RESUMO
Thioredoxin (Trx) family proteins are key players in redox signaling. Here, we have analyzed glutaredoxin (Grx) 1 and Grx2 in age-related macular degeneration (AMD) and in retinal pigment epithelial (ARPE-19) cells. We hypothesized that these redoxins regulate cellular functions and signaling circuits such as cell proliferation, Wnt signaling and VEGF release that have been correlated to the pathophysiology of AMD. ARPE-19 cells were transfected with specific siRNAs to silence the expression of Grx1 and Grx2 and were analyzed for proliferation/viability, migration capacity, ß-catenin activation, and VEGF release. An active site-mutated C-X-X-S Grx1 was utilized to trap interacting proteins present in ARPE-19 cell extracts. In both, AMD retinas and in ARPE-19 cells incubated under hypoxia/reoxygenation conditions, Grx1 showed an increased nuclear localization. Grx1-silenced ARPE-19 cells showed a significantly reduced proliferation and migration rate. Our trapping approach showed that Grx1 interacts with ß-catenin in a dithiol-disulfide exchange reaction. Knock-down of Grx1 led to a reduction in both total and active ß-catenin levels. These findings add redox control to the regulatory mechanisms of ß-catenin signaling in the retinal pigment epithelium and open the door to novel therapeutic approaches in AMD that is currently treated with VEGF-inhibitors.
Assuntos
Glutarredoxinas , Degeneração Macular , Epitélio Pigmentado da Retina , beta Catenina , Proliferação de Células/fisiologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Humanos , Degeneração Macular/genética , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Pigmentos da Retina/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo , beta Catenina/metabolismoRESUMO
Ischemia and reperfusion events, such as myocardial infarction (MI), are reported to induce remote organ damage severely compromising patient outcomes. Tissue survival and functional restoration relies on the activation of endogenous redox regulatory systems such as the oxidoreductases of the thioredoxin (Trx) family. Trxs and peroxiredoxins (Prxs) are essential for the redox regulation of protein thiol groups and for the reduction of hydrogen peroxide, respectively. Here, we determined whether experimental MI induces changes in Trxs and Prxs in the heart as well as in secondary organs. Levels and localization of Trx1, TrxR1, Trx2, Prx1, and Prx2 were analyzed in the femur, vertebrae, and kidneys of rats following MI or sham surgery. Trx1 levels were significantly increased in the heart (P = 0.0017) and femur (P < 0.0001) of MI animals. In the femur and lumbar vertebrae, Trx1 upregulation was detected in bone-lining cells, osteoblasts, megakaryocytes, and other hematopoietic cells. Serum levels of Trx1 increased significantly 2 days after MI compared to sham animals (P = 0.0085). Differential regulation of Trx1 in the bone was also detected by immunohistochemistry 1 month after MI. N-Acetyl-cysteine treatment over a period of 1 month induced a significant reduction of Trx1 levels in the bone of MI rats compared to sham and to MI vehicle. This study provides first evidence that MI induces remote organ upregulation of the redox protein Trx1 in the bone, as a response to ischemia-reperfusion injury in the heart.
Assuntos
Medula Óssea/metabolismo , Osso e Ossos/metabolismo , Infarto do Miocárdio/metabolismo , Tiorredoxinas/metabolismo , Regulação para Cima , Animais , Medula Óssea/patologia , Osso e Ossos/patologia , Masculino , Infarto do Miocárdio/patologia , Ratos , Ratos Endogâmicos F344 , Tiorredoxinas/análiseRESUMO
Processing of and responding to various signals is an essential cellular function that influences survival, homeostasis, development, and cell death. Extra- or intracellular signals are perceived via specific receptors and transduced in a particular signalling pathway that results in a precise response. Reversible post-translational redox modifications of cysteinyl and methionyl residues have been characterised in countless signal transduction pathways. Due to the low reactivity of most sulfur-containing amino acid side chains with hydrogen peroxide, for instance, and also to ensure specificity, redox signalling requires catalysis, just like phosphorylation signalling requires kinases and phosphatases. While reducing enzymes of both cysteinyl- and methionyl-derivates have been characterised in great detail before, the discovery and characterisation of MICAL proteins evinced the first examples of specific oxidases in signal transduction. This article provides an overview of the functions of MICAL proteins in the redox regulation of cellular functions.
Assuntos
Proteínas dos Microfilamentos/fisiologia , Oxigenases de Função Mista/fisiologia , Oxirredução , Transdução de Sinais , Animais , Catálise , Cisteína/química , Proteínas do Citoesqueleto/metabolismo , Genoma , Humanos , Peróxido de Hidrogênio/química , Cinética , Oxirredutases/metabolismo , Oxigênio/metabolismo , Fosforilação , Ligação ProteicaRESUMO
Demyelinated brain lesions, a hallmark of autoimmune neuroinflammatory diseases like multiple sclerosis, result from oligodendroglial cell damage. Activated microglia are considered a major source of nitric oxide and subsequent peroxynitrite-mediated damage of myelin. Here, we provide biochemical and biophysical evidence that the oxidoreductase glutaredoxin 2 inhibits peroxynitrite formation by transforming nitric oxide into dinitrosyl-diglutathionyl-iron-complexes. Glutaredoxin 2 levels influence both survival rates of primary oligodendrocyte progenitor cells and preservation of myelin structure in cerebellar organotypic slice cultures challenged with activated microglia or nitric oxide donors. Of note, glutaredoxin 2-mediated protection is not linked to its enzymatic activity as oxidoreductase, but to the disassembly of its uniquely coordinated iron-sulfur cluster using glutathione as non-protein ligand. The protective effect of glutaredoxin 2 is connected to decreased protein carbonylation and nitration. In line, brain lesions of mice suffering from experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, show decreased glutaredoxin 2 expression and increased nitrotyrosine formation indicating that this type of protection is missing in the inflamed central nervous system. Our findings link inorganic biochemistry to neuroinflammation and identify glutaredoxin 2 as a protective factor against neuroinflammation-mediated myelin damage. Thus, improved availability of glutathione-coordinated iron-sulfur clusters emerges as a potential therapeutic approach in inflammatory demyelination.
Assuntos
Encefalomielite Autoimune Experimental/metabolismo , Glutarredoxinas/metabolismo , Microglia/metabolismo , Óxido Nítrico/metabolismo , Oligodendroglia/metabolismo , Animais , Cerebelo/metabolismo , Cerebelo/patologia , Encefalomielite Autoimune Experimental/patologia , Escherichia coli , Feminino , Glutarredoxinas/genética , Glutationa Transferase/metabolismo , Células HeLa , Humanos , Inflamação/metabolismo , Inflamação/patologia , Camundongos Endogâmicos C57BL , Microglia/patologia , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Neuroproteção/fisiologia , Oligodendroglia/patologia , Ácido Peroxinitroso/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Schistosoma japonicum , Técnicas de Cultura de TecidosRESUMO
The mechanism by which oxidative stress induces inflammation and vice versa is unclear but is of great importance, being apparently linked to many chronic inflammatory diseases. We show here that inflammatory stimuli induce release of oxidized peroxiredoxin-2 (PRDX2), a ubiquitous redox-active intracellular enzyme. Once released, the extracellular PRDX2 acts as a redox-dependent inflammatory mediator, triggering macrophages to produce and release TNF-α. The oxidative coupling of glutathione (GSH) to PRDX2 cysteine residues (i.e., protein glutathionylation) occurs before or during PRDX2 release, a process central to the regulation of immunity. We identified PRDX2 among the glutathionylated proteins released in vitro by LPS-stimulated macrophages using mass spectrometry proteomic methods. Consistent with being part of an inflammatory cascade, we find that PRDX2 then induces TNF-α release. Unlike classical inflammatory cytokines, PRDX2 release does not reflect LPS-mediated induction of mRNA or protein synthesis; instead, PRDX2 is constitutively present in macrophages, mainly in the reduced form, and is released in the oxidized form on LPS stimulation. Release of PRDX2 is also observed in human embryonic kidney cells treated with TNF-α. Importantly, the PRDX2 substrate thioredoxin (TRX) is also released along with PRDX2, enabling an oxidative cascade that can alter the -SH status of surface proteins and thereby facilitate activation via cytokine and Toll-like receptors. Thus, our findings suggest a model in which the release of PRDX2 and TRX from macrophages can modify the redox status of cell surface receptors and enable induction of inflammatory responses. This pathway warrants further exploration as a potential novel therapeutic target for chronic inflammatory diseases.
Assuntos
Glutationa/metabolismo , Inflamação/metabolismo , Macrófagos/metabolismo , Estresse Oxidativo , Peroxirredoxinas/metabolismo , Animais , Western Blotting , Linhagem Celular , Humanos , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , CamundongosRESUMO
BACKGROUND: The cytoskeleton, unlike the bony vertebrate skeleton or the exoskeleton of invertebrates, is a highly dynamic meshwork of protein filaments that spans through the cytosol of eukaryotic cells. Especially actin filaments and microtubuli do not only provide structure and points of attachments, but they also shape cells, they are the basis for intracellular transport and distribution, all types of cell movement, and--through specific junctions and points of adhesion--join cells together to form tissues, organs, and organisms. SCOPE OF REVIEW: The fine tuned regulation of cytoskeletal dynamics is thus indispensible for cell differentiation and all developmental processes. Here, we discussed redox signalling mechanisms that control this dynamic remodeling. Foremost, we emphasised recent discoveries that demonstrated reversible thiol and methionyl switches in the regulation of actin dynamics. MAJOR CONCLUSIONS: Thiol and methionyl switches play an essential role in the regulation of cytoskeletal dynamics. GENERAL SIGNIFICANCE: The dynamic remodeling of the cytoskeleton is controlled by various redox switches. These mechanisms are indispensible during development and organogenesis and might contribute to numerous pathological conditions. This article is part of a Special Issue entitled Redox regulation of differentiation and de-differentiation.
Assuntos
Desdiferenciação Celular/fisiologia , Diferenciação Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Animais , Humanos , Modelos Biológicos , Neoplasias/metabolismo , Neoplasias/fisiopatologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/fisiopatologia , OxirreduçãoRESUMO
BACKGROUND: Thioredoxin (Trx) family proteins are crucial mediators of cell functions via regulation of the thiol redox state of various key proteins and the levels of the intracellular second messenger hydrogen peroxide. Their expression, localization and functions are altered in various pathologies. Here, we have analyzed the impact of Trx family proteins in neuronal development and recovery, following hypoxia/ischemia and reperfusion. METHODS: We have analyzed the regulation and potential functions of Trx family proteins during hypoxia/ischemia and reoxygenation of the developing brain in both an animal and a cellular model of perinatal asphyxia. We have analyzed the distribution of 14 Trx family and related proteins in the cerebellum, striatum, and hippocampus, three areas of the rat brain that are especially susceptible to hypoxia. Using SH-SY5Y cells subjected to hypoxia and reoxygenation, we have analyzed the functions of some redoxins suggested by the animal experiment. RESULTS AND CONCLUSIONS: We have described/discovered a complex, cell-type and tissue-specific expression pattern following the hypoxia/ischemia and reoxygenation. Particularly, Grx2 and Trx1 showed distinct changes during tissue recovery following hypoxia/ischemia and reoxygenation. Silencing of these proteins in SH-SY5Y cells subjected to hypoxia-reoxygenation confirmed that these proteins are required to maintain the normal neuronal phenotype. GENERAL SIGNIFICANCE: These findings demonstrate the significance of redox signaling in cellular pathways. Grx2 and Trx1 contribute significantly to neuronal integrity and could be clinically relevant in neuronal damage following perinatal asphyxia and other neuronal disorders.
Assuntos
Asfixia Neonatal/enzimologia , Encéfalo/enzimologia , Glutarredoxinas/metabolismo , Hipóxia-Isquemia Encefálica/enzimologia , Neurônios/enzimologia , Tiorredoxinas/metabolismo , Animais , Asfixia Neonatal/patologia , Encéfalo/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Glutarredoxinas/genética , Humanos , Hipóxia-Isquemia Encefálica/patologia , Masculino , Neurônios/patologia , Oxirredução , Oxigênio/metabolismo , Fenótipo , Interferência de RNA , Ratos Sprague-Dawley , Transdução de Sinais , Tiorredoxinas/genética , Fatores de Tempo , TransfecçãoRESUMO
Nonclassical protein secretion is of major importance as a number of cytokines and inflammatory mediators are secreted via this route. Current evidence indicates that there are several mechanistically distinct methods of nonclassical secretion. We have shown recently that peroxiredoxin (Prdx) 1 and Prdx2 are released by various cells upon exposure to inflammatory stimuli such as lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF-α). The released Prdx then acts to induce production of inflammatory cytokines. However, Prdx1 and 2 do not have signal peptides and therefore must be secreted by alternative mechanisms, as has been postulated for the inflammatory mediators interleukin-1ß (IL-1ß) and high mobility group box-1 (HMGB1). We show here that circulating Prdx1 and 2 are present exclusively as disulfide-linked homodimers. Inflammatory stimuli also induce in vitro release of Prdx1 and 2 as disulfide-linked homodimers. Mutation of cysteines Cys51 or Cys172 (but not Cys70) in Prdx2, and Cys52 or Cys173 (but not Cys71 or Cys83) in Prdx1 prevented dimer formation and this was associated with inhibition of their TNF-α-induced release. Thus, the presence and oxidation of key cysteine residues in these proteins are a prerequisite for their secretion in response to TNF-α, and this release can be induced with an oxidant. By contrast, the secretion of the nuclear-associated danger signal HMGB1 is independent of cysteine oxidation, as shown by experiments with a cysteine-free HMGB1 mutant. Release of Prdx1 and 2 is not prevented by inhibitors of the classical secretory pathway, instead, both Prdx1 and 2 are released in exosomes from both human embryonic kidney (HEK) cells and monocytic cells. Serum Prdx1 and 2 also are associated with the exosomes. These results describe a novel pathway of protein secretion mediated by cysteine oxidation that underlines the importance of redox-dependent signaling mechanisms in inflammation.
Assuntos
Cisteína/metabolismo , Exossomos/metabolismo , Oxirredução , Peroxirredoxinas/metabolismo , Animais , Técnicas de Cultura de Células , Linhagem Celular , Sobrevivência Celular , Humanos , Oxidantes/metabolismo , Oxidantes/farmacologia , Peroxirredoxinas/sangue , Peroxirredoxinas/química , Peroxirredoxinas/genética , Multimerização Proteica , Estabilidade Proteica , Ratos , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The spatiotemporal modification of specific cysteinyl residues in proteins has emerged as a novel concept in signal transduction. Such modifications alter the redox state of the cysteinyl thiol group, with implications for the structure and biological function of the protein. Regulatory cysteines are therefore classified as 'thiol switches'. In this review we emphasize the relevance of enzymes for specific and efficient redox sensing, evaluate prerequisites and general properties of redox switches, and highlight mechanistic principles for toggling thiol switches. Moreover, we provide an overview of potential mechanisms for the initial formation of regulatory disulfide bonds. In brief, we address the three basic questions (i) what defines a thiol switch, (ii) which parameters confer signal specificity, and (iii) how are thiol switches oxidized?
Assuntos
Cisteína/química , Espécies Reativas de Oxigênio/metabolismo , Humanos , Oxirredução , Estresse Oxidativo , Transdução de SinaisRESUMO
Vertebrate-specific glutaredoxin 2 (Grx2) is expressed in at least two isoforms, mitochondrial Grx2a and cytosolic Grx2c. We have previously shown that cytosolic Grx2 is essential for embryonic development of the brain. In particular, we identified collapsin response mediator protein 2 (CRMP2/DPYSL2), a mediator of the semaphorin-plexin signaling pathway, as redox-regulated target of Grx2c and demonstrated that this regulation is required for normal axonal outgrowth. In this study, we demonstrate the molecular mechanism of this regulation, a specific and reversible intermolecular Cys-504-Cys-504 dithiol-disulfide switch in homotetrameric CRMP2. This switch determines two conformations of the quaternary CRMP2 complex that controls axonal outgrowth and thus neuronal development.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Modelos Neurológicos , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Sequência de Aminoácidos , Diferenciação Celular , Linhagem Celular , Cisteína/química , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Neurogênese , Oxirredução , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Subunidades Proteicas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
BACKGROUND: Glutaredoxins (Grxs) catalyze the reduction of protein disulfides via the dithiol mechanism and the de-/glutathionylation of substrates via the monothiol mechanism. These rapid, specific, and generally also reversible modifications are part of various signaling cascades regulating for instance cell proliferation, differentiation and apoptosis. Even though crucial functions of the conserved, mitochondrial Grx2a and the cytosolic/nuclear Grx2c isoforms have been proposed, only a few substrates have been identified in vitro or in vivo. The significance of redox signaling is emerging, yet a general lack of methods for the time-resolved analysis of these distinct and rapid modifications in vivo constitutes the biggest challenge in the redox signaling field. METHODS AND RESULTS: Here, we have identified potential interaction partners for Grx2 isoforms in human HeLa cells and mouse tissues by an intermediate trapping approach. Some of the 50 potential substrates are part of the cytoskeleton or act in protein folding, cellular signaling and metabolism. Part of these interactions were further verified by immunoprecipitation or a newly established 2-D redox blot. CONCLUSIONS: Our study demonstrates that Grx2 catalyzes both the specific oxidation and the reduction of cysteinyl residues in the same compartment at the same time and without affecting the global cellular thiol-redox state. GENERAL SIGNIFICANCE: The knowledge of specific targets will be helpful in understanding the functions of Grx2. The 2-D redox blot may be useful for the analysis of the overall thiol-redox state of proteins with high molecular weight and numerous cysteinyl residues, that evaded analysis by previously described methods.
Assuntos
Dissulfetos/metabolismo , Glutarredoxinas/metabolismo , Proteínas/metabolismo , Tolueno/análogos & derivados , Animais , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , Citosol/metabolismo , Células HeLa , Humanos , Mamíferos/metabolismo , Camundongos , Oxirredução , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas , Tolueno/metabolismoRESUMO
Cellular functions and survival are dependent on a tightly controlled redox potential. Currently, an increasing amount of data supports the concept of local changes in the redox environment and specific redox signaling events controlling cell function. Specific protein thiol groups are the major targets of redox signaling and regulation. Thioredoxins and glutaredoxins catalyze reversible thiol-disulfide exchange reactions and are primary regulators of the protein thiol redox state. Here, we demonstrate that embryonic brain development depends on the enzymatic activity of glutaredoxin 2. Zebrafish with silenced expression of glutaredoxin 2 lost virtually all types of neurons by apoptotic cell death and the ability to develop an axonal scaffold. As demonstrated in zebrafish and in a human cellular model for neuronal differentiation, glutaredoxin 2 controls axonal outgrowth via thiol redox regulation of collapsin response mediator protein 2, a central component of the semaphorin pathway. This study provides an example of a specific thiol redox regulation essential for vertebrate embryonic development.
Assuntos
Encéfalo/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Glutarredoxinas/química , Peixe-Zebra/embriologia , Animais , Apoptose , Axônios/fisiologia , Linhagem Celular Tumoral , Biologia do Desenvolvimento , Glutarredoxinas/genética , Humanos , Neuritos/metabolismo , Oxirredução , Proteínas Recombinantes/química , Transdução de Sinais , VertebradosRESUMO
Redox modifications of specific cysteinyl and methionyl residues regulate key enzymes and signal-transducing proteins in various pathways. Here, we analyzed the effect of redox modifications on protein structure screening the RCSB protein data bank for oxidative modifications of proteins, i.e. protein disulfides, mixed disulfides with glutathione, cysteinyl sulfenic acids, cysteinyl S-nitrosylation, and methionyl sulfoxide residues. When available, these structures were compared to the structures of the same proteins in the reduced state with respect to both pre-requirements for the oxidative modifications as well as the structural consequences of the modifications. In general, the conformational changes induced by the redox modification are small, i.e. within the range of normal fluctuations. Some redox modifications, disulfides in particular, induces alterations in the electrostatic properties of the proteins. Solvent accessibility does not seem to be a strict pre-requirement for the redox modification of a particular residue. We identified an enrichment of certain other amino acid residues in the vicinity of the susceptible residues, for disulfide and sulfenic acid modifications, for instance, histidyl and tyrosyl residues. These motifs, as well as the specific features of the susceptible sulfur-containing amino acids, may become helpful for the prediction of redox modifications.
Assuntos
Glutationa , Fatores de Transcrição , Oxirredução , Fatores de Transcrição/metabolismo , Glutationa/metabolismo , Dissulfetos/metabolismo , Cisteína/metabolismoRESUMO
The reversible reduction and oxidation of protein thiols was first described as mechanism to control light/dark-dependent metabolic regulation in photosynthetic organisms. Today, it is recognized as an essential mechanism of regulation and signal transduction in all kingdoms of life. Proteins of the thioredoxin (Trx) family, Trxs and glutaredoxins (Grxs) in particular, catalyze thiol-disulfide exchange reactions and are vital players in the operation of thiol switches. Various Trx and Grx isoforms are present in all compartments of the cell. These proteins have a rather broad but at the same time distinct substrate specificity. Understanding the molecular basis of their target specificity is central to the understanding of physiological and pathological redox signaling. Electrostatic complementarity of the redoxins with their target proteins has been proposed as a major reason. Here, we analyzed the electrostatic similarity of all Arabidopsis thaliana Trxs, Grxs, and proteins containing such domains. Clustering of the redoxins based on this comparison suggests overlapping and also distant target specificities and thus functions of the different sub-classes including all Trx isoforms as well as the three classes of Grxs, i.e. CxxC-, CGFS-, and CC-type Grxs. Our analysis also provides a rationale for the tuned substrate specificities of both the ferredoxin- and NADPH-dependent Trx reductases.
Assuntos
Arabidopsis , Glutarredoxinas , Eletricidade Estática , Tiorredoxinas , Compostos de Sulfidrila , Tiorredoxina Dissulfeto RedutaseRESUMO
BACKGROUND: Oxidoreductases of the thioredoxin family of proteins have been thoroughly studied in numerous cellular and animal models mimicking human diseases. Despite of their well documented role in various disease conditions, no systematic information on the presence of these proteins is available. METHODS: Here, we have systematically analyzed the presence of some of the major constituents of the glutaredoxin (Grx)-, peroxiredoxin (Prx)-, and thioredoxin (Trx)-systems, i.e. Grx1, Grx2, Grx3 (TXNL-2/PICOT), Grx5, nucleoredoxin (Nrx), Prx1, Prx2, Prx3, Prx4, Prx5, Prx6, Trx1, thioredoxin reductase 1 (TrxR1), Trx2, TrxR2, and γ-glutamyl cysteine synthetase (γ-GCS) in various tissues of the mouse using immunohistochemistry. RESULTS: The identification of the Trx family proteins in the central nervous system, sensory organs, digestive system, lymphatic system, reproductive system, urinary system, respiratory system, endocrine system, skin, heart, and muscle revealed a number of significant differences between these proteins with respect to their distribution in these tissues. CONCLUSION: Our results imply more specific functions and interactions between the proteins of this family than previously assumed. GENERAL SIGNIFICANCE: Crucial functions of Trx family proteins have been demonstrated in various disease conditions. A detailed overview on their distribution in various tissues will be helpful to fully comprehend their potential role and the interactions of these proteins in the most thoroughly studied model for human diseases-the laboratory mouse. This article is part of a Special Issue entitled Human and Murine Redox Protein Atlases.
Assuntos
Glutarredoxinas/metabolismo , Camundongos/metabolismo , Peroxirredoxinas/metabolismo , Tiorredoxinas/metabolismo , Animais , Atlas como Assunto , Feminino , Glutarredoxinas/genética , Glutarredoxinas/imunologia , Humanos , Imuno-Histoquímica , Masculino , Camundongos/genética , Camundongos/imunologia , Modelos Biológicos , Oxirredução , Peroxirredoxinas/genética , Peroxirredoxinas/imunologia , Gravidez , Tiorredoxinas/genética , Tiorredoxinas/imunologia , Distribuição TecidualRESUMO
BACKGROUND: The oxidoreductases of the thioredoxin (Trx) family of proteins play a major role in the cellular response to oxidative stress. Redox imbalance is a major feature of brain damage. For instance, neuronal damage and glial reaction induced by a hypoxic-ischemic episode is highly related to glutamate excitotoxicity, oxidative stress and mitochondrial dysfunction. Most animal models of hypoxia-ischemia in the central nervous system (CNS) use rats to study the mechanisms involved in neuronal cell death, however, no comprehensive study on the localization of the redox proteins in the rat CNS was available. METHODS: The aim of this work was to study the distribution of the following proteins of the thioredoxin and glutathione/glutaredoxin (Grx) systems in the rat CNS by immunohistochemistry: Trx1, Trx2, TrxR1, TrxR2, Txnip, Grx1, Grx2, Grx3, Grx5, and γ-GCS, peroxiredoxin 1 (Prx1), Prx2, Prx3, Prx4, Prx5, and Prx6. We have focused on areas most sensitive to a hypoxia-ischemic insult: Cerebellum, striatum, hippocampus, spinal cord, substantia nigra, cortex and retina. RESULTS AND CONCLUSIONS: Previous studies implied that these redox proteins may be distributed in most cell types and regions of the CNS. Here, we have observed several remarkable differences in both abundance and regional distribution that point to a complex interplay and crosstalk between the proteins of this family. GENERAL SIGNIFICANCE: We think that these data might be helpful to reveal new insights into the role of thiol redox pathways in the pathogenesis of hypoxia-ischemia insults and other disorders of the CNS. This article is part of a Special Issue entitled Human and Murine Redox Protein Atlases.
Assuntos
Sistema Nervoso Central/metabolismo , Glutarredoxinas/metabolismo , Tiorredoxinas/metabolismo , Animais , Atlas como Assunto , Sistema Nervoso Central/anatomia & histologia , Humanos , Hipóxia-Isquemia Encefálica/metabolismo , Hipóxia-Isquemia Encefálica/patologia , Imuno-Histoquímica , Masculino , Oxirredução , RatosRESUMO
Nucleoredoxin (Nrx) belongs to the Thioredoxin protein family and functions in redox-mediated signal transduction. It contains the dithiol active site motif Cys-Pro-Pro-Cys and interacts and regulates different proteins in distinct cellular pathways. Nrx was shown to be catalytically active in the insulin assay and recent findings indicate that Nrx functions, in fact, as oxidase. Here, we have analyzed Nrx in the mammalian retina exposed to (perinatal) hypoxia-ischemia/reoxygenation, combining ex vivo and in vitro models. Our data show that Nrx regulates cell differentiation, which is important to (i) increase the number of glial cells and (ii) replenish neurons that are lost following the hypoxic insult. Nrx is essential to maintain cell morphology. These regulatory changes are related to VEGF but do not seem to be linked to the Wnt/ß-catenin pathway, which is not affected by Nrx knock-down. In conclusion, our results strongly suggest that hypoxia-ischemia could lead to alterations in the organization of the retina, related to changes in RPE cell differentiation. Nrx may play an essential role in the maintenance of the RPE cell differentiation state via the regulation of VEGF release.
RESUMO
The proteins from the thioredoxin family are crucial actors in redox signaling and the cellular response to oxidative stress. The major intracellular source for oxygen radicals are the components of the respiratory chain in mitochondria. Here, we show that the mitochondrial 2-Cys peroxiredoxin (Prx3) is not only substrate for thioredoxin 2 (Trx2), but can also be reduced by glutaredoxin 2 (Grx2) via the dithiol reaction mechanism. Grx2 reduces Prx3 exhibiting catalytic constants (K(m), 23.8 µmol·liter(-1); V(max), 1.2 µmol·(mg·min)(-1)) similar to Trx2 (K(m), 11.2 µmol·liter(-1); V(max), 1.1 µmol·(mg·min)(-1)). The reduction of the catalytic disulfide of the atypical 2-Cys Prx5 is limited to the Trx system. Silencing the expression of either Trx2 or Grx2 in HeLa cells using specific siRNAs did not change the monomer:dimer ratio of Prx3 detected by a specific 2-Cys Prx redox blot. Only combined silencing of the expression of both proteins led to an accumulation of oxidized protein. We further demonstrate that the distribution of Prx3 in different mouse tissues is either linked to the distribution of Trx2 or Grx2. These results introduce Grx2 as a novel electron donor for Prx3, providing further insights into pivotal cellular redox signaling mechanisms.
Assuntos
Glutarredoxinas/metabolismo , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Peroxirredoxinas/metabolismo , Tiorredoxinas/metabolismo , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Inativação Gênica , Glutarredoxinas/genética , Células HeLa , Humanos , Camundongos , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Especificidade de Órgãos/fisiologia , Oxirredução , Estresse Oxidativo/fisiologia , Peroxirredoxina III , Peroxirredoxinas/genética , Multimerização Proteica/fisiologia , Ratos , Tiorredoxinas/genéticaRESUMO
The mammalian cytosolic thioredoxin (Trx) system consists of Trx1 and its reductase, the NADPH-dependent seleno-enzyme TrxR1. These proteins function as electron donor for metabolic enzymes, for instance in DNA synthesis, and the redox regulation of numerous processes. In this work, we analysed the interactions between these two proteins. We proposed electrostatic complementarity as major force controlling the formation of encounter complexes between the proteins and thus the efficiency of the subsequent electron transfer reaction. If our hypothesis is valid, formation of the encounter complex should be independent of the redox reaction. In fact, we were able to confirm that also a redox inactive mutant of Trx1 lacking both active site cysteinyl residues (C32,35S) binds to TrxR1 in a similar manner and with similar kinetics as the wild-type protein. We have generated a number of mutants with alterations in electrostatic properties and characterised their interaction with TrxR1 in kinetic assays. For human Trx1 and TrxR1, complementary electrostatic surfaces within the area covered in the encounter complex appear to control the affinity of the reductase for its substrate Trx. Electrostatic compatibility was even observed in areas that do not form direct molecular interactions in the encounter complex, and our results suggest that the electrostatic complementarity in these areas influences the catalytic efficiency of the reduction. The human genome encodes ten cytosolic Trx-like or Trx domain-containing proteins. In agreement with our hypothesis, the proteins that have been characterised as TrxR1 substrates also show the highest similarity in their electrostatic properties.