RESUMO
Laminin receptor (Lamr) in shrimp was previously proposed to be a potential receptor protein for Taura syndrome virus (TSV) based on yeast two-hybrid assays. Since shrimp Lamr bound to the VP1 capsid protein of TSV, we were interested to know whether capsid/envelope proteins from other shrimp viruses would also bind to Lamr. Thus, capsid/envelope encoding genes from 5 additional shrimp viruses were examined. These were Penaeus stylirostris densovirus (PstDNV), white spot syndrome virus (WSSV), infectious myonecrosis virus (IMNV), Macrobrachium rosenbergii nodavirus (MrNV), and yellow head virus (YHV). Protein interaction analysis using yeast two-hybrid assay revealed that Lamr specifically interacted with capsid/envelope proteins of RNA viruses IMNV and YHV but not MrNV and not with the capsid/envelope proteins of DNA viruses PstDNV and WSSV. In vitro pull-down assay also confirmed the interaction between Lamr and YHV gp116 envelope protein, and injection of recombinant Lamr (rLamr) protein produced in yeast cells protected shrimp against YHV in laboratory challenge tests.
Assuntos
Proteínas do Capsídeo/metabolismo , Penaeidae/imunologia , Vírus de RNA/metabolismo , Receptores de Laminina/metabolismo , Roniviridae/metabolismo , Proteínas do Envelope Viral/metabolismo , Sequência de Aminoácidos , Animais , Regulação da Expressão Gênica , Penaeidae/metabolismo , Penaeidae/virologia , Vírus de RNA/fisiologia , Proteínas Recombinantes/metabolismo , Roniviridae/fisiologia , Técnicas do Sistema de Duplo-HíbridoRESUMO
BACKGROUND: Fortilin negatively regulates apoptosis and is overexpressed in cancer. However, the role of fortilin in mammalian development is not clear. METHODS AND RESULTS: In order to evaluate the physiological role of fortilin in vivo, we performed a targeted disruption of the fortilin gene in mice. Fortilin(+/-) mice have the ability to survive and exhibit normal growth, while fortilin(-/-) mice are embryonically lethal around the 3.5 days post-coital (dpc). Cultured blastocysts from fortilin(+/-) embryos undergo normal outgrowth to produce inner cell mass (ICM) and trophoblasts (TB), while ICM of fortilin(-/-) embryos either fails to outgrow or prematurely disintegrates. Mouse embryonic fibroblasts (MEF) derived from fortilin(+/-) embryos are more susceptible to noxious stimuli than are wild type embryos. It has been consistently shown in Xenopus embryos that the depletion of fortilin's message severely compromises the formation of neural tissue, even in the brain, while overexpression of fortilin induces the partial double body axis in embryos and is capable of blocking BMP4-induced transcription of Vent1, Vent2, and Msx1 genes. This suggests that fortilin is an inhibitor of the BMP pathway. Strikingly, when fortilin levels are reduced by siRNA, BMP4 causes MEF to undergo extensive DNA-fragmentation, while DNA fragmentation is minimal in the presence of fortilin. In addition, BMP4 induces more Msx2 in the absence of fortilin than in its presence. Furthermore, Msx2 overexpression causes MEF to undergo apoptotic cell death. CONCLUSION: We conclude that in early phase of development, fortilin functions as an inhibitor of the BMP pathway. The presence of fortilin in the very early stages of development is required for the survival of embryos. GENERAL SIGNIFICANCE: Abnormalities in the fortilin gene may be associated with early pregnancy loss.
Assuntos
Biomarcadores Tumorais/deficiência , Proteínas Morfogenéticas Ósseas/metabolismo , Perda do Embrião/metabolismo , Transdução de Sinais , Animais , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Blastocisto/citologia , Blastocisto/metabolismo , Proteína Morfogenética Óssea 4/metabolismo , Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Cruzamentos Genéticos , Perda do Embrião/patologia , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Fertilidade , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Saúde , Humanos , Camundongos , Camundongos Mutantes , Neurônios/citologia , Neurônios/metabolismo , Análise de Sobrevida , Proteína Tumoral 1 Controlada por Tradução , Xenopus/embriologia , beta-Galactosidase/metabolismoRESUMO
BACKGROUND: In-stent restenosis, or renarrowing within a coronary stent, is the most ominous complication of percutaneous coronary intervention, caused by vascular smooth muscle cell (VSMC) migration into and proliferation in the intima. Although drug-eluting stents reduce restenosis, they delay the tissue healing of the injured arteries. No promising alternative anti-restenosis treatments are currently on the horizon. METHODS: In endothelium-denudated mouse carotid arteries, oral morelloflavone-an active ingredient of the Thai medicinal plant Garcinia dulcis-significantly decreased the degree of neointimal hyperplasia, without affecting neointimal cell cycle progression or apoptosis as evaluated by Ki-67 and TUNEL staining, respectively. At the cellular level, morelloflavone robustly inhibited VSMC migration as shown by both scratch wound and invasion assays. In addition, morelloflavone prevented VSMCs from forming lamellipodia, a VSMC migration apparatus. Mechanistically, the inhibition by morelloflavone of VSMC migration was through its negative regulatory effects on several migration-related kinases, including FAK, Src, ERK, and RhoA. Consistently with the animal data, morelloflavone did not affect VSMC cell cycle progression or induce apoptosis. RESULTS: These data suggest that morelloflavone blocks injury-induced neointimal hyperplasia via the inhibition of VSMC migration, without inducing apoptosis or cell cycle arrest. GENERAL SIGNIFICANCE: We propose morelloflavone to be a viable oral agent for the prevention of restenosis, without compromising effects on the integrity and healing of the injured arteries.
Assuntos
Biflavonoides/farmacologia , Garcinia/química , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Túnica Íntima/efeitos dos fármacos , Animais , Apolipoproteínas E/genética , Apoptose , Biflavonoides/isolamento & purificação , Biflavonoides/uso terapêutico , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/patologia , Estenose das Carótidas/patologia , Estenose das Carótidas/prevenção & controle , Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Quimiotaxia/efeitos dos fármacos , Hiperplasia , Masculino , Camundongos , Camundongos Knockout , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/fisiologia , Pseudópodes/efeitos dos fármacos , Túnica Íntima/patologiaRESUMO
A gene encoding a receptor for activated protein kinase C1 homolog has been isolated from the shrimp, Penaeus monodon (Pm-RACK1). The full length Pm-RACK1 cDNA has 957 bp, and an open reading frame encoding a protein of 318 amino acid residues. The protein contains seven WD40 repeats and shares approximately 78% identity with vertebrate RACK1. In adult shrimp, Pm-RACK1 transcripts were detected in all tissues. During WSSV infection, Pm-RACK1 was upregulated in hepatopancreas, stomach and hemocytes. We identified Pm-RACK1 as a specific cellular target protein for VP9, a nonstructural protein of WSSV. The interaction of these two proteins may be involved in mediating intracellular VP9 functions.
Assuntos
Penaeidae/genética , Penaeidae/virologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Proteínas Virais/metabolismo , Vírus da Síndrome da Mancha Branca 1/fisiologia , Sequência de Aminoácidos , Animais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Dados de Sequência Molecular , Penaeidae/metabolismo , Receptores de Quinase C Ativada , Receptores de Superfície Celular/química , Alinhamento de Sequência , Técnicas do Sistema de Duplo-HíbridoRESUMO
Fortilin, a 172-amino-acid polypeptide present both in the cytosol and nucleus, possesses potent anti-apoptotic activity. Although fortilin is known to bind Ca2+, the biochemistry and biological significance of such an interaction remains unknown. In the present study we report that fortilin must bind Ca2+ in order to protect cells against Ca2+-dependent apoptosis. Using a standard Ca2+-overlay assay, we first validated that full-length fortilin binds Ca2+ and showed that the N-terminus (amino acids 1-72) is required for its Ca2+-binding. We then used flow dialysis and CD spectropolarimetry assays to demonstrate that fortilin binds Ca2+ with a dissociation constant (Kd) of approx. 10 mM and that the binding of fortilin to Ca2+ induces a significant change in the secondary structure of fortilin. In order to evaluate the impact of the binding of fortilin to Ca2+ in vivo, we measured intracellular Ca2+ levels upon thapsigargin challenge and found that the lack of fortilin in the cell results in the exaggerated elevation of intracellular Ca2+ in the cell. We then tested various point mutants of fortilin for their Ca2+ binding and identified fortilin(E58A/E60A) to be a double-point mutant of fortilin lacking the ability of Ca2+-binding. We then found that wild-type fortilin, but not fortilin(E58A/E60A), protected cells against thapsigargin-induced apoptosis, suggesting that the binding of fortilin to Ca2+ is required for fortilin to protect cells against Ca2+-dependent apoptosis. Together, these results suggest that fortilin is an intracellular Ca2+ scavenger, protecting cells against Ca2+-dependent apoptosis by binding and sequestering Ca2+ from the downstream Ca2+-dependent apoptotic pathways.
Assuntos
Apoptose/fisiologia , Biomarcadores Tumorais/metabolismo , Cálcio/antagonistas & inibidores , Cálcio/metabolismo , Animais , Biomarcadores Tumorais/química , Biomarcadores Tumorais/genética , Cálcio/fisiologia , Cátions Bivalentes/metabolismo , Linhagem Celular , Regulação para Baixo/fisiologia , Líquido Intracelular/química , Líquido Intracelular/metabolismo , Camundongos , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína/fisiologia , Transdução de Sinais/fisiologia , Proteína Tumoral 1 Controlada por TraduçãoRESUMO
Shrimp exhibit a diverse response to viral infection that is manifested in drastic up- and down-regulations of a variety of genes. In our previous work, we identified syntenin of the shrimp Penaeus monodon (Pm) as a dynamic responder to white spot syndrome virus (WSSV) infection, its message being greatly upregulated in the acute phase of the infection. In order to further explore the link between Pm-syntenin and viral infection, we performed a yeast two-hybrid screening of a P. monodon cDNA library, using Pm-syntenin as bait. One of the molecules that specifically interacted with Pm-syntenin was the receptor-binding domain of alpha-2-macroglobulin (alpha2M). A GST pull-down assay showed that GST-alpha2M, but not GST alone, was capable of co-precipitating syntenin. Another GST pull-down assay showed that GST-syntenin, but not GST alone, was capable of co-precipitating alpha2M. In addition, mutant analyses showed that the N-terminal 131 amino acids of syntenin were both necessary and sufficient to bind the C-terminus receptor-binding domain of alpha2M. Furthermore, WSSV-infected Pm showed a significant upregulation of the alpha2M message, suggesting that both syntenin and its protein partner alpha2M are upregulated in the acute phase of a WSSV infection. Taken together with a previous report showing the co-localization of alpha2M and syntenin in the exosome of a dendritic cell line, it is likely that syntenin, through its interaction with alpha2M, plays an important role in the immune defense mechanisms of viral infections of shrimps.
Assuntos
Proteínas de Transporte/metabolismo , Penaeidae/metabolismo , Animais , Sequência de Bases , Biotecnologia , Proteínas de Transporte/genética , DNA/genética , Infecções por Vírus de DNA/genética , Infecções por Vírus de DNA/metabolismo , Infecções por Vírus de DNA/virologia , Expressão Gênica , Hemócitos/metabolismo , Penaeidae/genética , Penaeidae/virologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Vírus da Síndrome da Mancha Branca 1/patogenicidade , alfa-Macroglobulinas/genética , alfa-Macroglobulinas/metabolismoRESUMO
Crustacean fortilin or the product of the translationally controlled tumor protein (TCTP) gene isolated from Penaeus monodon, is well conserved and has a Ca(++) binding domain. Pm-fortilin has anti-apoptotic properties and is present at high levels during the onset of viral infections in P. monodon. The possibility of using rFortilin to protect against white spot syndrome virus (WSSV) infection was tested. Injection of shrimp with rFortilin, after infection with WSSV, resulted in 80-100% survival and detection of very low levels of WSSV by PCR, whereas in moribund samples WSSV levels were very high. This result implies that injection of recombinant rFortilin decreases viral infection by an unknown mechanism, but probably by inhibiting viral replication. Using a yeast two-hybrid screen for cellular protein partners to rFortilin we identified an unknown protein that bound to fortilin. This is a novel polypeptide of 93 amino acids with a number of XPPX signature sequences that are often reported to have a function in antiviral peptides.