RESUMO
Replication of the mitochondrial genome and expression of the genes it encodes both depend on a sufficient supply of nucleotides to mitochondria. Accordingly, dysregulated nucleotide metabolism not only destabilises the mitochondrial genome, but also affects its transcription. Here, we report that a mitochondrial nucleoside diphosphate kinase, NME6, supplies mitochondria with pyrimidine ribonucleotides that are necessary for the transcription of mitochondrial genes. Loss of NME6 function leads to the depletion of mitochondrial transcripts, as well as destabilisation of the electron transport chain and impaired oxidative phosphorylation. These deficiencies are rescued by an exogenous supply of pyrimidine ribonucleosides. Moreover, NME6 is required for the maintenance of mitochondrial DNA when the access to cytosolic pyrimidine deoxyribonucleotides is limited. Our results therefore reveal an important role for ribonucleotide salvage in mitochondrial gene expression.
Assuntos
Genes Mitocondriais , Pirimidinas , Pirimidinas/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Nucleotídeos , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Ribonucleotídeos/genéticaRESUMO
During embryonic development, cardiomyocytes undergo differentiation and maturation, processes that are tightly regulated by tissue-specific signaling cascades. Although redox signaling pathways involved in cardiomyogenesis are established, the exact sources responsible for reactive oxygen species (ROS) formation remain elusive. The present study investigates whether ROS produced by the mitochondrial flavoenzyme monoamine oxidase A (MAO-A) play a role in cardiomyocyte differentiation from human induced pluripotent stem cells (hiPSCs). Wild type (WT) and MAO-A knock out (KO) hiPSCs were generated by CRISPR/Cas9 genome editing and subjected to cardiomyocyte differentiation. Mitochondrial ROS levels were lower in MAO-A KO compared to the WT cells throughout the differentiation process. MAO-A KO hiPSC-derived cardiomyocytes (hiPSC-CMs) displayed sarcomere disarray, reduced α- to ß-myosin heavy chain ratio, GATA4 upregulation and lower macroautophagy levels. Functionally, genetic ablation of MAO-A negatively affected intracellular Ca2+ homeostasis in hiPSC-CMs. Mechanistically, MAO-A generated ROS contributed to the activation of AKT signaling that was considerably attenuated in KO cells. In addition, MAO-A ablation caused a reduction in WNT pathway gene expression consistent with its reported stimulation by ROS. As a result of WNT downregulation, expression of MESP1 and NKX2.5 was significantly decreased in MAO-A KO cells. Finally, MAO-A re-expression during differentiation rescued expression levels of cardiac transcription factors, contractile structure, and intracellular Ca2+ homeostasis. Taken together, these results suggest that MAO-A mediated ROS generation is necessary for the activation of AKT and WNT signaling pathways during cardiac lineage commitment and for the differentiation of fully functional human cardiomyocytes.
Assuntos
Células-Tronco Pluripotentes Induzidas , Miócitos Cardíacos , Humanos , Miócitos Cardíacos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Monoaminoxidase/genética , Monoaminoxidase/metabolismo , Diferenciação Celular/fisiologia , Via de Sinalização WntRESUMO
Coenzyme Q (or ubiquinone) is a redox-active lipid that serves as universal electron carrier in the mitochondrial respiratory chain and antioxidant in the plasma membrane limiting lipid peroxidation and ferroptosis. Mechanisms allowing cellular coenzyme Q distribution after synthesis within mitochondria are not understood. Here we identify the cytosolic lipid transfer protein STARD7 as a critical factor of intracellular coenzyme Q transport and suppressor of ferroptosis. Dual localization of STARD7 to the intermembrane space of mitochondria and the cytosol upon cleavage by the rhomboid protease PARL ensures the synthesis of coenzyme Q in mitochondria and its transport to the plasma membrane. While mitochondrial STARD7 preserves coenzyme Q synthesis, oxidative phosphorylation function and cristae morphogenesis, cytosolic STARD7 is required for the transport of coenzyme Q to the plasma membrane and protects against ferroptosis. A coenzyme Q variant competes with phosphatidylcholine for binding to purified STARD7 in vitro. Overexpression of cytosolic STARD7 increases ferroptotic resistance of the cells, but limits coenzyme Q abundance in mitochondria and respiratory cell growth. Our findings thus demonstrate the need to coordinate coenzyme Q synthesis and cellular distribution by PARL-mediated STARD7 processing and identify PARL and STARD7 as promising targets to interfere with ferroptosis.
Assuntos
Mitocôndrias , Ubiquinona , Transporte Biológico , Transporte de Elétrons , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Oxirredução , Ubiquinona/farmacologia , Ubiquinona/metabolismo , Proteínas de Transporte/metabolismoRESUMO
Reactive oxygen species (ROS) have been correlated with almost every human disease. Yet clinical exploitation of these hypotheses by pharmacological modulation of ROS has been scarce to nonexistent. Are ROS, thus, irrelevant for disease? No. One key misconception in the ROS field has been its consideration as a rather detrimental metabolic by-product of cell metabolism, and thus, any approach eliminating ROS to a certain tolerable level would be beneficial. We now know, instead, that ROS at every concentration, low or high, can serve many essential signaling and metabolic functions. This likely explains why systemic, nonspecific antioxidants have failed in the clinic, often with neutral and sometimes even detrimental outcomes. Recently, drug development has focused, instead, on identifying and selectively modulating ROS enzymatic sources that in a given constellation cause disease while leaving ROS physiologic signaling and metabolic functions intact. As sources, the family of NADPH oxidases stands out as the only enzyme family solely dedicated to ROS formation. Selectively targeting disease-relevant ROS-related proteins is already quite advanced, as evidenced by several phase II/III clinical trials and the first drugs having passed registration. The ROS field is expanding by including target enzymes and maturing to resemble more and more modern, big data-enhanced drug discovery and development, including network pharmacology. By defining a disease based on a distinct mechanism, in this case ROS dysregulation, and not by a symptom or phenotype anymore, ROS pharmacology is leaping forward from a clinical underperformer to a proof of concept within the new era of mechanism-based precision medicine. SIGNIFICANCE STATEMENT: Despite being correlated to almost every human disease, nearly no ROS modulator has been translated to the clinics yet. Here, we move far beyond the old-fashioned misconception of ROS as detrimental metabolic by-products and suggest 1) novel pharmacological targeting focused on selective modulation of ROS enzymatic sources, 2) mechanism-based redefinition of diseases, and 3) network pharmacology within the ROS field, altogether toward the new era of ROS pharmacology in precision medicine.
Assuntos
Antioxidantes/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Animais , Antioxidantes/uso terapêutico , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Humanos , Oxirredução/efeitos dos fármacos , Ensaios Clínicos Controlados Aleatórios como AssuntoRESUMO
Mitochondria are essential metabolic hubs that dynamically adapt to physiological demands. More than 40 proteases residing in different compartments of mitochondria, termed mitoproteases, preserve mitochondrial proteostasis and are emerging as central regulators of mitochondrial plasticity. These multifaceted enzymes limit the accumulation of short-lived, regulatory proteins within mitochondria, modulate the activity of mitochondrial proteins by protein processing, and mediate the degradation of damaged proteins. Various signaling cascades coordinate the activity of mitoproteases to preserve mitochondrial homeostasis and ensure cell survival. Loss of mitoproteases severely impairs the functional integrity of mitochondria, is associated with aging, and causes pleiotropic diseases. Understanding the dual function of mitoproteases as regulatory and quality control enzymes will help unravel the role of mitochondrial plasticity in aging and disease.
Assuntos
Envelhecimento/genética , Mitocôndrias/genética , Proteínas Mitocondriais/química , Neoplasias/genética , Doenças Neurodegenerativas/genética , Peptídeo Hidrolases/química , Envelhecimento/metabolismo , Animais , Apoptose/genética , Regulação da Expressão Gênica , Homeostase/genética , Humanos , Metabolismo dos Lipídeos/genética , Mitocôndrias/enzimologia , Dinâmica Mitocondrial/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mitofagia/genética , Neoplasias/enzimologia , Neoplasias/patologia , Doenças Neurodegenerativas/enzimologia , Doenças Neurodegenerativas/patologia , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Fosfolipídeos/metabolismo , Proteólise , Proteostase/genéticaRESUMO
Reactive oxygen species (ROS) have an equivocal role in myocardial ischaemia reperfusion injury. Within the cardiomyocyte, mitochondria are both a major source and target of ROS. We evaluate the effects of a selective, dose-dependent increase in mitochondrial ROS levels on cardiac physiology using the mitochondria-targeted redox cycler MitoParaquat (MitoPQ). Low levels of ROS decrease the susceptibility of neonatal rat ventricular myocytes (NRVMs) to anoxia/reoxygenation injury and also cause profound protection in an in vivo mouse model of ischaemia/reperfusion. However higher doses of MitoPQ resulted in a progressive alteration of intracellular [Ca2+] homeostasis and mitochondrial function in vitro, leading to dysfunction and death at high doses. Our data show that a primary increase in mitochondrial ROS can alter cellular function, and support a hormetic model in which low levels of ROS are cardioprotective while higher levels of ROS are cardiotoxic.
Assuntos
Modelos Animais de Doenças , Hormese , Mitocôndrias Cardíacas/metabolismo , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/citologia , Paraquat/farmacologia , Superóxidos/metabolismo , Animais , Animais Recém-Nascidos , Apoptose , Herbicidas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/patologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Ratos , Ratos WistarAssuntos
Pesquisa Biomédica/normas , Cardiologia/normas , Fármacos Cardiovasculares/uso terapêutico , Avaliação Pré-Clínica de Medicamentos/normas , Precondicionamento Isquêmico Miocárdico/normas , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Projetos de Pesquisa/normas , Animais , Pesquisa Biomédica/métodos , Cardiologia/métodos , Confiabilidade dos Dados , Interpretação Estatística de Dados , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Precondicionamento Isquêmico Miocárdico/métodos , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Reprodutibilidade dos TestesRESUMO
Reactive oxygen species (ROS) are involved in both physiological and pathological processes. This widely accepted concept is based more on the effects of antioxidant interventions than on reliable assessments of rates and sites of intracellular ROS formation. This argument applies also to mitochondria that are generally considered the major site for ROS formation, especially in skeletal and cardiac myocytes.Detection of oxidative modifications of intracellular or circulating molecules is frequently used as a marker of ROS formation. However, this approach provides limited information on spatiotemporal aspects of ROS formation that have to be defined in order to elucidate the role of ROS in a given pathophysiological condition. This information can be obtained by means of fluorescent probes that allow monitoring ROS formation in cell-free extracts and isolated cells. Thus, this approach can be used to characterize ROS formation in both isolated mitochondria and mitochondria within intact cells. This chapter describes three major examples of the use of fluorescent probes for monitoring mitochondrial ROS formation. Detailed methods description is accompanied by a critical analysis of the limitations of each technique, highlighting the possible sources of errors in performing the assay and results interpretation.
Assuntos
Corantes Fluorescentes/química , Fluorometria/métodos , Mitocôndrias Cardíacas/metabolismo , Cultura Primária de Células/métodos , Espécies Reativas de Oxigênio/análise , Animais , Animais Recém-Nascidos , Calibragem , Células Cultivadas , Fluorometria/instrumentação , Processamento de Imagem Assistida por Computador/instrumentação , Processamento de Imagem Assistida por Computador/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Miócitos Cardíacos , Oxirredução , Cultura Primária de Células/instrumentação , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , SoftwareRESUMO
Monoamine oxidase (MAO) inhibitors ameliorate contractile function in diabetic animals, but the mechanisms remain unknown. Equally elusive is the interplay between the cardiomyocyte alterations induced by hyperglycemia and the accompanying inflammation. Here we show that exposure of primary cardiomyocytes to high glucose and pro-inflammatory stimuli leads to MAO-dependent increase in reactive oxygen species that causes permeability transition pore opening and mitochondrial dysfunction. These events occur upstream of endoplasmic reticulum (ER) stress and are abolished by the MAO inhibitor pargyline, highlighting the role of these flavoenzymes in the ER/mitochondria cross-talk. In vivo, streptozotocin administration to mice induced oxidative changes and ER stress in the heart, events that were abolished by pargyline. Moreover, MAO inhibition prevented both mast cell degranulation and altered collagen deposition, thereby normalizing diastolic function. Taken together, these results elucidate the mechanisms underlying MAO-induced damage in diabetic cardiomyopathy and provide novel evidence for the role of MAOs in inflammation and inter-organelle communication. MAO inhibitors may be considered as a therapeutic option for diabetic complications as well as for other disorders in which mast cell degranulation is a dominant phenomenon.
Assuntos
Degranulação Celular/efeitos dos fármacos , Estresse do Retículo Endoplasmático , Mitocôndrias/metabolismo , Inibidores da Monoaminoxidase/farmacologia , Monoaminoxidase/metabolismo , Remodelação Ventricular/efeitos dos fármacos , Animais , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/patologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Glucose/farmacologia , Interleucina-1beta/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Monoaminoxidase/química , Monoaminoxidase/genética , Células Musculares/citologia , Células Musculares/metabolismo , Células Musculares/fisiologia , Miocárdio/metabolismo , Miocárdio/patologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismoRESUMO
In the past decade, accumulating evidence highlighted the role of monoamine oxidases (MAOs) in cardiovascular disease (CVD). MAOs are flavoenzymes located in the outer mitochondrial membrane, responsible for the degradation of neurotransmitters and biogenic amines. During this process they generate hydrogen peroxide, aldehydes and ammonia, species that can target mitochondria and induce mitochondrial dysfunction and cardiomyocyte death. Indeed, MAO inhibition affords cardioprotection in several models of CVD, such as ischemia/reperfusion, heart failure and diabetes. Importantly, a few studies provided encouraging results suggesting that MAO inhibition might be beneficial also in patients with CVD. Thus, selective and reversible MAO inhibitors, currently used as therapy for depression and neurodegenerative disorders, might be considered as candidate drugs for the treatment of CVD.
Assuntos
Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/metabolismo , Inibidores da Monoaminoxidase/farmacologia , Inibidores da Monoaminoxidase/uso terapêutico , Monoaminoxidase/metabolismo , Animais , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismoRESUMO
Reactive oxygen species (ROS) formation and signaling are of major importance and regulate a number of processes in physiological conditions. A disruption in redox status regulation, however, has been associated with numerous pathological conditions. In recent years it has become increasingly clear that oxidative and reductive modifications are confined in a spatio-temporal manner. This makes ROS signaling similar to that of Ca(2+) or other second messengers. Some subcellular compartments are more oxidizing (such as lysosomes or peroxisomes) whereas others are more reducing (mitochondria, nuclei). Moreover, although more reducing, mitochondria are especially susceptible to oxidation, most likely due to the high number of exposed thiols present in that compartment. Recent advances in the development of redox probes allow specific measurement of defined ROS in different cellular compartments in intact living cells or organisms. The availability of these tools now allows simultaneous spatio-temporal measurements and correlation between ROS generation and organelle and/or cellular function. The study of ROS compartmentalization and microdomains will help elucidate their role in physiology and disease. Here we will examine redox probes currently available and how ROS generation may vary between subcellular compartments. Furthermore, we will discuss ROS compartmentalization in physiological and pathological conditions focusing our attention on mitochondria, since their vulnerability to oxidative stress is likely at the basis of several diseases.
RESUMO
Bumblebees, amongst the most important of pollinators, are under enormous population pressures. One of these is disease. The bumblebee and its gut trypanosome Crithidia bombi are one of the fundamental models of ecological immunology. Although there is previous evidence of increased immune gene expression upon Crithidia infection, recent work has focussed on the bumblebee's gut microbiota. Here, by knocking down gene expression using RNAi, we show for the first time that antimicrobial peptides (AMPs) have a functional role in anti-Crithidia defense.