RESUMO
The clustered protocadherins are a subfamily of neuronal cell adhesion molecules that play an important role in development of the nervous systems in vertebrates. The clustered protocadherin genes exhibit complex expression patterns in the central nervous system. In this study, we have investigated the molecular mechanism underlying neuronal expression of protocadherin genes using the protocadherin gene cluster in fugu as a model. By in silico prediction, we identified multiple neuron-restrictive silencer elements (NRSEs) scattered in the fugu protocadherin cluster and demonstrated that these elements bind specifically to NRSF/REST in vitro and in vivo. By using a transgenic Xenopus approach, we show that these NRSEs regulate neuronal specificity of protocadherin promoters by suppressing their activity in non-neuronal tissues. We provide evidence that protocadherin genes that do not contain an NRSE in their 5' intergenic region are regulated by NRSEs in the regulatory region of their neighboring genes. We also show that protocadherin clusters in other vertebrates such as elephant shark, zebrafish, coelacanth, lizard, mouse and human, contain different sets of multiple NRSEs. Taken together, our data suggest that the neuronal specificity of protocadherin cluster genes in vertebrates is regulated by the NRSE-NRSF/REST system.
Assuntos
Caderinas/genética , Inativação Gênica , Família Multigênica , Neurônios/metabolismo , Elementos Silenciadores Transcricionais , Animais , Linhagem Celular , Humanos , Camundongos , Regiões Promotoras Genéticas , Takifugu/genética , Xenopus laevis , Peixe-Zebra/genéticaRESUMO
The mitochondrial respiratory chain (MRC) plays crucial roles in cellular energy production. However, its function in early embryonic development remains largely unknown. To address this issue, GRIM-19, a newly identified MRC complex I subunit, was knocked down in Xenopus laevis embryos. A severe deficiency in heart formation was observed, and the deficiency could be rescued by reintroducing human GRIM-19 mRNA. The mechanism involved was further investigated. We found that the activity of NFAT, a transcription factor family that contributes to early organ development, was downregulated in GRIM-19 knockdown embryos. Furthermore, the expression of a constitutively active form of mouse NFATc4 in these embryos rescued the heart developmental defects. NFAT activity is controlled by a calcium-dependent protein phosphatase, calcineurin, which suggests that calcium signaling may be disrupted by GRIM-19 knockdown. Indeed, both the calcium response and calcium-induced NFAT activity were impaired in the GRIM-19 or NDUFS3 (another complex I subunit) knockdown cell lines. We also showed that NFAT can rescue expression of Nkx2.5, which is one of the key genes for early heart development. Our data demonstrated the essential role of MRC in heart formation and revealed the signal transduction and gene expression cascade involved in this process.
Assuntos
Sinalização do Cálcio , Coração/embriologia , Mitocôndrias/fisiologia , Fatores de Transcrição NFATC/metabolismo , Xenopus laevis/embriologia , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose/biossíntese , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/genética , Neoplasias da Mama/patologia , Calcineurina/metabolismo , Cálcio/análise , Linhagem Celular Tumoral , DNA Complementar , Transporte de Elétrons , Embrião não Mamífero , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Células HeLa , Humanos , Hibridização In Situ , Células Jurkat , Luciferases/metabolismo , Dados de Sequência Molecular , Músculo Esquelético/ultraestrutura , Miocárdio/ultraestrutura , NADH NADPH Oxirredutases/biossíntese , NADH NADPH Oxirredutases/química , NADH NADPH Oxirredutases/genética , Fatores de Transcrição NFATC/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Homologia de Sequência de Aminoácidos , Transcrição Gênica , TransfecçãoRESUMO
Angiotensin-converting enzyme 2 (ACE2), a newly identified member in the renin-angiotensin system (RAS), acts as a negative regulator of ACE. It is mainly expressed in cardiac blood vessels and the tubular epithelia of kidneys and abnormal expression has been implicated in diabetes, hypertension and heart failure. The mechanism and physiological function of this zinc metallopeptidase in mammals are not yet fully understood. Non-mammalian vertebrate models offer attractive and simple alternatives that could facilitate the exploration of ACE2 function. In this paper we report the in silico analysis of Ace2 genes from the Gallus (chicken), Xenopus (frog), Fugu and Tetraodon (pufferfish) genome assembly databases, and from the Danio (zebrafish) cDNA library. Exon ambiguities of Danio and Xenopus Ace2s were resolved by RT-PCR and 3'RACE. Analyses of the exon-intron structures, alignment, phylogeny and hydrophilicity plots, together with the conserved synteny among these vertebrates, support the orthologous relationship between mammalian and non-mammalian ACE2s. The putative promoters of Ace2 from human, Tetraodon and Xenopus tropicalis drove the expression of enhanced green fluorescent protein (EGFP) specifically in the heart tissue of transgenic Xenopus thus making it a suitable model for future functional genomic studies. Additionally, the search for conserved cis-elements resulted in the discovery of WGATAR motifs in all the putative Ace2 promoters from 7 different animals, suggesting a possible role of GATA family transcriptional factors in regulating the expression of Ace2.
Assuntos
Peptidil Dipeptidase A/genética , Vertebrados/genética , Enzima de Conversão de Angiotensina 2 , Animais , Animais Geneticamente Modificados , Sequência de Bases , Galinhas/genética , Mapeamento Cromossômico , DNA Complementar/genética , Éxons , Proteínas de Fluorescência Verde/genética , Humanos , Íntrons , Dados de Sequência Molecular , Peptidil Dipeptidase A/química , Filogenia , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Takifugu/genética , Tetraodontiformes/genética , Xenopus/genética , Peixe-Zebra/genéticaRESUMO
Severe acute respiratory syndrome (SARS), a life-threatening disease, is caused by the newly identified virus SARS coronavirus (SARS-CoV). In order to study the spike (S) protein of this highly contagious virus, we established a clonal cell-line, CHO-SG, from the Chinese hamster ovary cells that stably expresses C-terminally EGFP-tagged SARS-CoV S protein (S-EGFP). The ectodomain of the S glycoprotein is localized on the surface of CHO-SG cells with N-acetyl-glucosamine-terminated carbohydrate structure. CHO-SG cells associated tightly with Vero E6 cells, a SARS-CoV receptor (ACE2) expressing cell-line, and the interaction remained stable under highly stringent condition (1M NaCl). This interaction could be blocked by either the serum from a SARS convalescent patient or a goat anti-ACE2 antibody, indicating that the interaction is specific. A binding epitope with lesser degree of glycosylation and native conformation was localized by using rabbit anti-sera raised against five denatured recombinant S protein fragments expressed in Escherichia coli. One of the sera obtained from the fragment encompassing amino acids 48-358 significantly blocked the interaction between CHO-SG and Vero E6 cells. The region is useful for studying neutralizing antibodies in future vaccine development. This paper describes an easy and safe cell-based assay suitable for studying the binding between SARS-CoV S protein and its receptor.
Assuntos
Glicoproteínas de Membrana/metabolismo , Receptores Virais/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/metabolismo , Proteínas do Envelope Viral/metabolismo , Animais , Células CHO , Chlorocebus aethiops , Cricetinae , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteína da Espícula de Coronavírus , Células Vero , Proteínas do Envelope Viral/genéticaRESUMO
Rhodopsin genes in most vertebrate species, with the exception of teleost fishes, contain introns. Despite differences in the gene structure, similar regulatory motifs have been identified in fish, amphibian and mammalian rhodopsin promoters, suggesting that rhodopsin gene regulation may be conserved in vertebrates. However, there is no direct evidence to support this notion. To address this, the rhodopsin promoter from the pufferfish, Fugu rubripes, was isolated and tested in transgenic mice and frogs. A 6.5 kb Fugu genomic fragment containing the rhodopsin gene and 4.5 kb 5' flanking region was able to direct expression of the Fugu rhodopsin gene to the retina of transgenic mice. In transgenic tadpoles, photoreceptor rod cell-specific expression of a reporter gene was achieved using only 500 bp Fugu rhodopsin promoter fragment. Mutagenesis of this promoter fragment revealed that a conserved NRE-like motif is crucial for the retina-specific expression. Our investigation suggests that the regulation of retinal specific expression is conserved in the pufferfish, frog and mouse and that the ancestral intron-containing rhodopsin gene has been displaced by an intronless copy in teleosts.
Assuntos
Retina/metabolismo , Rodopsina/genética , Vertebrados/genética , Animais , Animais Geneticamente Modificados , Sequência de Bases , Olho/citologia , Olho/metabolismo , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde , Larva/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutação , Células Fotorreceptoras/citologia , Células Fotorreceptoras/metabolismo , Regiões Promotoras Genéticas/genética , Sequências Reguladoras de Ácido Nucleico/genética , Retina/citologia , Rodopsina/metabolismo , Homologia de Sequência do Ácido Nucleico , Takifugu , Xenopus laevisRESUMO
S-phase transcription of the histone 2B (H2B) gene is dependent on Octamer-binding factor 1 (Oct-1) and Oct-1 Co-Activator in S-phase (OCA-S), a protein complex comprising glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase (p38/GAPDH and p36/LDH) along with other components. H2B transcription in vitro is modulated by NAD(H). This potentially links the cellular redox status to histone expression. Here, we show that H2B transcription requires a proper NAD(+)/NADH redox status in vitro and in vivo. Therefore, perturbing a properly balanced redox impairs H2B transcription. A redox-modulated direct p38/GAPDH-Oct-1 interaction nucleates the occupancy of the H2B promoter by the OCA-S complex, in which p36/LDH plays a critical role in the hierarchical organization of the complex. As for p38/GAPDH, p36/LDH is essential for the OCA-S function in vivo, and OCA-S-associated p36/LDH possesses an LDH enzyme activity that impacts H2B transcription. These studies suggest that the cellular redox status (metabolic states) can directly feedback to gene switching in higher eukaryotes as is commonly observed in prokaryotes.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica/fisiologia , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Histonas/biossíntese , L-Lactato Desidrogenase/metabolismo , Nitrosaminas/metabolismo , Fator 1 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Gliceraldeído-3-Fosfato Desidrogenases/genética , Células HeLa , Histonas/genética , Humanos , L-Lactato Desidrogenase/genética , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Fator 1 de Transcrição de Octâmero/genética , Oxirredução , Regiões Promotoras Genéticas/fisiologia , Fase S/fisiologia , Fatores de Transcrição/genética , Transcrição Gênica/fisiologia , XenopusRESUMO
We have previously shown that an Escherichia coli-expressed, denatured spike (S) protein fragment of the severe acute respiratory coronavirus, containing residues 1029 to 1192 which include the heptad repeat 2 (HR2) domain, was able to induce neutralizing polyclonal antibodies (C. T. Keng, A. Zhang, S. Shen, K. M. Lip, B. C. Fielding, T. H. Tan, C. F. Chou, C. B. Loh, S. Wang, J. Fu, X. Yang, S. G. Lim, W. Hong, and Y. J. Tan, J. Virol. 79:3289-3296, 2005). In this study, monoclonal antibodies (MAbs) were raised against this fragment to identify the linear neutralizing epitopes in the functional domain and to investigate the mechanisms involved in neutralization. Eighteen hybridomas secreting the S protein-specific MAbs were obtained. Binding sites of these MAbs were mapped to four linear epitopes. Two of them were located within the HR2 region and two immediately upstream of the HR2 domain. MAbs targeting these epitopes showed in vitro neutralizing activities and were able to inhibit cell-cell membrane fusion. These results provide evidence of novel neutralizing epitopes that are located in the HR2 domain and the spacer region immediately upstream of the HR2 of the S protein.
Assuntos
Anticorpos Antivirais/imunologia , Glicoproteínas de Membrana/imunologia , Síndrome Respiratória Aguda Grave/virologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Linhagem Celular , Epitopos/imunologia , Humanos , Fusão de Membrana/imunologia , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Dados de Sequência Molecular , Testes de Neutralização , Estrutura Terciária de Proteína , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/metabolismo , Síndrome Respiratória Aguda Grave/imunologia , Glicoproteína da Espícula de Coronavírus , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genéticaRESUMO
The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) interacts with cellular receptors to mediate membrane fusion, allowing viral entry into host cells; hence it is recognized as the primary target of neutralizing antibodies, and therefore knowledge of antigenic determinants that can elicit neutralizing antibodies could be beneficial for the development of a protective vaccine. Here, we expressed five different fragments of S, covering the entire ectodomain (amino acids 48 to 1192), as glutathione S-transferase fusion proteins in Escherichia coli and used the purified proteins to raise antibodies in rabbits. By Western blot analysis and immunoprecipitation experiments, we showed that all the antibodies are specific and highly sensitive to both the native and denatured forms of the full-length S protein expressed in virus-infected cells and transfected cells, respectively. Indirect immunofluorescence performed on fixed but unpermeabilized cells showed that these antibodies can recognize the mature form of S on the cell surface. All the antibodies were also able to detect the maturation of the 200-kDa form of S to the 210-kDa form by pulse-chase experiments. When the antibodies were tested for their ability to inhibit SARS-CoV propagation in Vero E6 culture, it was found that the anti-SDelta10 antibody, which was targeted to amino acid residues 1029 to 1192 of S, which include heptad repeat 2, has strong neutralizing activities, suggesting that this region of S carries neutralizing epitopes and is very important for virus entry into cells.
Assuntos
Mapeamento de Epitopos , Glicoproteínas de Membrana/imunologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Antivirais/imunologia , Western Blotting , Células Cultivadas , Chlorocebus aethiops , Clonagem Molecular , Epitopos/genética , Epitopos/imunologia , Escherichia coli/genética , Imunofluorescência , Imunoprecipitação , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiologia , Testes de Neutralização , Coelhos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Glicoproteína da Espícula de Coronavírus , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/fisiologiaRESUMO
A new coronavirus (severe acute respiratory syndrome coronavirus [SARS-CoV]) has been identified to be the etiological agent of severe acute respiratory syndrome. Given the highly contagious and acute nature of the disease, there is an urgent need for the development of diagnostic assays that can detect SARS-CoV infection. For determination of which of the viral proteins encoded by the SARS-CoV genome may be exploited as diagnostic antigens for serological assays, the viral proteins were expressed individually in mammalian and/or bacterial cells and tested for reactivity with sera from SARS-CoV-infected patients by Western blot analysis. A total of 81 sera, including 67 from convalescent patients and seven pairs from two time points of infection, were analyzed, and all showed immunoreactivity towards the nucleocapsid protein (N). Sera from some of the patients also showed immunoreactivity to U274 (59 of 81 [73%]), a protein that is unique to SARS-CoV. In addition, all of the convalescent-phase sera showed immunoreactivity to the spike (S) protein when analyzed by an immunofluorescence method utilizing mammalian cells stably expressing S. However, samples from the acute phase (2 to 9 days after the onset of illness) did not react with S, suggesting that antibodies to N may appear earlier than antibodies to S. Alternatively, this could be due to the difference in the sensitivities of the two methods. The immunoreactivities to these recombinant viral proteins are highly specific, as sera from 100 healthy donors did not react with any of them. These results suggest that recombinant N, S, and U274 proteins may be used as antigens for the development of serological assays for SARS-CoV.