntos
Injúria Renal Aguda/patologia , Asma/patologia , Lesões Encefálicas Traumáticas/patologia , Morte Celular , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Injúria Renal Aguda/metabolismo , Animais , Apoptose , Asma/metabolismo , Lesões Encefálicas Traumáticas/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular , Humanos , Isoenzimas/metabolismo , Lipoxigenase/química , Lipoxigenase/metabolismo , Camundongos , Modelos Moleculares , Oxazolidinonas/farmacologia , Oxirredução , Proteína de Ligação a Fosfatidiletanolamina/químicaRESUMO
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.
Assuntos
Ferroptose/fisiologia , Fosfolipases A2 do Grupo VI/metabolismo , Animais , Araquidonato 15-Lipoxigenase/metabolismo , Modelos Animais de Doenças , Feminino , Fosfolipases A2 do Grupo VI/fisiologia , Humanos , Ferro/metabolismo , Leucotrienos/metabolismo , Metabolismo dos Lipídeos/fisiologia , Peróxidos Lipídicos/metabolismo , Lipídeos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oxirredução , Doença de Parkinson/metabolismo , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Fosfolipases/metabolismo , Fosfolipídeos/metabolismo , Ratos , Ratos Endogâmicos LewRESUMO
For targeted protein panels, the ability to specifically assay post-translational modifications (PTMs) in a quantitative, sensitive, and straightforward manner would substantially advance biological and pharmacological studies. The present study highlights the effectiveness of the Affi-BAMS™ epitope-directed affinity bead capture/MALDI MS platform for quantitatively defining complex PTM marks of H3 and H4 histones. Using H3 and H4 histone peptides and isotopically labelled derivatives, this affinity bead and MALDI MS platform achieves a range of >3 orders of magnitude with a technical precision CV of <5%. Using nuclear cellular lysates, Affi-BAMS PTM-peptide capture resolves heterogeneous histone N-terminal PTMs with as little as 100 µg of starting material. In an HDAC inhibitor and MCF7 cell line model, the ability to monitor dynamic histone H3 acetylation and methylation events is further demonstrated (including SILAC quantification). Affi-BAMS (and its capacity for the multiplexing of samples and target PTM-proteins) thus provides a uniquely efficient and effective approach for analyzing dynamic epigenetic histone marks, which is critical for the regulation of chromatin structure and gene expression.
Assuntos
Histonas , Proteômica , Histonas/metabolismo , Espectrometria de Massas em Tandem , Processamento de Proteína Pós-Traducional , Código das Histonas , Peptídeos/metabolismo , AcetilaçãoRESUMO
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.
Assuntos
Ferroptose/fisiologia , Macrófagos/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Animais , Araquidonato 15-Lipoxigenase/metabolismo , Araquidonato 15-Lipoxigenase/fisiologia , Morte Celular , Feminino , Ferro/metabolismo , Ferro/fisiologia , Leucotrienos/metabolismo , Peroxidação de Lipídeos/fisiologia , Peróxidos Lipídicos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Óxido Nítrico Sintase Tipo II/fisiologia , Oxirredução , Espécies Reativas de Oxigênio/metabolismoRESUMO
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.
Assuntos
Neoplasias da Mama , Feminino , Humanos , Íons , Lipídeos , Espectrometria de Massa de Íon Secundário , Microambiente TumoralRESUMO
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.
Assuntos
Ferroptose/fisiologia , Óxido Nítrico/metabolismo , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Araquidonato 15-Lipoxigenase/metabolismo , Morte Celular/fisiologia , Humanos , Lipidômica , Macrófagos/metabolismo , Simulação de Dinâmica Molecular , Oxirredução , Fosfatidiletanolaminas , Fosfolipídeos/metabolismoRESUMO
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 70â keV (H2 O)n+ (n>28 000) cluster ion beam, we were able to map PEox with 1.2â 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 (20â pmol µmol-1 lipid) peroxidized lipids in subcellular compartments and their accumulation in disease conditions.
Assuntos
Ferroptose/fisiologia , Peroxidação de Lipídeos/fisiologia , Fosfatidiletanolaminas/metabolismo , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Lesões Encefálicas Traumáticas/metabolismo , Carbolinas/farmacologia , Linhagem Celular , Ferroptose/efeitos dos fármacos , Masculino , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos Sprague-Dawley , Espectrometria de Massa de Íon Secundário/métodosRESUMO
OBJECTIVES: Traumatic brain injury triggers multiple cell death pathways, possibly including ferroptosis-a recently described cell death pathway that results from accumulation of 15-lipoxygenase-mediated lipid oxidation products, specifically oxidized phosphatidylethanolamine containing arachidonic or adrenic acid. This study aimed to investigate whether ferroptosis contributed to the pathogenesis of in vitro and in vivo traumatic brain injury, and whether inhibition of 15-lipoxygenase provided neuroprotection. DESIGN: Cell culture study and randomized controlled animal study. SETTING: University research laboratory. SUBJECTS: HT22 neuronal cell line and adult male C57BL/6 mice. INTERVENTIONS: HT22 cells were subjected to pharmacologic induction of ferroptosis or mechanical stretch injury with and without administration of inhibitors of ferroptosis. Mice were subjected to sham or controlled cortical impact injury. Injured mice were randomized to receive vehicle or baicalein (12/15-lipoxygenase inhibitor) at 10-15 minutes postinjury. MEASUREMENTS AND MAIN RESULTS: Pharmacologic inducers of ferroptosis and mechanical stretch injury resulted in cell death that was rescued by prototypical antiferroptotic agents including baicalein. Liquid chromatography tandem-mass spectrometry revealed the abundance of arachidonic/adrenic-phosphatidylethanolamine compared with other arachidonic/adrenic acid-containing phospholipids in the brain. Controlled cortical impact resulted in accumulation of oxidized phosphatidylethanolamine, increased expression of 15-lipoxygenase and acyl-CoA synthetase long-chain family member 4 (enzyme that generates substrate for the esterification of arachidonic/adrenic acid into phosphatidylethanolamine), and depletion of glutathione in the ipsilateral cortex. Postinjury administration of baicalein attenuated oxidation of arachidonic/adrenic acid-containing-phosphatidylethanolamine, decreased the number of terminal deoxynucleotidyl transferase dUTP nick-end labeling positive cells in the hippocampus, and improved spatial memory acquisition versus vehicle. CONCLUSIONS: Biomarkers of ferroptotic death were increased after traumatic brain injury. Baicalein decreased ferroptotic phosphatidylethanolamine oxidation and improved outcome after controlled cortical impact, suggesting that 15-lipoxygenase pathway might be a valuable therapeutic target after traumatic brain injury.
Assuntos
Lesões Encefálicas Traumáticas , Ferroptose , Neurônios , Animais , Masculino , Lesões Encefálicas Traumáticas/complicações , Lesões Encefálicas Traumáticas/patologia , Linhagem Celular , Modelos Animais de Doenças , Cromatografia Gasosa-Espectrometria de Massas , Aprendizagem em Labirinto , Camundongos Endogâmicos C57BL , Neurônios/patologia , CamundongosRESUMO
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.
Assuntos
Lesões Encefálicas/metabolismo , Cardiolipinas/sangue , Cardiomiopatias/metabolismo , Mitocôndrias Cardíacas/metabolismo , Animais , Reanimação Cardiopulmonar/métodos , Estudos de Casos e Controles , Feminino , Cromatografia Gasosa-Espectrometria de Massas/métodos , Parada Cardíaca/metabolismo , Humanos , Masculino , Oxirredução , Ratos , Ratos Sprague-DawleyRESUMO
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.
Assuntos
Ácido Araquidônico/metabolismo , Ácidos Graxos Insaturados/metabolismo , Fosfolipídeos/metabolismo , Animais , Ácido Araquidônico/antagonistas & inibidores , Morte Celular/efeitos dos fármacos , Linhagem Celular , Coenzima A Ligases/antagonistas & inibidores , Coenzima A Ligases/deficiência , Coenzima A Ligases/metabolismo , Ácidos Graxos Insaturados/antagonistas & inibidores , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
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 â¼100 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.
Assuntos
Araquidonato 15-Lipoxigenase/metabolismo , Morte Celular/fisiologia , Fosfatidiletanolaminas/metabolismo , Animais , Araquidonato 15-Lipoxigenase/química , Catálise , Linhagem Celular , Camundongos , Mutação , Oxirredução , Proteína de Ligação a Fosfatidiletanolamina/genética , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Fosfatidiletanolaminas/química , Especificidade por SubstratoRESUMO
The Tat machinery catalyzes the transport of folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane in plants. Using fluorescence quenching and cross-linking approaches, we demonstrate that the Escherichia coli TatBC complex catalyzes insertion of a pre-SufI signal peptide hairpin that penetrates about halfway across the membrane bilayer. Analysis of 512 bacterial Tat signal peptides using secondary structure prediction and docking algorithms suggest that this hairpin interaction mode is generally conserved. An internal cross-link in the signal peptide that blocks transport but does not affect binding indicates that a signal peptide conformational change is required during translocation. These results suggest, to our knowledge, a novel hairpin-hinge model in which the signal peptide hairpin unhinges during movement of the mature domain across the membrane. Thus, in addition to enabling the necessary recognition, the interaction of Tat signal peptides with the receptor complex plays a critical role in the transport process itself.
Assuntos
Produtos do Gene tat/química , Produtos do Gene tat/metabolismo , Sinais Direcionadores de Proteínas , Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Conformação Proteica , Transporte Proteico , Água/químicaRESUMO
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.
Assuntos
Lesões Encefálicas Traumáticas/metabolismo , Encéfalo/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Mediadores da Inflamação/metabolismo , Animais , Araquidonato 15-Lipoxigenase/imunologia , Araquidonato 15-Lipoxigenase/metabolismo , Encéfalo/imunologia , Encéfalo/patologia , Química Encefálica/imunologia , Lesões Encefálicas Traumáticas/imunologia , Lesões Encefálicas Traumáticas/patologia , Ácidos Graxos não Esterificados/imunologia , Mediadores da Inflamação/imunologia , Masculino , Oxirredução , Estresse Oxidativo , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
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.
Assuntos
Cardiolipinas/metabolismo , Transdução de Sinais/fisiologia , Animais , Citocromos c/metabolismo , Humanos , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Oxirredução , Fosfolipídeos/metabolismoRESUMO
PURPOSE OF REVIEW: Lipid peroxidation has long been established as a key player in the pathophysiology of critical illness. Recent developments in oxidative lipidomics have aided in deciphering the molecular mechanisms of lipid oxidation in health and disease. This review discusses recent achievements and recent developments in oxidative lipidomics and its contribution to the understanding of critical illness. RECENT FINDINGS: Most studies involving acute injury focus on identifying the end products of lipid peroxidation. This misses the early events and targets of peroxidation mechanisms. Recent developments in liquid chromatography tandem mass spectrometry-based oxidative lipidomics have enabled the identification of a wide variety of enzymatically generated lipid oxidation products. Such lipid mediators have been found to play an important role in injury, inflammation, and recovery in disease states such as sepsis or head trauma. SUMMARY: Multiple lipid oxidation products are formed either through enzymatic pathways or through random chemical reactions. These products are often biologically active and can contribute to the regulation of cellular signaling. Oxidative lipidomics has contributed to the identification and quantification of lipid peroxidation products, the mechanism and time course of their production after injury, and synergistic functioning with other regulatory processes in the body. These advances in knowledge will help guide the future development of interventions in critical illness.
Assuntos
Cuidados Críticos , Peroxidação de Lipídeos , Lipídeos/análise , Biomarcadores/análise , Cromatografia Líquida de Alta Pressão , Estado Terminal , Humanos , Inflamação , Metabolismo dos Lipídeos/fisiologia , Lipídeos/química , Oxirredução , Espectrometria de Massas em TandemRESUMO
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.
Assuntos
Lesões Encefálicas Traumáticas/diagnóstico por imagem , Cardiolipinas , Córtex Cerebral/diagnóstico por imagem , Hipocampo/diagnóstico por imagem , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tálamo/diagnóstico por imagem , Animais , Contusão Encefálica/diagnóstico por imagem , Contusão Encefálica/metabolismo , Lesões Encefálicas Traumáticas/metabolismo , Cardiolipinas/metabolismo , Córtex Cerebral/metabolismo , Hipocampo/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Tálamo/metabolismoRESUMO
MOTIVATION: Quantification of lipids is a primary goal in lipidomics. In direct infusion/injection (or shotgun) lipidomics, accurate downstream identification and quantitation requires accurate summarization of repetitive peak measurements. Imprecise peak summarization multiplies downstream error by propagating into species identification and intensity estimation. To our knowledge, this is the first analysis of direct infusion peak summarization in the literature. RESULTS: We present two novel peak summarization algorithms for direct infusion samples and compare them with an off-machine ad hoc summarization algorithm as well as with the propriety Xcalibur algorithm. Our statistical agglomeration algorithm reduces peakwise error by 38% mass/charge (m/z) and 44% (intensity) compared with the ad hoc method over three datasets. Pointwise error is reduced by 23% (m/z). Compared with Xcalibur, our statistical agglomeration algorithm produces 68% less m/z error and 51% less intensity error on average on two comparable datasets. AVAILABILITY: The source code for Statistical Agglomeration and the datasets used are freely available for non-commercial purposes at https://github.com/optimusmoose/statistical_agglomeration. Modified Bin Aggolmeration is freely available in MSpire, an open source mass spectrometry package at https://github.com/princelab/mspire/.
Assuntos
Genômica/métodos , Lipídeos/análise , Algoritmos , Internet , Lipídeos/química , Espectrometria de Massas , Linguagens de Programação , SoftwareRESUMO
Ceramide is a sphingolipid that serves as an important second messenger in an increasing number of stress-induced pathways. Ceramide has long been known to affect the mitochondria, altering both morphology and physiology. We sought to assess the impact of ceramide on skeletal muscle mitochondrial structure and function. A primary observation was the rapid and dramatic division of mitochondria in ceramide-treated cells. This effect is likely to be a result of increased Drp1 (dynamin-related protein 1) action, as ceramide increased Drp1 expression and Drp1 inhibition prevented ceramide-induced mitochondrial fission. Further, we found that ceramide treatment reduced mitochondrial O2 consumption (i.e. respiration) in cultured myotubes and permeabilized red gastrocnemius muscle fibre bundles. Ceramide treatment also increased H2O2 levels and reduced Akt/PKB (protein kinase B) phosphorylation in myotubes. However, inhibition of mitochondrial fission via Drp1 knockdown completely protected the myotubes and fibre bundles from ceramide-induced metabolic disruption, including maintained mitochondrial respiration, reduced H2O2 levels and unaffected insulin signalling. These data suggest that the forced and sustained mitochondrial fission that results from ceramide accrual may alter metabolic function in skeletal muscle, which is a prominent site not only of energy demand (via the mitochondria), but also of ceramide accrual with weight gain.
Assuntos
Ceramidas/toxicidade , Peróxido de Hidrogênio/metabolismo , Mitocôndrias Musculares/metabolismo , Dinâmica Mitocondrial/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Animais , Linhagem Celular , Dinaminas/metabolismo , Insulina/metabolismo , Masculino , Camundongos , Mitocôndrias Musculares/patologia , Fibras Musculares Esqueléticas/patologia , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Unlike chronological age, biological age is a strong indicator of health of an individual. However, the molecular fingerprint associated with biological age is ill-defined. To define a high-resolution signature of biological age, we analyzed metabolome, circulating senescence-associated secretome (SASP)/inflammation markers and the interaction between them, from a cohort of healthy and rapid agers. The balance between two fatty acid oxidation mechanisms, ß-oxidation and ω-oxidation, associated with the extent of functional aging. Furthermore, a panel of 25 metabolites, Healthy Aging Metabolic (HAM) index, predicted healthy agers regardless of gender and race. HAM index was also validated in an independent cohort. Causal inference with machine learning implied three metabolites, ß-cryptoxanthin, prolylhydroxyproline, and eicosenoylcarnitine as putative drivers of biological aging. Multiple SASP markers were also elevated in rapid agers. Together, our findings reveal that a network of metabolic pathways underlie biological aging, and the HAM index could serve as a predictor of phenotypic aging in humans.
Assuntos
Senescência Celular , Secretoma , Humanos , Envelhecimento/genética , Envelhecimento/metabolismo , Metaboloma , Biomarcadores/metabolismoRESUMO
Mitochondrial trifunctional protein (TFP) deficiency is an inherited metabolic disorder leading to a block in long-chain fatty acid ß-oxidation. Mutations in either HADHA and HADHB, which encode the TFPα and ß subunits, respectively, usually result in combined TFP deficiency. A single common mutation, HADHA c.1528G>C (p.E510Q), leads to isolated 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency. TFP also catalyzes a step in the remodeling of cardiolipin (CL), a phospholipid critical to mitochondrial membrane stability and function. We explored the effect of mutations in TFP subunits on CL and other phospholipid content and composition and the consequences of these changes on mitochondrial bioenergetics in patient-derived fibroblasts. Abnormalities in these parameters varied extensively among different fibroblasts, and some cells were able to maintain basal oxygen consumption rates similar to controls. Although CL reduction was universally identified, a simultaneous increase in monolysocardiolipins was discrepant among cells. A similar profile was seen in liver mitochondria isolates from a TFP-deficient mouse model. Response to new potential drugs targeting cardiolipin metabolism might be dependent on patient genotype.