RESUMEN
Peroxiredoxins (Prxs) are a family of thiol peroxidases that participate in hydroperoxide detoxification and regulates H2O2 signaling. In mammals, the four typical 2-Cys Prxs (Prxs 1, 2, 3 and 4) are known to regulate H2O2-mediated intracellular signaling. The 2 catalytic cysteines of 2-Cys Prxs, the so-called peroxidatic and resolving cysteines, are regulatory switches that are prone to react with redox signaling molecules. We investigated the respective modifications induced by H2O2, NO and H2S in the murine macrophage cell line RAW264.7 by mass spectrometry and immunoblotting after separating 2-Cys Prxs by one-dimensional or two-dimensional PAGE. We found that H2S, unlike NO, does not prevent H2O2-mediated sulfinylation of 2-Cys Prxs and that Prx2 is more sensitive to NO-mediated protection against sulfinylation by peroxides. We also observed that cells exposed to exogenous NO, released by Cys-SNO or DETA-NO, or producing NO upon stimulation by IFN-γ and LPS, present an acidic form of Prx1 whose modification is consistent with S-homocysteinylation of its peroxidatic cysteine.
Asunto(s)
Peroxirredoxinas/metabolismo , Animales , Línea Celular , Cromatografía Líquida de Alta Presión , Cisteína/química , Cisteína/metabolismo , Electroforesis en Gel Bidimensional , Peróxido de Hidrógeno/toxicidad , Sulfuro de Hidrógeno/toxicidad , Interferón gamma/farmacología , Lipopolisacáridos/toxicidad , Ratones , Óxido Nítrico/toxicidad , Peroxirredoxinas/análisis , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Compuestos de Sulfhidrilo/químicaRESUMEN
In eukaryotes, mitochondrial iron-sulfur cluster (ISC), export and cytosolic iron-sulfur cluster assembly (CIA) machineries carry out biogenesis of iron-sulfur (Fe-S) clusters, which are critical for multiple essential cellular pathways. However, little is known about their export out of mitochondria. Here we show that Fe-S assembly of mitoNEET, the first identified Fe-S protein anchored in the mitochondrial outer membrane, strictly depends on ISC machineries and not on the CIA or CIAPIN1. We identify a dedicated ISC/export pathway in which augmenter of liver regeneration, a mitochondrial Mia40-dependent protein, is specific to mitoNEET maturation. When inserted, the Fe-S cluster confers mitoNEET folding and stability in vitro and in vivo. The holo-form of mitoNEET is resistant to NO and H2O2 and is capable of repairing oxidatively damaged Fe-S of iron regulatory protein 1 (IRP1), a master regulator of cellular iron that has recently been involved in the mitochondrial iron supply. Therefore, our findings point to IRP1 as the missing link to explain the function of mitoNEET in the control of mitochondrial iron homeostasis.
Asunto(s)
Proteína 1 Reguladora de Hierro/química , Hierro/metabolismo , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/química , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/química , Animales , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación de la Expresión Génica , Células HeLa , Células Hep G2 , Homeostasis , Humanos , Peróxido de Hidrógeno/química , Proteína 1 Reguladora de Hierro/genética , Proteína 1 Reguladora de Hierro/metabolismo , Ratones , Ratones Transgénicos , Mitocondrias/química , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Membranas Mitocondriales/química , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Óxido Nítrico/química , Oxidación-Reducción , Pliegue de Proteína , Estabilidad Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de SeñalRESUMEN
Peroxiredoxins (Prxs) are a family of peroxidases that maintain thiol homeostasis by catalyzing the reduction of organic hydroperoxides, H2O2, and peroxynitrite. Eukaryotic 2-Cys-Prxs, also referred to as typical Prxs, can be inactivated by oxidation of the catalytic cysteine to sulfinic acid, which may regulate the intracellular messenger function of H2O2. A small redox protein, sulfiredoxin (Srx), has been shown to reduce sulfinylated 2-Cys-Prxs and thus to regenerate active 2-Cys-Prxs. We previously reported that cytokine-induced nitric oxide (NO) intervenes in this pathway by decreasing the level of 2-Cys overoxidation and by upregulating Srx through the activation of the transcription factor nuclear factor erythroid 2-related factor (Nrf2). Here, we describe the methods used to monitor the interplay between NO and H2O2 in the regulation of the Prx/Srx system in immunostimulated macrophages, which produce both reactive oxygen species and NO.
Asunto(s)
Proteínas de Homeodominio/metabolismo , Peróxido de Hidrógeno/metabolismo , Óxido Nítrico/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismo , Transducción de Señal , Animales , Western Blotting , Línea Celular , Medios de Cultivo , Activadores de Enzimas/farmacología , Glucosa Oxidasa/química , Macrófagos/enzimología , Ratones , Oxidación-Reducción , Procesamiento Proteico-Postraduccional , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Benzylidenemalononitrile (BMN) tyrphostins are well known as potent tyrosine kinase inhibitors. Moreover, in recent years it has been recognized that members of the tyrphostin family possess additional biological activities independent of their ability to inhibit protein tyrosine kinases. In this study, we examined the relationship between the structure of 49 BMNs and related compounds, and their capacity to induce heme oxygenase 1 (HO-1) gene expression in U937 human monocytic cells, to activate upstream signaling pathways and to protect cells against menadione-induced oxidative stress. It was found that the electron-withdrawing (NO(2), CN, halogen) groups in BMN molecules and double meta-MeO substituents increased the HO-1 gene induction, while the electron-donating groups in ortho/para position (OH, MeO and N-morpholino) significantly decreased it. The magnitude of activation of c-Jun, Nrf2, p38 MAPK, and p70S6K correlated with specific substitution patterns in the BMN structure. BMN-dependent maximal up-regulation of HO-1 required parallel increase in Nrf2 and phospho-c-Jun cellular levels. Liquid chromatography mass spectrometry (LC-MS) analysis revealed that BMNs can generate conjugates with one or two glutathione equivalent(s). This study supports the hypothesis that BMNs induce the expression of protective genes by alkylating sensitive cysteine residues of regulatory factors.
Asunto(s)
Compuestos de Bencilideno/farmacología , Nitrilos/farmacología , Estrés Oxidativo , Transducción de Señal/efectos de los fármacos , Compuestos de Bencilideno/metabolismo , Cromatografía Liquida , Glutatión/metabolismo , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Humanos , Nitrilos/metabolismo , ARN Mensajero/análisis , Transducción de Señal/fisiología , Espectrometría de Masa por Ionización de Electrospray , Relación Estructura-Actividad , Tirfostinos/farmacología , Células U937RESUMEN
In mammals, iron regulatory proteins (IRPs) 1 and 2 posttranscriptionally regulate expression of genes involved in iron metabolism, including transferrin receptor 1, the ferritin (Ft) H and L subunits, and ferroportin by binding mRNA motifs called iron responsive elements (IREs). IRP1 is a bifunctional protein that mostly exists in a non-IRE-binding, [4Fe-4S] cluster aconitase form, whereas IRP2, which does not assemble an Fe-S cluster, spontaneously binds IREs. Although both IRPs fulfill a trans-regulatory function, only mice lacking IRP2 misregulate iron metabolism. NO stimulates the IRE-binding activity of IRP1 by targeting its Fe-S cluster. IRP2 has also been reported to sense NO, but the intrinsic function of IRP1 and IRP2 in NO-mediated regulation of cellular iron metabolism is controversial. In this study, we exposed bone marrow macrophages from Irp1(-/-) and Irp2(-/-) mice to NO and showed that the generated apo-IRP1 was entirely responsible for the posttranscriptional regulation of transferrin receptor 1, H-Ft, L-Ft, and ferroportin. The powerful action of NO on IRP1 also remedies the defects of iron storage found in IRP2-null bone marrow macrophages by efficiently reducing Ft overexpression. We also found that NO-dependent IRP1 activation, resulting in increased iron uptake and reduced iron sequestration and export, maintains enough intracellular iron to fuel the Fe-S cluster biosynthetic pathway for efficient restoration of the citric acid cycle aconitase in mitochondria. Thus, IRP1 is the dominant sensor and transducer of NO for posttranscriptional regulation of iron metabolism and participates in Fe-S cluster repair after exposure to NO.
Asunto(s)
Células de la Médula Ósea/metabolismo , Factores Relajantes Endotelio-Dependientes/farmacología , Proteína 1 Reguladora de Hierro/metabolismo , Proteína 2 Reguladora de Hierro/metabolismo , Hierro/metabolismo , Macrófagos/metabolismo , Óxido Nítrico/farmacología , Animales , Apoferritinas/genética , Apoferritinas/metabolismo , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Proteína 1 Reguladora de Hierro/genética , Proteína 2 Reguladora de Hierro/genética , Ratones , Ratones Noqueados , Receptores de Transferrina/genética , Receptores de Transferrina/metabolismoRESUMEN
Peroxiredoxins (Prx's) are a family of peroxidases that maintain thiol homeostasis by catalyzing the reduction of organic hydroperoxides, H2O2, and peroxynitrite. Under conditions of oxidative stress, eukaryotic Prx's can be inactivated by the substrate-dependent oxidation of the catalytic cysteine to sulfinic acid, which may regulate the intracellular messenger function of H2O2. A small redox protein, sulfiredoxin (Srx), conserved only in eukaryotes, has been shown to reduce sulfinylated 2-Cys Prx's, adding to the complexity of the H2O2 signaling network. In this study, we addressed the regulation of Srx expression in immunostimulated primary macrophages that produce both reactive oxygen species (ROS) and nitric oxide (NO(â¢)). We present genetic evidence that NO-mediated Srx up-regulation is mediated by the transcription factor nuclear factor erythroid 2-related factor (Nrf2). We also show that the NO(â¢)/Srx pathway inhibits generation of ROS. These results reveal a link between innate immunity and H2O2 signaling. We propose that an NO(â¢)/Nrf2/Srx pathway participates in the maintenance of redox homeostasis in cytokine-activated macrophages and other inflammatory settings.
Asunto(s)
Antioxidantes/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Óxido Nítrico/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismo , Animales , Células Cultivadas , Peróxido de Hidrógeno/metabolismo , Inmunidad Innata , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oxidación-Reducción , Estrés Oxidativo , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/genética , Reacción en Cadena de la Polimerasa , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Peroxiredoxins constitute a major family of cysteine-based peroxide-scavenging enzymes. They carry an intriguing redox switch by undergoing substrate-mediated inactivation via overoxidation of their catalytic cysteine to the sulfinic acid form that is reverted by reduction catalyzed by the sulfinic acid reductase sulfiredoxin (Srx). The biological significance of such inactivation is not understood, nor is the function of Srx1. To address this question, we generated a mouse line with a null deletion of the Srx1-encoding Srxn1 gene. We show here that Srxn1(-/-) mice are perfectly viable and do not suffer from any apparent defects under laboratory conditions, but have an abnormal response to lipopolysaccharide that manifests by increased mortality during endotoxic shock. Microarray-based mRNA profiles show that although the response of Srxn1(-/-) mice to lipopolysaccharide is typical, spanning all spectrum and all pathways of innate immunity, it is delayed by several hours and remains intense when the response of Srxn1(+/+) mice has already dissipated. These data indicate that Srx1 activity protects mice from the lethality of endotoxic shock, adding this enzyme to other host factors, as NRF2 and peroxiredoxin 2, which by regulating cellular reactive oxygen species levels act as important modifiers in the pathogenesis of sepsis.
Asunto(s)
Lipopolisacáridos/farmacología , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismo , Choque Séptico/metabolismo , Alanina Transaminasa/sangre , Animales , Aspartato Aminotransferasas/sangre , Células Cultivadas , Femenino , Ingeniería Genética , Proteínas de Homeodominio/metabolismo , Inmunidad Innata , Estimación de Kaplan-Meier , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/genética , Especies Reactivas de Oxígeno/metabolismo , Choque Séptico/inmunología , Transducción de Señal , Transcripción GenéticaRESUMEN
We examined early and late alterations in gene expression patterns and phosphorylation levels of key regulators of selected signaling pathways in U937 cells exposed to various (*)NO fluxes. cDNA microarray analysis and real-time quantitative PCR identified 45 NO-sensitive genes (>or=2-fold change), among which KLF2, KLF6, TSC22D3, DDIT4, MKP-5 (up-regulated), KIF23, histone H4, ARL6IP2, CLNS1A, SLC7A6, CDKN3, SRP19, and BCL11A (down-regulated) have not been reported before. For two selected genes, KLF2 and DDIT4, the sensitivity to (.)NO was also proven at the protein level. Among the examined genes, only KLF2 had a higher sensitivity to slow release of NO (DETA-NO) than to high-dose, short-duration exposure (DPTA-NO), reaching an about 50-fold increase in mRNA level. Our study revealed that fast and slow NO donors activate similar signaling pathways and induce phosphorylation of MAP kinases and downstream transcription factors ATF2 and c-Jun. Inhibitory analysis of major signaling pathways showed that activity of p38 MAPK and tyrosine kinases is indispensable for gene induction in cells exposed to DPTA-NO, whereas G-protein Rho suppression caused superinduction of KLF2 in (*)NO-stimulated cells. Finally, we showed that both (*)NO donors caused a marked decrease in phosphorylation of p70S6K, an mTOR substrate and regulator of mRNA translation, and protein kinase Akt, an upstream positive regulator of mTOR.
Asunto(s)
Factores de Transcripción de Tipo Kruppel/biosíntesis , Monocitos/metabolismo , Proteínas Proto-Oncogénicas/biosíntesis , Factores de Transcripción/biosíntesis , Factor de Transcripción Activador 2/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Reguladores de Proteínas de Unión al GTP/metabolismo , Perfilación de la Expresión Génica , Humanos , Factor 6 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Análisis por Micromatrices , Monocitos/patología , Óxido Nítrico/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas/genética , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal , Factores de Transcripción/genética , Células U937RESUMEN
Peroxiredoxins (PRXs) are thiol peroxidases associated with many cellular functions including proliferation, cell cycle, apoptosis, and differentiation. There is also increasing evidence that these ubiquitous antioxidant enzymes control H(2)O(2) signaling in eukaryotes. Here, we provide evidence that the LPS/TLR4 and the Th1 cytokine IFN-gamma pathways induce expression of PRX5, a potent peroxide and peroxynitrite reductase, in primary macrophages. Furthermore, deletion of TRIF, MyD88, or type I IFN receptor revealed that the LPS/TLR4-dependent increase in PRX5 expression is mediated by a TRIF-dependent/IFN-beta-independent pathway. IFN-gamma-dependent induction of the PRX5 gene was markedly reduced in MyD88(-/-) and TNF(-/-) macrophages. Moreover, addition of exogenous TNF allowed the recovery of full PRX5 expression in both MyD88(-/-) and TNF(-/-) cells stimulated with IFN-gamma, suggesting that basal TNF produced in an MyD88-dependent manner contributes to PRX5 induction. Downstream of the TLR pathways, we have explored the role of MAPK activation and found that p38 and JNK mainly contribute to PRX5 up-regulation in immunostimulated macrophages. Expression of PRX5 is thus responsive to innate immunity signals, and we propose that PRX5 is an additional host defense weapon of activated macrophages.
Asunto(s)
Interferón gamma/metabolismo , Macrófagos/metabolismo , Peroxirredoxinas/metabolismo , Receptor Toll-Like 4/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Animales , Células Cultivadas , Inmunidad Innata , Interferón gamma/inmunología , Lipopolisacáridos/metabolismo , MAP Quinasa Quinasa 4/metabolismo , Activación de Macrófagos , Macrófagos/inmunología , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/genética , Óxido Nítrico Sintasa de Tipo II/genética , Peroxirredoxinas/genética , Peroxirredoxinas/inmunología , Receptores de Interferón/genética , Transducción de Señal/inmunología , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunología , Factor de Necrosis Tumoral alfa/genética , Regulación hacia Arriba , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Tyrphostins are well-established selective inhibitors of protein tyrosine kinase activity of EGF receptor and other growth factor receptors. Unexpectedly, we found that, in U-937 monocytic cells, tyrphostin AG-126 augments the sensitivity of the corresponding genes to NO, in contrast to other protein tyrosine kinase inhibitors like genistein, PD 168393, PP2, and SU 11652. Moreover, by itself AG-126 appeared to be a potent activator of the expression of heme oxygenase 1 (HO-1), H-ferritin, activating transcription factor 3 (ATF3), interleukin 8 (IL-8), and several other NO- and redox-regulated genes. The most sensitive to AG-126 was the HO-1 gene, with a fold-change of expression reaching 300. Besides, we showed that AG-126 stimulated key elements of upstream signaling systems as p38 MAP kinase and AP-1 and Nrf2 transcription factors. Together with AG-126, structurally related benzylidenemalononitrile tyrphostins AG-9, AG-10, AG-18, and AG-1288 were able to up-regulate the expression of HO-1 and several other genes, although with relatively less efficacy. Conversely, tyrphostins AG-30 and AG-490 were ineffective regulators of gene expression. Comparison of the chemical structures of these compounds indicates that most important for transcriptional activation of target genes is the presence of either the 4-nitro or 4-methoxy group in the benzene ring and two CN-groups of the malononitrile residue. Several lines of evidence indicate that the gene induction capacity of AG-126-like tyrphostins is not related to the inhibition of protein tyrosine kinases.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Óxido Nítrico/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Tirfostinos/farmacología , Animales , Humanos , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Células U937RESUMEN
Friedreich ataxia (FRDA) is a rare hereditary neurodegenerative disease characterized by progressive ataxia and cardiomyopathy. The cause of the disease is a defect in mitochondrial frataxin, an iron chaperone involved in the maturation of Fe-S cluster proteins. Several human diseases, including cardiomyopathies, have been found to result from deficiencies in the activity of specific proteases, which have important roles in protein turnover and in the removal of damaged or unneeded protein. In this study, using the muscle creatine kinase mouse heart model for FRDA, we show a clear progressive increase in protein levels of two important mitochondrial ATP-dependent proteases, Lon and ClpP, in the hearts of muscle creatine kinase mutants. These proteases have been shown to degrade unfolded and damaged proteins in the matrix of mitochondria. Their upregulation, which was triggered at a mid-stage of the disease through separate pathways, was accompanied by an increase in proteolytic activity. We also demonstrate a simultaneous and significant progressive loss of mitochondrial Fe-S proteins with no substantial change in their mRNA level. The correlative effect of Lon and ClpP upregulation on loss of mitochondrial Fe-S proteins during the progression of the disease may suggest that Fe-S proteins are potential targets of Lon and ClpP proteases in FRDA.
Asunto(s)
Forma MM de la Creatina-Quinasa/fisiología , Endopeptidasa Clp/biosíntesis , Proteínas de Unión a Hierro/fisiología , Proteínas Hierro-Azufre/metabolismo , Proteínas Mitocondriales/fisiología , Proteasa La/biosíntesis , Adenosina Trifosfato/metabolismo , Animales , Forma MM de la Creatina-Quinasa/genética , Ataxia de Friedreich/genética , Ataxia de Friedreich/metabolismo , Proteínas de Unión a Hierro/genética , Ratones , Ratones Transgénicos , Mutación , Miocardio/enzimología , Regulación hacia Arriba , FrataxinaRESUMEN
Peroxiredoxins participate in the antioxidant response by reducing H(2)O(2), organic peroxides and peroxynitrite. Peroxiredoxins have a conserved NH(2)-terminal cysteine residue that is oxidized to sulfenic acid during catalysis of peroxide reduction. In eukaryotes, the sulfenic acid can be further oxidized to a sulfinic acid. Resulting inactivation of peroxiredoxins favors H(2)O(2) signaling but may eventually result in oxidative stress. Interestingly, it has recently been shown that overoxidized peroxiredoxins progressively recover activity owing to sulfiredoxin, an enzyme recently characterized in yeast and mammals. This reversible peroxide-sensitive switch represents a new type of regulation that controls reactive oxygen species-mediated cytoxicity and signaling. This report presents a brief overview of the regulation by peroxiredoxins of the messenger function of H(2)O(2) and comments on the results of recent studies that addressed the consequence of nitric oxide production on both expression and redox state of peroxiredoxins in various physiopathological processes including macrophage immunostimulation, the response of dopaminergic neurons to N-methyl-d-aspartate-stimulation and the plant hypersensitive response.
Asunto(s)
Macrófagos/metabolismo , Óxido Nítrico/metabolismo , Peroxirredoxinas/metabolismo , Animales , Regulación de la Expresión Génica , Humanos , Peróxido de Hidrógeno/metabolismo , Macrófagos/enzimología , Ratones , Oxidación-Reducción , Estrés Oxidativo/fisiología , Peroxirredoxinas/genética , Isoformas de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Ácidos Sulfínicos/metabolismoRESUMEN
Macrophages are key cells of the immune system. Immunologically activated macrophages are known to release a cocktail of reactive oxygen and nitrogen species. In this work, RAW 264.7 macrophages were activated by interferon-gamma and lipopolysaccharide, and the reactive mixture released by single cells was analyzed, in real time, by amperometry at platinized carbon microelectrodes. In comparison with untreated macrophages, significant increases in amperometric responses were observed for activated macrophages. Nitric oxide (NO*), nitrite (NO2*-), and peroxynitrite (ONOO-) were the main reactive species detected. The amounts of these reactive species were quantified, and their average fluxes released by a single, activated macrophage were evaluated. The detection of ONOO- is of particular interest, as its role and implications in various physiological conditions have been widely debated. Herein, direct evidence for the formation of ONOO- in stimulated macrophages is presented. Finally, the presence of 1400W, a selective inducible nitric oxide synthase (iNOS) inhibitor, led to an almost complete attenuation of the amperometric response of activated RAW 264.7 cells. The majority of the reactive species released by a macrophage are thus likely to be derived from NO* and superoxide (O2*-) co-produced by iNOS.
Asunto(s)
Macrófagos/inmunología , Macrófagos/metabolismo , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Línea Celular , Electroquímica , Fluorometría , Depuradores de Radicales Libres/metabolismo , Inmunización , Interferón gamma/metabolismo , Lipopolisacáridos/metabolismo , Ratones , Óxido Nítrico/metabolismo , Nitroprusiato/análisis , Nitroprusiato/metabolismo , Ácido Peroxinitroso/metabolismo , Especies de Nitrógeno Reactivo/análisis , Especies Reactivas de Oxígeno/análisis , Factores de TiempoRESUMEN
Reactive oxygen species and nitric oxide (NO) are capable of both mediating redox-sensitive signal transduction and eliciting cell injury. The interplay between these messengers is quite complex, and intersection of their signaling pathways as well as regulation of their fluxes requires tight control. In this regard, peroxiredoxins (Prxs), a recently identified family of six thiol peroxidases, are central because they reduce H2O2, organic peroxides, and peroxynitrite. Here we provide evidence that endogenously produced NO participates in protection of murine primary macrophages against oxidative and nitrosative stress by inducing Prx I and VI expression at mRNA and protein levels. We also show that NO prevented the sulfinylation-dependent inactivation of 2-Cys Prxs, a reversible overoxidation that controls H2O2 signaling. In addition, studies using macrophages from sulfiredoxin (Srx)-deficient mice indicated that regeneration of 2-Cys Prxs to the active form was dependent on Srx. Last, we show that NO increased Srx expression and hastened Srx-dependent recovery of 2-Cys Prxs. We therefore propose that modulation by NO of Prx expression and redox state, as well as up-regulation of Srx expression, constitutes a novel pathway that contributes to antioxidant response and control of H2O2-mediated signal transduction in mammals.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/fisiología , Macrófagos/enzimología , Óxido Nítrico/metabolismo , Estrés Oxidativo/fisiología , Peroxiredoxina VI/biosíntesis , Peroxirredoxinas/biosíntesis , Transducción de Señal/fisiología , Animales , Línea Celular , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Peróxido de Hidrógeno/inmunología , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/farmacología , Macrófagos/citología , Macrófagos/inmunología , Ratones , Ratones Noqueados , Óxido Nítrico/inmunología , Oxidantes/inmunología , Oxidantes/metabolismo , Oxidantes/farmacología , Oxidación-Reducción/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Peroxiredoxina VI/genética , Peroxiredoxina VI/inmunología , Peroxirredoxinas/genética , Peroxirredoxinas/inmunología , Ácido Peroxinitroso/inmunología , Ácido Peroxinitroso/metabolismo , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
Biogenesis of iron-sulfur (Fe-S) clusters in mammals involves a complex mitochondrial machinery that provides inorganic sulfide and iron for their assembly and insertion into apo-proteins. Mechanisms of Fe-S cluster assembly are just being unraveled, and regulation of the genes of this machinery remains unknown. In this study, we report that expression of two essential components of the Fe-S machinery, the cysteine desulfurase Nfs1 and its scaffold protein partner IscU, is down-regulated at both mRNA and protein levels when murine macrophages are physiologically stimulated with IFN-gamma and LPS. Regulation did not rely on cluster disassembly or NO production because exposure of cells to exogenous sources of NO did not alter Nfs1 expression, while it converted cytosolic Fe-S aconitase into its apo-form and because macrophages from NOS2 deficient mice displayed Nfs1 down-regulation. While IFN-gamma alone induced Nfs1 protein instability, LPS triggered a delayed decline of Nfs1, rather involving transcriptional events or mRNA instability. Also, the expression of IscU was down-regulated in IFN-gamma- and/or LPS-stimulated macrophages independently of NO, pointing to a general mechanism for marshalling the regulation of the Fe-S cluster assembly machinery in macrophages exposed to inflammatory stimuli.
Asunto(s)
Liasas de Carbono-Azufre/metabolismo , Interferón gamma/administración & dosificación , Proteínas Hierro-Azufre/metabolismo , Lipopolisacáridos/administración & dosificación , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Animales , Liasas de Carbono-Azufre/administración & dosificación , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/fisiología , Ratones , Ratones Endogámicos C57BLRESUMEN
The effects and the mode of action of hypericin (1) were studied, in the dark, on the action potential (AP) and the L-type Ca2+ channel of frog atrial heart muscle, using intracellular microelectrode and patch-clamp techniques, respectively. In the presence of Ca2+ in Ringer solution, hypericin (1 to 4 microM) did not markedly modify the AP. Total replacement of Ca2+ by Sr2+ in the solution (Ringer Sr2+) revealed that hypericin (4 microM) prolonged the AP duration (APD). Hypericin dose-dependently increased the magnitude of the Sr2+current, which develops through L-type Ca2+ channels in the Ringer solution containing tetrodotoxin (0.7 microM) and tetraethylammonium (10 mM), but did not modify the kinetics of activation and inactivation. This revealed that hypericin increased L-type Ca2+ channel conductance, which accounted for the APD lengthening. The hypericin-induced APD lengthening recorded in the Ringer Sr2+ was not prevented by (i) a blockade of alpha- and beta-adrenoceptors by yohimbine (1 microM), urapidil (1 microM), and propanolol (50 microM), respectively, and (ii) PKC blockade by staurosporine (1 microM). The hypericin-induced APD lengthening recorded in the Ringer Sr2+ was prevented by blocking soluble guanylate cyclase (sGC) activity by 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (13 microM), which mimicked the effects of hypericin. Hypericin decreased the cellular cGMP level by 69% in atrial myocytes. The compound also decreased the cellular cGMP level by inhibiting sGC, thus cancelling the nucleotide inhibitory effect on the cardiac L-type Ca2+ channel.
Asunto(s)
Canales de Calcio Tipo L/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Perileno/análogos & derivados , Potenciales de Acción/efectos de los fármacos , Animales , Antracenos , GMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Guanilato Ciclasa/antagonistas & inhibidores , Corazón/efectos de los fármacos , Estructura Molecular , Óxido Nítrico Sintasa de Tipo II/metabolismo , Oxadiazoles/farmacología , Perileno/química , Perileno/farmacología , Fosfoproteínas Fosfatasas/metabolismo , Ranidae , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Guanilil Ciclasa SolubleRESUMEN
RNA-binding activity of IRP1 (iron regulatory protein 1) is regulated by the insertion/extrusion of a [4Fe-4S] cluster into/from the IRP1 molecule. NO (nitic oxide), whose ability to activate IRP1 by removing its [4Fe-4S] cluster is well known, has also been shown to down-regulate expression of the IRP1 gene. In the present study, we examine whether this regulation occurs at the transcriptional level. Analysis of the mouse IRP1 promoter sequence revealed two conserved putative binding sites for transcription factor(s) regulated by NO and/or changes in intracellular iron level: Sp1 (promoter-selective transcription factor 1) and MTF1 (metal transcription factor 1), plus GAS (interferon-gamma-activated sequence), a binding site for STAT (signal transducer and activator of transcription) proteins. In order to define the functional activity of these sequences, reporter constructs were generated through the insertion of overlapping fragments of the mouse IRP1 promoter upstream of the luciferase gene. Transient expression assays following transfection of HuH7 cells with these plasmids revealed that while both the Sp1 and GAS sequences are involved in basal transcriptional activity of the IRP1 promoter, the role of the latter is predominant. Analysis of protein binding to these sequences in EMSAs (electrophoretic mobility-shift assays) using nuclear extracts from mouse RAW 264.7 macrophages stimulated to synthesize NO showed a significant decrease in the formation of Sp1-DNA and STAT-DNA complexes, compared with controls. We have also demonstrated that the GAS sequence is involved in NO-dependent down-regulation of IRP1 transcription. Further analysis revealed that levels of STAT5a and STAT5b in the nucleus and cytosol of NO-producing macrophages are substantially lower than in control cells. These findings provide evidence that STAT5 proteins play a role in NO-mediated down-regulation of IRP1 gene expression.
Asunto(s)
Proteína 1 Reguladora de Hierro/biosíntesis , Óxido Nítrico/fisiología , Factor de Transcripción STAT5/metabolismo , Animales , Sitios de Unión , Línea Celular , Núcleo Celular/metabolismo , Citosol/metabolismo , ADN/genética , ADN/metabolismo , Regulación hacia Abajo/fisiología , Humanos , Interferón gamma/farmacología , Hierro/metabolismo , Proteína 1 Reguladora de Hierro/genética , Lipopolisacáridos/farmacología , Macrófagos/metabolismo , Macrófagos/fisiología , Ratones , Óxido Nítrico/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Factor de Transcripción STAT5/antagonistas & inhibidores , Factor de Transcripción STAT5/biosíntesis , Factor de Transcripción STAT5/genética , Factor de Transcripción Sp1/antagonistas & inhibidores , Factor de Transcripción Sp1/metabolismo , TransfecciónRESUMEN
In prokaryotes and yeast, the general mechanism of biogenesis of iron-sulfur (Fe-S) clusters involves activities of several proteins among which IscS and Nfs1p provide, through cysteine desulfuration, elemental sulfide for Fe-S core formation. Although these proteins have been well characterized, the role of their mammalian homolog in Fe-S cluster biogenesis has never been evaluated. We report here the first functional study that implicates the putative cysteine desulfurase m-Nfs1 in the biogenesis of both mitochondrial and cytosolic mammalian Fe-S proteins. Depletion of m-Nfs1 in cultured fibroblasts through small interfering RNA-based gene silencing significantly inhibited the activities of mitochondrial NADH-ubiquinone oxidoreductase (complex I) and succinate-ubiquinone oxidoreductase (complex II) of the respiratory chain, as well as aconitase of the Krebs cycle, with no alteration in their protein levels. Activity of cytosolic xanthine oxidase, which holds a [2Fe-2S] cluster, was also specifically reduced, and iron-regulatory protein-1 was converted from its [4Fe-4S] aconitase form to its apo- or RNA-binding form. Reduction of Fe-S enzyme activities occurred earlier and more markedly in the cytosol than in mitochondria, suggesting that there is a mechanism that primarily dedicates m-Nfs1 to the biogenesis of mitochondrial Fe-S clusters in order to maintain cell survival. Finally, depletion of m-Nfs1, which conferred on apo-IRP-1 a high affinity for ferritin mRNA, was associated with the down-regulation of the iron storage protein ferritin.
Asunto(s)
Liasas de Carbono-Azufre/fisiología , Citosol/metabolismo , Proteínas Hierro-Azufre/química , Mitocondrias/metabolismo , Interferencia de ARN , Animales , Liasas de Carbono-Azufre/química , Liasas de Carbono-Azufre/genética , Regulación hacia Abajo , Complejo I de Transporte de Electrón/química , Complejo II de Transporte de Electrones/química , Ferritinas/química , Ratones , Mitocondrias/enzimología , Células 3T3 NIH , Xantina Oxidasa/químicaRESUMEN
Macrophages are key cells of the immune system. During phagocytosis, the macrophage engulfs a foreign bacterium, virus, or particle into a vacuole, the phagosome, wherein oxidants are produced to neutralize and decompose the threatening element. These oxidants derive from in situ production of superoxide and nitric oxide by specific enzymes. However, the chemical nature and sequence of release of these compounds is far from being completely determined. The aim of the present work was to study the fundamental mechanism of oxidant release by macrophages at the level of a single cell, in real time and quantitatively. The tip of a microelectrode was positioned at a micrometric distance from a macrophage in a culture to measure oxidative-burst release by the cell when it was submitted to physical stimulation. The ensuing release of electroactive reactive oxygen and nitrogen species was detected by amperometry and the exact nature of the compounds was characterized through comparison with in vitro electrochemical oxidation of H2O2, ONOO-, NO*, and NO2(-) solutions. These results enabled the calculation of time variations of emission flux for each species and the reconstruction of the original flux of production of primary species, O2*- and NO*, by the macrophage.
Asunto(s)
Macrófagos/fisiología , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Línea Celular , Membrana Celular/fisiología , Células Cultivadas , Radicales Libres/análisis , Radicales Libres/metabolismo , Peróxido de Hidrógeno/metabolismo , Técnicas In Vitro , Potenciales de la Membrana/fisiología , Ratones , Microelectrodos , Óxido Nítrico/metabolismo , Oxidación-Reducción , Ácido Peroxinitroso/metabolismo , Estimulación Física , Factores de TiempoRESUMEN
In this study we examined the gene expression pattern of *NO-dependent genes in U937 and Mono Mac 6 monocytes exposed to the synthetic NO-donor DPTA-NO using microarray technology. cDNA microarray data were validated by Northern blot analysis and quantitative real-time PCR. This approach allowed the identification of 17 *NO-sensitive genes that showed at least a twofold difference in expression, in both U937 cells and Mono Mac 6 cells exposed to 500 microM DPTA-NO for 4 h. NO-stimulated genes belong to various functional groups, including transcription factors, signaling molecules, and cytokines. Among the selected genes, 11 (ATF-4, c-maf, SGK-1, PBEF, ATPase 8, NADH dehydrogenase 4, STK6, TRAF4-associated factor 1, molybdopterin synthase, CKS1, and CIDE-B) have not been previously reported to be sensitive to *NO. Because several *NO-stimulated genes are transcription factors, we analyzed the mRNA expression profile in U937 cells exposed to DPTA-NO for 14 h. We found that long-term *NO treatment influenced transcription rates of a rather limited set of genes, including CIDE-B, BNIP3, p21/Cip1, molybdopterin synthase, and TRAF4-associated factor 1. To accelerate formation of nitrosating species, U937 cells were exposed to DPTA-NO along with suboptimal concentrations of 2-phenyl-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide (PTIO). PTIO-mediated increase in nitrosating species remarkably enhanced *NO-dependent induction of IL-8, p21/Cip1, and MKP-1 and built a specific gene expression profile.