RESUMO
Neuroglobin (NGB) is an oxygen-binding protein that is mainly expressed in nervous tissues where it is considered to be neuroprotective during ischemic brain injury. Interestingly, transgenic mice overexpressing NGB reveal cytoprotective effects on tissues lacking endogenous NGB, which might indicate a therapeutic role for NGB in a broad range of ischemic conditions. In the present study, we investigated the effect of NGB overexpression on survival as well as on the size and occurrence of myocardial infarctions (MI) in a mouse model of acute MI (AMI) and a model of advanced atherosclerosis (ApoE -/- Fbn1 C1039G+/- mice), in which coronary plaques and MI develop in mice being fed a Western-type diet. Overexpression of NGB significantly enhanced post-AMI survival and reduced MI size by 14% 1 week after AMI. Gene expression analysis of the infarction border showed reduction of tissue hypoxia and attenuation of hypoxia-induced inflammatory pathways, which might be responsible for these beneficial effects. In contrast, NGB overexpression did not affect survival or occurrence of MI in the atherosclerotic mice although the incidence of coronary plaques was significantly reduced. In conclusion, NGB proved to act cytoprotectively during MI in the acute setting while this effect was less pronounced in the atherosclerosis model.
Assuntos
Citoproteção/genética , Regulação da Expressão Gênica , Globinas/genética , Isquemia Miocárdica/genética , Miocárdio/patologia , Proteínas do Tecido Nervoso/genética , RNA/genética , Doença Aguda , Animais , Doença Crônica , Modelos Animais de Doenças , Globinas/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patologia , Miocárdio/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Neuroglobina , Estresse Oxidativo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
This study aimed to investigate the mechanism underlying the neuroprotective effect of hemin in oxygen-glucose deprivation (OGD)-treated neurons. OGD-treated SH-SY5Y cells (human neuroblastoma cells) were used in the study. The cellular viability of SH-SY5Y cells was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and the cell apoptosis rate was determined by flow cytometry analysis with Annexin V-fluorescein isothiocyanate and propidium iodide staining with or without hemin pretreatment. Cell viability and apoptotic activation were detected after hemin administration combined with neuroglobin (Nqb), thioredoxin-1, peroxiredoxin-2, or heme oxygenase-1 siRNA transient transfection. The release of cytochrome c from mitochondria and the interaction between Ngb and cytochrome c were examined with hemin pretreatment. Hemin had a neuroprotective effect in OGD-treated SH-SY5Y cells, which was mainly mediated by the upregulation of Ngb. Moreover, the release of cytochrome c from mitochondria was inhibited by hemin-induced Ngb expression through facilitating the interaction of Ngb with cytochrome c in mitochondria. The present findings provided new insights into the neuroprotective mechanisms of hemin. It was concluded that low-dose hemin pretreatment had a neuroprotective effect in OGD-treated SH-SY5Y cells, through inhibiting cell apoptosis. The neuroprotective effects of hemin following hypoxic-ischemic neuronal damage were mainly mediated by Ngb. One underlying mechanism was hemin-induced overexpression of mitochondrial Ngb, which inhibited endogenous apoptosis via the association with cytochrome c.
Assuntos
Apoptose/fisiologia , Globinas/biossíntese , Glucose/deficiência , Hemina/farmacologia , Proteínas do Tecido Nervoso/biossíntese , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Apoptose/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Humanos , Neuroglobina , Neurônios/efeitos dos fármacosRESUMO
Biological significance of the globin protein family could be ascertained by their conservation through archaea to human. Globin(s) have been "classically" studied as oxygen binding protein(s), with recent implications in a host of other physiological functions. Drosophila melanogaster possesses three globin genes (glob1, glob2, glob3) located at different cytogenetic positions. We have performed a comprehensive investigation on the cellular expression profile and functional relevance of glob1 in Drosophila development. A profound level of maternally contributed glob1 gene products was found during early embryogenesis. Subsequently, commencement of zygotic transcription leads to its strong expression in somatic muscles, gut primordia, fat bodies, tracheal cells, etc. Similarly, dynamic expression of glob1 was evident in most of the larval tissues, interestingly with high expression in dividing cells. Reduced expression of glob1 leads to various impairments and lethality during embryogenesis and larval development. A substantial increase in level of cellular ROS was also evident due to reduced expression of glob1 which consequently leads to locomotor impairment and early aging in surviving adult flies. To best of our knowledge, this is the first report which demonstrates that in addition to oxygen management, globin gene(s) are also involved in regulating various aspects of development in Drosophila.
Assuntos
Proteínas de Drosophila/biossíntese , Drosophila melanogaster/fisiologia , Globinas/biossíntese , Estresse Oxidativo/fisiologia , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Desenvolvimento Embrionário , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Globinas/genética , Hemoglobinas , Larva , Masculino , Espécies Reativas de Oxigênio/metabolismoRESUMO
Thyroid hormones (THs) are essential and crucial for brain development, playing a role in growth and differentiation. Two globins named neuroglobin (Ngb) and cytoglobin (Cygb) are located in the brain, and each one has different distribution and function: They seem to have similar action by providing O(2) for respiratory chain, and detoxification of reactive oxygen species (ROS) and nitric oxide (NO) protecting tissues against irreversible lesions. We aimed to investigate the influence of thyroid state in Ngb and Cygb metabolism in different brain regions and evaluate their responses in cerebellum, hippocampus and cerebral cortex (hereafter called as cortex) after supraphysiological doses at different time points of TH administration. Experiments were carried out in rats, divided in eight experimental groups Control (C), thyroidectomy (Tx), and thyroidectomy treated with jugular intravenous injection (i.v). T3 (100 µl/100 g) injection and sacrificed after 30, 60, 120 min and 6, 12 and 24 h. In cortex, we found increase in Ngb gene and protein expression in different time points compared to C group, however Cygb gene and protein expression were decreased. In hippocampus, Ngb and Cygb protein expression increased 24 h after i.v. T3 injection in comparison to Tx. In cerebellum, we found increased Ngb gene expression after 120 min, 6, 12 and 24 h after T3 administration compared to Tx, and in contrast, protein expression was found to be significantly increased only 12 and 24 h compared to Tx. Ngb and Cygb expression in brain is influenced by thyroid hormone state both by its lack or excess.
Assuntos
Química Encefálica/fisiologia , Globinas/biossíntese , Globinas/genética , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Hormônios Tireóideos/fisiologia , Animais , Química Encefálica/efeitos dos fármacos , Cerebelo/metabolismo , Córtex Cerebral/metabolismo , Citoglobina , Hipocampo/metabolismo , Masculino , Neuroglobina , Ratos , Ratos Wistar , Hormônios Tireóideos/sangue , Hormônios Tireóideos/farmacologia , Tireoidectomia , Tri-Iodotironina/farmacologiaRESUMO
Chromatin remodeling is essential for controlling the expression of genes during development. The histone-modifying enzyme G9a/KMT1C can act both as a coactivator and a corepressor of transcription. Here, we show that the dual function of G9a as a coactivator vs. a corepressor entails its association within two distinct protein complexes, one containing the coactivator Mediator and one containing the corepressor Jarid1a/KDM5A. Functionally, G9a is important in stabilizing the Mediator complex for gene activation, whereas its repressive function entails a coordinate action with the histone H3 lysine 4 (H3K4) demethylase Jarid1a for the maintenance of gene repression. The essential nature of cross-talk between the histone methyltransferase G9a and the demethylase Jarid1a is demonstrated on the embryonic E(y)-globin gene, where the concurrent introduction of repressive histone marks (dimethylated H3K9 and dimethylated H3K27) and removal of activating histone mark (trimethylated H3K4) is required for maintenance of gene silencing. Taken together with our previous demonstration of cross-talk between UTX and MLL2 to mediate activation of the adult ß(maj)-globin gene, these data suggest a model where "active" and "repressive" cross-talk between histone-modifying enzymes coexist on the same multigene locus and play a crucial role in the precise control of developmentally regulated gene expression.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Inativação Gênica/fisiologia , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Proteína 2 de Ligação ao Retinoblastoma/metabolismo , Animais , Proteínas de Ligação a DNA , Loci Gênicos/fisiologia , Globinas/biossíntese , Globinas/genética , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/genética , Histonas/genética , Histona Desmetilases com o Domínio Jumonji , Complexo Mediador/genética , Complexo Mediador/metabolismo , Metilação , Camundongos , Camundongos Knockout , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteína 2 de Ligação ao Retinoblastoma/genéticaRESUMO
PURPOSE OF REVIEW: This review will provide an overview of the translational regulation of globin mRNAs and integrated stress response (ISR) during erythropoiesis by heme-regulated eIF2α kinase (HRI). HRI is an intracellular heme sensor that coordinates heme and globin synthesis in erythropoiesis by inhibiting protein synthesis of globins and heme biosynthetic enzymes during heme deficiency. RECENT FINDINGS: It has been demonstrated recently that HRI also activates the eIF2αP-activating transcription factor 4 (ATF4) ISR in primary erythroid precursors to combat oxidative stress. During chronic iron/heme deficiency in vivo, this HRI-eIF2αP-ATF4 signaling is necessary both to reduce oxidative stress and to promote erythroid differentiation. Augmenting eIF2αP signaling by the small molecule salubrinal, which inhibits dephosphorylation of eIF2αP, reduces excess α-globin synthesis and enhances translation of ATF4 mRNA in mouse ß-thalassemic erythroid precursors. Intriguingly, salubrinal treatment of differentiating human CD34⺠cells in culture increases fetal hemoglobin production with a concomitant decrease of adult hemoglobin by a posttranscriptional mechanism. SUMMARY: HRI-eIF2αP-ATF4 stress signaling is important not only to inhibit excess globin synthesis during erythropoiesis, but is also critical for adaptation to oxidative stress and for enhancing effective erythropoiesis. Modulation of this signaling pathway with small chemicals may provide a novel therapy for hemoglobinopathy.
Assuntos
Eritropoese/fisiologia , Regulação da Expressão Gênica , Globinas/biossíntese , eIF-2 Quinase/fisiologia , Animais , Globinas/metabolismo , Humanos , Camundongos , Estresse Oxidativo/fisiologia , RNA Mensageiro/metabolismo , Transdução de Sinais/fisiologiaRESUMO
This study was designed to investigate the expression profile of CYGB, its potential neuroprotective function, and underlying molecular mechanisms using a model of neonatal hypoxia-ischemia (HI) brain injury. Cygb mRNA and protein expression were evaluated within the first 36 h after the HI model was induced using RT-PCR and Western blotting. Cygb mRNA expression was increased at 18 h in a time-dependent manner, and its level of protein expression increased progressively in 24 h. To verify the neuroprotective effect of CYGB, a gene transfection technique was employed. Cygb cDNA and shRNA delivery adenovirus systems were established (Cygb-cDNA-ADV and Cygb-shRNA-ADV, respectively) and injected into the brains of 3-day-old rats 4 days before they were induced with HI treatment. Rats from different groups were euthanized 24 h post-HI, and brain samples were harvested. 2,3,5-Triphenyltetrazolium chloride, TUNEL, and Nissl staining indicated that an up-regulation of CYGB resulted in reduced acute brain injury. The superoxide dismutase level was found to be dependent on expression of CYGB. The Morris water maze test in 28-day-old rats demonstrated that CYGB expression was associated with improvement of long term cognitive impairment. Studies also demonstrated that CYGB can up-regulate mRNA and protein levels of VEGF and increase both the density and diameter of the microvessels but inhibits activation of caspase-2 and -3. Thus, this is the first in vivo study focusing on the neuroprotective role of CYGB. The reduction of neonatal HI injury by CYGB may be due in part to antioxidant and antiapoptotic mechanisms and by promoting angiogenesis.
Assuntos
Lesões Encefálicas/metabolismo , Isquemia Encefálica/metabolismo , Globinas/biossíntese , Proteínas do Tecido Nervoso/metabolismo , Regulação para Cima , Doença Aguda , Adenoviridae , Animais , Animais Recém-Nascidos , Antioxidantes/metabolismo , Apoptose/genética , Lesões Encefálicas/genética , Lesões Encefálicas/patologia , Lesões Encefálicas/fisiopatologia , Lesões Encefálicas/terapia , Isquemia Encefálica/genética , Isquemia Encefálica/patologia , Isquemia Encefálica/fisiopatologia , Isquemia Encefálica/terapia , Caspase 3/genética , Caspase 3/metabolismo , Circulação Cerebrovascular/genética , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Citoglobina , Feminino , Globinas/genética , Masculino , Aprendizagem em Labirinto , Neovascularização Fisiológica/genética , Proteínas do Tecido Nervoso/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Transdução Genética , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Neuroglobin (NGB) is a neuron-specific vertebrate globin shown to protect against hypoxia, ischemia, oxidative stress and the toxic effects of Amyloid-beta. Following on our and others' results highlighting the importance of NGB expression in disease, we searched for genetic determinants of its expression. We found that a microRNA expressed with the NGB transcript shows significant target enrichments in the angiogenesis pathway and the Alzheimer disease/presenilin pathway. Using reporter constructs we identified potential promoter/enhancer elements between the transcription start site and 1,142 bp upstream. Using 184 post-mortem temporal lobe samples we replicated the reported negative effect of age, and after genotyping tagging SNPs we found one (rs981471) showing a significant correlation with the gene's expression and another (rs8014408) showing an interaction with age, the rare C allele being correlated with higher expression and faster decline. The two SNPs are towards the 3' end of NGB within the same LD block, 52 Kb apart and modestly correlated (r (2) = 0.5). Next generation sequencing of the same 184 temporal lobe samples and 79 confirmed AD patients across the entire gene region (including >12 Kb on the 3' and 5' flank) revealed limited coding variation, suggesting purifying selection of NGB, but did not identify regulatory or disease associated rare variants. A dinucleotide repeat in intron 1 with extensive evidence of functionality showed interesting but inconclusive results, as it was not amenable to further molecular analysis.
Assuntos
Globinas/biossíntese , Globinas/genética , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Transcrição Gênica , Idoso , Alelos , Doença de Alzheimer/genética , Sequência de Aminoácidos , Animais , Encéfalo/patologia , Galinhas , Biologia Computacional , Feminino , Regulação da Expressão Gênica , Genes Reporter , Variação Genética , Genoma , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Desequilíbrio de Ligação , Masculino , Camundongos , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Dados de Sequência Molecular , Neuroglobina , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Homologia de Sequência de Aminoácidos , Lobo Temporal/metabolismo , Peixe-ZebraRESUMO
OBJECTIVES: To determine the role of neuroglobin in the pathology of sepsis-associated encephalopathy and ascertain if neuroglobin has any protective effects against sepsis-associated encephalopathy. DESIGN: Randomized laboratory animal study. SETTING: Research university animal laboratory. SUBJECTS: Two hundred and forty adult male Sprague-Dawley rats. INTERVENTIONS: Rats received cecal puncture and ligation (or sham) surgery to induce sepsis, then broken up into groups based on whether or not the rat developed sepsis-associated encephalopathy as determined by electroencephalograph and evoked potential recordings. The rats were then left untreated to examine the effect of sepsis-associated encephalopathy on neuroglobin, treated with a neuroglobin antisense nucleotide to block gene expression, or given hemin, a neuroglobin inducer. MEASUREMENTS AND MAIN RESULTS: Following sepsis induction, diagnosis, and treatment, the brains were analyzed for both gross and ultrastructural morphology. Also, neuronal neuroglobin immunoreactivity and apoptosis (via terminal uridine nucleotide end-labeling) were examined. Blood serum levels were then analyzed for neuroglobin, superoxide dismutase, and malondialdehyde levels. We determined that sepsis-associated encephalopathy induces damage evident when examining both gross and ultrastructural morphology, as well as induces neuronal neuroglobin expression. Also, blockade of neuroglobin expression via antisense treatment will exacerbate these pathological effects, while increasing neuroglobin levels via hemin will ameliorate them. Blood analysis found that levels of superoxide dismutase and malondialdehyde mirrored the level of pathology found in the brain, while plasma neuroglobin levels reflected the amount of neuronal neuroglobin immunoreactivity. CONCLUSIONS: We conclude that neuroglobin is involved in the pathogenesis of sepsis-associated encephalopathy and has neuroprotective effects. We also determined that hemin has protective effects against sepsis-associated encephalopathy as well, most probably due to its effect on neuroglobin.
Assuntos
Encefalopatias/etiologia , Globinas/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Sepse/complicações , Animais , Apoptose/fisiologia , Encéfalo/patologia , Encéfalo/ultraestrutura , Encefalopatias/patologia , Encefalopatias/fisiopatologia , Modelos Animais de Doenças , Globinas/biossíntese , Masculino , Malondialdeído/sangue , Microscopia Eletrônica de Transmissão , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/sangue , Neuroglobina , Ratos , Ratos Sprague-Dawley , Sepse/patologia , Sepse/fisiopatologia , Superóxido Dismutase/sangueRESUMO
Heme-regulated eIF2α kinase (Hri) is necessary for balanced synthesis of heme and globin. In addition, Hri deficiency exacerbates the phenotypic severity of ß-thalassemia intermedia in mice. Activation of Hri during heme deficiency and in ß-thalassemia increases eIF2α phosphorylation and inhibits globin translation. Under endoplasmic reticulum stress and nutrient starvation, eIF2α phosphorylation also induces the Atf4 signaling pathway to mitigate stress. Although the function of Hri in regulating globin translation is well established, its role in Atf4 signaling in erythroid precursors is not known. Here, we report the role of the Hri-activated Atf4 signaling pathway in reducing oxidative stress and in promoting erythroid differentiation during erythropoiesis. On acute oxidative stress, Hri(-/-) erythroblasts suffered from increased levels of reactive oxygen species (ROS) and apoptosis. During chronic iron deficiency in vivo, Hri is necessary both to reduce oxidative stress and to promote erythroid differentiation. Hri(-/-) mice developed ineffective erythropoiesis during iron deficiency with inhibition of differentiation at the basophilic erythroblast stage. This inhibition is recapitulated during ex vivo differentiation of Hri(-/-) fetal liver erythroid progenitors. Importantly, the Hri-eIF2αP-Atf4 pathway was activated and required for erythroid differentiation. We further demonstrate the potential of modulating Hri-eIF2αP-Atf4 signaling with chemical compounds as pharmaceutical therapies for ß-thalassemia.
Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Eritroblastos/metabolismo , Eritropoese , Estresse Oxidativo , Transdução de Sinais , eIF-2 Quinase/metabolismo , Fator 4 Ativador da Transcrição/genética , Animais , Diferenciação Celular/genética , Células Cultivadas , Eritroblastos/patologia , Feto/embriologia , Feto/metabolismo , Feto/patologia , Globinas/biossíntese , Globinas/genética , Ferro/metabolismo , Deficiências de Ferro , Fígado/embriologia , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Knockout , Biossíntese de Proteínas/genética , Espécies Reativas de Oxigênio/metabolismo , Talassemia beta/genética , Talassemia beta/metabolismo , Talassemia beta/patologia , Talassemia beta/terapia , eIF-2 Quinase/genéticaRESUMO
Emerging new and intriguing roles of cytoglobin (Cygb) have attracted considerable attention of cancer researchers in recent years. Hypoxic upregulation of Cygb as well as its altered expression in various human cancers suggest another possible role of this newly discovered globin in tumor cell response under low oxygen tension. Since tumor hypoxia is strongly associated with malignant progression of disease and poor treatment response, it constitutes an area of paramount importance for rational design of cancer selective therapies. However, the mechanisms involved during this process are still elusive. This review outlines the current understanding of Cygb's involvement in tumor hypoxia and discusses its role in tumorigenesis. A better perception of Cygb in tumor hypoxia response is likely to open novel perspectives for future tumor therapy.
Assuntos
Carcinogênese/genética , Globinas/genética , Neoplasias/genética , Neoplasias/terapia , Hipóxia Celular/genética , Citoglobina , Regulação Neoplásica da Expressão Gênica , Globinas/biossíntese , Humanos , Neoplasias/patologia , Oxigênio/metabolismoRESUMO
Loss of cytoglobin is found to be involved in the progression of several human cancers. However, its expression pattern and biological roles in human ovarian cancers are not clear. In this study, we examined cytoglobin expression in 118 archived ovarian cancer specimens using immunohistochemistry. A total of 72 specimens (61.0 %) showed cytoglobin downregulation. cytoglobin downregulation positively correlated with advanced FIGO stage and tumor grade. Cytoglobin plasmid transfection was performed in SKOV3 cell line and siRNA knockdown was carried out in SW626 cell line. MTT, colony formation assay and matrigel invasion assay were carried out to assess the role of cytoglobin on cell proliferation and invasion. Cytoglobin overexpression inhibited cell growth, invasion, cell cycle progression and cyclin D1 expression in SKOV3 cell line and its depletion promoted cell proliferation, invasion, cell cycle transition and cyclin D1 expression. In conclusion, cytoglobin is downregulated in ovarian cancers and associated with advanced stage. Our data provides evidence that cytoglobin regulates the ovarian cancer cell proliferation and invasion.
Assuntos
Globinas/biossíntese , Invasividade Neoplásica/genética , Neoplasias Ovarianas/genética , Idoso , Movimento Celular/genética , Proliferação de Células , Ciclina D1/biossíntese , Citoglobina , Feminino , Regulação Neoplásica da Expressão Gênica , Globinas/genética , Humanos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias Ovarianas/patologia , RNA Interferente PequenoRESUMO
For the past fifty-five years, much of my research has focused on the function and biogenesis of red blood cells, including the cloning and study of many membrane proteins such as glucose and anion transporters and the erythropoietin receptor. We have also elucidated the mechanisms of membrane insertion, folding, and maturation of many plasma membrane and secreted proteins. Despite all of this work and more, I remain extremely proud of our very early work on the regulation of mRNA translation: work on bacteriophage f2 RNA in the 1960s and on translation of α- and ß-globin mRNAs in the early 1970s. Using techniques hopelessly antiquated by today's standards, we correctly elucidated many important aspects of translational control, and I thought readers would be interested in learning how we did these experiments.
Assuntos
Biologia Molecular/história , Biossíntese de Proteínas/fisiologia , Animais , Bacteriófagos/metabolismo , Membrana Celular/metabolismo , Globinas/biossíntese , História do Século XX , Humanos , Proteínas de Membrana/biossíntese , Biologia Molecular/métodos , Dobramento de Proteína , RNA Mensageiro/metabolismoRESUMO
Neuroglobin (Ngb) is an endogenous neuroprotective molecule against hypoxic/ischemic brain injury, but the underlying mechanisms remain largely undefined. Our recent study revealed that Ngb can bind to voltage-dependent anion channel (VDAC), a regulator of mitochondria permeability transition (MPT). In this study we examined the role of Ngb in MPT pore (mPTP) opening following oxygen-glucose deprivation (OGD) in primary cultured mouse cortical neurons. Co-immunoprecipitation (Co-IP) and immunocytochemistry showed that the binding between Ngb and VDAC was increased after OGD compared to normoxia, indicating the OGD-enhanced Ngb-VDAC interaction. Ngb overexpression protected primary mouse cortical neurons from OGD-induced neuronal death, to an extent comparable to mPTP opening inhibitor, cyclosporine A (CsA) pretreatment. We further measured the role of Ngb in OGD-induced mPTP opening using Ngb overexpression and knockdown approaches in primary cultured neurons, and recombinant Ngb exposure to isolated mitochondria. Same as CsA pretreatment, Ngb overexpression significantly reduced OGD-induced mPTP opening markers including mitochondria swelling, mitochondrial NAD(+) release, and cytochrome c (Cyt c) release in primary cultured neurons. Recombinant Ngb incubation significantly reduced OGD-induced NAD(+) release and Cyt c release from isolated mitochondria. In contrast, Ngb knockdown significantly increased OGD-induced neuron death, and increased OGD-induced mitochondrial NAD(+) release and Cyt c release as well, and these outcomes could be rescued by CsA pretreatment. In summary, our results demonstrated that Ngb overexpression can inhibit OGD-induced mPTP opening in primary cultured mouse cortical neurons, which may be one of the molecular mechanisms of Ngb's neuroprotection.
Assuntos
Globinas/fisiologia , Glucose/deficiência , Hipóxia/patologia , Mitocôndrias/efeitos dos fármacos , Proteínas do Tecido Nervoso/fisiologia , Neurônios/efeitos dos fármacos , Animais , Western Blotting , Morte Celular/fisiologia , Córtex Cerebral/citologia , Córtex Cerebral/patologia , Ciclosporina/farmacologia , Citocromos c/metabolismo , Dependovirus/genética , Globinas/biossíntese , Imuno-Histoquímica , Imunoprecipitação , L-Lactato Desidrogenase/metabolismo , Camundongos , Dilatação Mitocondrial/efeitos dos fármacos , NAD/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Neuroglobina , Neurônios/patologia , Permeabilidade , Cultura Primária de Células , RNA Interferente Pequeno/genética , Proteínas Recombinantes/metabolismo , Canais de Ânion Dependentes de Voltagem/metabolismoRESUMO
Comparative genomic studies have led to the recent identification of several novel globin types in the Metazoa. They have revealed a surprising evolutionary diversity of functions beyond the familiar O(2) supply roles of hemoglobin and myoglobin. Here we report the discovery of a hitherto unrecognized family of proteins with a unique modular architecture, possessing an N-terminal calpain-like domain, an internal, circular permuted globin domain, and an IQ calmodulin-binding motif. Putative orthologs are present in the genomes of many metazoan taxa, including vertebrates. The calpain-like region is homologous to the catalytic domain II of the large subunit of human calpain-7. The globin domain satisfies the criteria of a myoglobin-like fold but is rearranged and split into two parts. The recombinantly expressed human globin domain exhibits an absorption spectrum characteristic of hexacoordination of the heme iron atom. Molecular evolutionary analyses indicate that this chimeric globin family is phylogenetically ancient and originated in the common ancestor to animals and choanoflagellates. In humans and mice, the gene is predominantly expressed in testis tissue, and we propose the name "androglobin" (Adgb). Expression is associated with postmeiotic stages of spermatogenesis and is insensitive to experimental hypoxia. Evidence exists for increased gene expression in fertile compared with infertile males.
Assuntos
Proteínas de Ligação a Calmodulina/biossíntese , Globinas/biossíntese , Globinas/genética , Testículo/fisiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Proteínas de Ligação a Calmodulina/química , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a Calmodulina/metabolismo , Calpaína/química , Calpaína/genética , Calpaína/metabolismo , Evolução Molecular , Globinas/química , Globinas/metabolismo , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Família Multigênica , Filogenia , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alinhamento de Sequência , Testículo/metabolismoRESUMO
KLF1 regulates a diverse suite of genes to direct erythroid cell differentiation from bipotent progenitors. To determine the local cis-regulatory contexts and transcription factor networks in which KLF1 operates, we performed KLF1 ChIP-seq in the mouse. We found at least 945 sites in the genome of E14.5 fetal liver erythroid cells which are occupied by endogenous KLF1. Many of these recovered sites reside in erythroid gene promoters such as Hbb-b1, but the majority are distant to any known gene. Our data suggests KLF1 directly regulates most aspects of terminal erythroid differentiation including production of alpha- and beta-globin protein chains, heme biosynthesis, coordination of proliferation and anti-apoptotic pathways, and construction of the red cell membrane and cytoskeleton by functioning primarily as a transcriptional activator. Additionally, we suggest new mechanisms for KLF1 cooperation with other transcription factors, in particular the erythroid transcription factor GATA1, to maintain homeostasis in the erythroid compartment.
Assuntos
Células Eritroides/metabolismo , Eritropoese/genética , Fatores de Transcrição Kruppel-Like/genética , Animais , Apoptose/genética , Sequência de Bases , Citoesqueleto/genética , Membrana Eritrocítica/genética , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Expressão Gênica , Globinas/biossíntese , Globinas/genética , Heme/biossíntese , Heme/genética , Camundongos , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras GenéticasRESUMO
Surges of nitric oxide compromise mitochondrial respiration primarily by competitive inhibition of oxygen binding to cytochrome c oxidase (complex IV) and are particularly injurious in neurons, which rely on oxidative phosphorylation for all their energy needs. Here, we show that transgenic overexpression of the neuronal globin protein, neuroglobin, helps diminish protein nitration, preserve mitochondrial function and sustain ATP content of primary cortical neurons challenged by extended nitric oxide exposure. Specifically, in transgenic neurons, elevated neuroglobin curtailed nitric oxide-induced alterations in mitochondrial oxygen consumption rates, including baseline oxygen consumption, consumption coupled with ATP synthesis, proton leak and spare respiratory capacity. Concomitantly, activation of genes involved in sensing and responding to oxidative/nitrosative stress, including the early-immediate c-Fos gene and the phase II antioxidant enzyme, heme oxygenase-1, was diminished in neuroglobin-overexpressing compared to wild-type neurons. Taken together, these differences reflect a lesser insult produced by similar concentrations of nitric oxide in neuroglobin-overexpressing compared to wild-type neurons, suggesting that abundant neuroglobin buffers nitric oxide and raises the threshold of nitric oxide-mediated injury in neurons.
Assuntos
Globinas/metabolismo , Mitocôndrias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Óxido Nítrico/metabolismo , Estresse Oxidativo/fisiologia , Análise de Variância , Animais , Córtex Cerebral/citologia , Globinas/biossíntese , Globinas/genética , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Neuroglobina , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Transdução de Sinais , Regulação para CimaRESUMO
A goal of molecular genetics is to understand the relationship between basic nuclear processes, epigenetic changes and the numerous proteins that orchestrate these effects. One such protein, ATRX, contains a highly conserved plant homeodomain (PHD)-like domain, present in many chromatin-associated proteins, and a carboxy-terminal domain which identifies it as a member of the SNF2 family of helicase/ATPases. Mutations in ATRX give rise to characteristic developmental abnormalities including severe mental retardation, facial dysmorphism, urogenital abnormalities and alpha-thalassaemia. This circumstantial evidence suggests that ATRX may act as a transcriptional regulator through an effect on chromatin. We have recently shown that ATRX is localized to pericentromeric heterochromatin during interphase and mitosis, suggesting that ATRX might exert other chromatin-mediated effects in the nucleus. Moreover, at metaphase, some ATRX is localized at or close to the ribosomal DNA (rDNA) arrays on the short arms of human acrocentric chromosomes. Here we show that mutations in ATRX give rise to changes in the pattern of methylation of several highly repeated sequences including the rDNA arrays, a Y-specific satellite and subtelomeric repeats. Our findings provide a potential link between the processes of chromatin remodelling, DNA methylation and gene expression in mammalian development.
Assuntos
DNA Helicases , Metilação de DNA , Proteínas de Ligação a DNA/genética , Mutação , Proteínas Nucleares , Fatores de Transcrição/genética , Southern Blotting , Ilhas de CpG/genética , Análise Mutacional de DNA , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , DNA Satélite/genética , Regulação para Baixo/genética , Técnica Indireta de Fluorescência para Anticorpo , Globinas/biossíntese , Globinas/genética , Humanos , Deficiência Intelectual/genética , Sequências Repetitivas de Ácido Nucleico/genética , Síndrome , Telômero/genética , Cromossomo X/genética , Proteína Nuclear Ligada ao X , Talassemia alfa/genéticaRESUMO
Mutations at the alpha-globin locus are the most common class of mutations in humans, with deletion of all four adult alpha-globin genes resulting in the perinatal lethal condition haemoglobin Barts hydrops fetalis. Using gene targeting in mice, we have deleted a 16 kilobase region encompassing both adult alpha-globin genes. Animals homozygous for this deletion become hydropic and die late in gestation mimicking humans with hydrops fetalis. Introduction of a human alpha-globin transgene rescued these animals from perinatal death thus demonstrating the utility of this murine model in the development of cellular and gene based approaches for treating this human genetic disease.
Assuntos
Modelos Animais de Doenças , Marcação de Genes , Genes Letais , Terapia Genética , Globinas/genética , Hidropisia Fetal/genética , Camundongos Knockout/genética , Animais , Sequência de Bases , Quimera , Morte Fetal/etiologia , Regulação da Expressão Gênica no Desenvolvimento , Idade Gestacional , Globinas/biossíntese , Hemoglobinas Anormais , Humanos , Hidropisia Fetal/sangue , Hidropisia Fetal/embriologia , Hidropisia Fetal/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Deleção de Sequência , Talassemia alfa/sangue , Talassemia alfa/genética , Talassemia alfa/terapiaRESUMO
BACKGROUND: Proliferative inflammatory atrophy (PIA) has been proposed as a potential precursor for prostate cancer. The precise molecular abnormalities in prostatic atrophy compared to high-grade prostatic intraepithelial neoplasia (HGPIN) and carcinoma have not been fully defined. METHODS: We utilized laser capture microdissection and microarray analysis to characterize cells of PIA, HGPIN, invasive prostatic carcinoma, and non-atrophic benign prostatic epithelium (NABE). Cytoglobin was selected for immunohistochemistry (IHC) validation. IHC stains were evaluated for proportion of positive glands, and intensity of cytoglobin staining. An immunoreactive score (IR score) was determined as the product of the percentage of positive staining and intensity. RESULTS: Microarray analysis revealed probe sets that separated the microdissected cell types. Several genes showed overlapping expression patterns between PIA and PIN, and HGPIN and invasive carcinoma. Cytoglobin protein expression was detected in 57/93 (61%) of NABE and BPH cases, 92/93 atrophy (99%), 3/34 (9%) of PIN, and 23/61 carcinoma (37%) samples. The highest IHC scores were calculated for atrophy foci. A subset (33%) of atrophy cases showed the same low-cytoglobin expression level as PIN and carcinoma. CONCLUSIONS: Prostatic epithelium can be stratified into normal, atrophic, PIN, and invasive carcinoma categories based on differential genetic signatures. Cytoglobin, a protein that can be induced in response to oxidative stress, was elevated in most atrophy foci, suggesting hypoxic, and/or oxidative damage. The lower level of cytoglobin seen in neoplastic cells and 33% of atrophy foci may indicate a shared susceptibility to oxidative damage for this subset of atrophy cases and prostatic neoplasia.