RESUMO
NEMO (nuclear factor-κB
Assuntos
NF-kappa B , Perciformes , Animais , NF-kappa B/metabolismo , Transdução de Sinais , Regulação da Expressão Gênica , Fator de Necrose Tumoral alfa/genéticaRESUMO
Toll-interacting protein (Tollip) plays an important role in the innate immune response by negative regulation of the TLR-IL-1R signaling pathway. MyD88 serves as a universal adaptor in TLR-mediated NF-κB activation. However, the regulation mechanisms of Tollip in piscine MyD88-mediated NF-κB activation is largely unknown. In the present study, the cDNA sequence of LcTollip was identified from the large yellow croaker (Larimichthys crocea). The putative LcTollip protein encoded 275 amino acid residues, containing a N-terminal TBD domain, a central C2 domain, and a C-terminal CUE domain. Quantitative PCR showed that the most predominant constitutive expression of LcTollip was detected in spleen. In addition, LcTollip transcripts enhanced significantly after LPS and poly I:C challenge (P < 0.05). Cellular localization revealed that LcTollip existed in the cytoplasm and nucleus. Furthermore, the overexpression plasmids of wild type LcTollip as well as its six domain truncated mutants of LcTollip were constructed by overlap PCR. Dual luciferase analysis showed that NF-κB activation could not be induced by overexpression of LcTollip or its domain truncated mutants alone. However, the LcMyD88-induced-NF-κB activation was significantly suppressed by overexpression with LcTollip, and the truncated mutants LcTollip-ΔTBD, LcTollip-ΔC2, LcTollip-ΔCUE and LcTollip-ΔTBDΔCUE while not by LcTollip-ΔLR and LcTollip-ΔTBDΔC2. Moreover, co-immunoprecipitation (Co-IP) assay revealed that the interaction between LcTollip and LcMyD88 was through CUE domain. More interesting, IP and immunoblotting examination of HEK293T cells co-transfected with LcMyD88, LcTollip and HA-ubiquitin showed that LcMyD88 induced a dose-dependent de-ubiquitination of LcTollip while LcTollip enhanced a dose-dependent ubiquitination of LcMyD88. However, protein degradation investigation displayed that the proteolysis and ubiquitination of LcMyD88 were not connected. Our findings suggested that the LcTollip might involve in negative regulation TLR pathway by suppressing LcMyD88-mediated immune activation and improving the ubiquitination level of LcMyD88.
Assuntos
Fator 88 de Diferenciação Mieloide , Perciformes , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Aminoácidos/metabolismo , Animais , DNA Complementar/genética , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Luciferases/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Poli I-C/farmacologia , Transdução de Sinais , Ubiquitinação , Ubiquitinas/genéticaRESUMO
Interleukin (IL)-11 is a multifunctional cytokine that exerts a series of important immunomodulatory effects and exists in many tissues and cells. A 1106-bp nucleotide sequence representing the complete cDNA of IL-11 was obtained from large yellow croaker (Larimichthys crocea), containing an open reading frame (ORF) of 603 bp encoding for 200 amino acids (aa). The predicted LcIL-11 protein included a 12aa signal peptide and a conserved IL-11 domain. The polypeptide sequence identities between LcIL-11 and its counterparts in mammals and other fish are from 84% to 92% with known fish IL-11a and 22%-27% with fish IL-11b. LcIL-11 mRNA existed in most tissues with the most predominant expression in the gill. After immune challenge, the expression levels of LcIL-11 were induced largely in vivo and in vitro, with the peak-value of 32 times as much as the control in the liver at 24 h after Vibrio parahaemolyticus injection (p < 0.05) and the greatest value of 13.9 times as much as the control in LCK cells at 12 h after poly I:C stimulation (p < 0.05). Furthermore, the overexpression vector pcDNA3.1-LcIL-11 was constructed and transfected to LCK cells. Our results showed that the transcriptional expression levels of tumor necrosis factor (TNF)-α and myxovirus resistant protein (Mx) significantly up-regulated in LCK cells after LcIL-11 overexpression (p < 0.05). However, no significant changes of IL-1ß, janus kinase (JAK)2 and signal transducers and activators of transcription (STAT)5 was detected. Our finding indicated that LcIL-11 might enhance TNF-α and antiviral protein Mx expression in large yellow croaker.
Assuntos
Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Interleucina-11/genética , Interleucina-11/imunologia , Perciformes/imunologia , Sequência de Aminoácidos , Animais , DNA Complementar/genética , Perfilação da Expressão Gênica , Brânquias/imunologia , Fatores Imunológicos , Inflamação , Perciformes/genética , Perciformes/microbiologia , Filogenia , RNA Mensageiro , Vibrio parahaemolyticusRESUMO
Phosphoglycerate kinase (EC 2.7.2.3, PGK) catalyses the reversible transfer of a phosphate group from 1,3-diphosphoglyceric acid and ADP to produce 3-phosphoglyceric acid and ATP, which represents the initial production of ATP during glycolysis; therefore, PGK is a key enzyme in the energy metabolism. To study the role of PGK in the resistance to WSSV infection in shrimp, the full-length cDNA of the PGK gene (LvPGK) from Litopenaeus vannamei was obtained by using homology cloning and RACE amplification. The tissue distribution of LvPGK and its expression changes in the main immune tissues after WSSV stimulation were obtained by quantitative real-time PCR. Furthermore, RNA interference (RNAi) was used to study the role of LvPGK in shrimp defending against WSSV infection. The results showed that the full-length cDNA sequence of LvPGK was 1855 bp, contained a 1248 bp open reading frame (ORF) encoding 415 amino acids, and included a conserved PGK domain. LvPGK presented ubiquitous expression in most examined tissues, with the most predominant expression in the muscle and the weakest expression in the intestine. LvPGK transcripts could be induced in the hemocytes and hepatopancreas by injection with WSSV. Both the replication of WSSV and the shrimp cumulative mortality decreased significantly after LvPGK knockdown (Pâ¯<â¯0.01). After challenging LvPGK RNAi shrimp with WSSV, the concentration of glucose in the hepatopancreas and muscle tissue did not show significant change; however, the content of pyruvate and lactate decreased significantly (Pâ¯<â¯0.05). Moreover, significant decreases in the expression levels of crustin, ALF1, ALF2 and ALF3 were also detected. The results suggested that LvPGK might be involved in WSSV replication by increasing host aerobic and anaerobic metabolism.
Assuntos
Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Penaeidae/genética , Penaeidae/imunologia , Fosfoglicerato Quinase/genética , Fosfoglicerato Quinase/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Perfilação da Expressão Gênica , Penaeidae/enzimologia , Fosfoglicerato Quinase/química , Filogenia , Alinhamento de Sequência , Vírus da Síndrome da Mancha Branca 1/fisiologiaRESUMO
Glycogen phosphorylase (GP, EC 2.4.1.1) catalyze the rate-limiting step in glycogenolysis in animals, forming glucose-1-phosphate from the terminal alpha-1,4-glycosidic bond. Therefore, GP plays a crucial role in carbohydrate metabolism. In the present study, the full-length cDNA sequence of GP (LvGP) was cloned from shrimp, Litopenaeus vannamei. The obtained 3242-bp LvGP cDNA sequence included a 5'-terminal untranslated region (UTR) of 48 bp, an open reading frame (ORF) of 2559 bp encoding a polypeptide of 852 amino acids (aa) and a 3'-UTR of 635 bp. The predicted LvGP protein sequence contained a typical phosphorylase domain (113-829 aa) and shared 72%-97% identities with GP from other species. Phylogenetic analysis revealed that LvGP showed the closest relationship with GP from Marsupenaeus japonicus. Tissue expression profiles showed that LvGP existed in most examined tissues, with the most predominant expression in the brain, followed by the muscles and stomach. LvGP transcripts in hepatopancreas and hemocytes were up regulated after the WSSV challenge. Furthermore, the role of LvGP in shrimp defending against WSSV infection was investigated by RNA interference (RNAi). Our findings showed that WSSV proliferation and shrimp accumulative mortality increased significantly after LvGP RNAi (Pâ¯<â¯0.01). The glycogen, glucose, and pyruvate content decreased in GP RNAi shrimp after WSSV injection, however, the lactate and ATP concentration enhanced. Moreover, lectin and anti-lipopolysaccharide factor2 (ALF2) were induced in LvGP silencing shrimp after WSSV infection, whereas the expression levels of crustin, ALF1 and ALF3 decreased. Our results suggested that the LvGP might play a crucial role in shrimp defending against WSSV infection by regulating metabolism and affecting the anti-infectious gene expression.
Assuntos
Regulação da Expressão Gênica/imunologia , Glicogênio Fosforilase/genética , Glicogênio Fosforilase/imunologia , Imunidade Inata/genética , Penaeidae/genética , Penaeidae/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Perfilação da Expressão Gênica , Glicogênio Fosforilase/química , Filogenia , Interferência de RNA , Regulação para Cima , Vírus da Síndrome da Mancha Branca 1/fisiologiaRESUMO
Transforming growth factor-ß-activating kinase 1 (TAK1) is triggered by foreign pathogenic infection and involves in proinflammatory response through the activation of nuclear factor-κB (NF-κB), which is specifically regulated by TAK1-binding protein 1 (TAB1). However, the expression and regulatory characterizations of TAK1 and TAB1 in fish immune response remain largely unknown. In the present study, the cDNA sequences of TAK1 (LcTAK1) and TAB1 (LcTAB1) were identified from large yellow croaker, Larimichthys crocea. The open reading frame (ORF) of LcTAK1 was 1725 bp in length, encoding 574 amino acids. The putative LcTAK1 protein contained a protein kinase domain and a C-terminal coiled-coil region. The ORF of LcTAB1 was 1518 bp encoding 505 amino acids. And a typical PP2Cc domain and a conserved sequence motif (PYVDFSQFYLLWGSDH) at C-terminal were identified in the predicted LcTAB1 protein. Multiple alignments showed that LcTAK1 shared 74.0-97.9% and LcTAB1 shared 37.4-95.8% sequence identities with TAK1 and TAB1 proteins from other species, respectively. Quantitative PCR analysis indicated that both LcTAK1 and LcTAB1 were broadly expressed in all examined tissues, with the most predominant expression in brain and the weakest expression in muscle, respectively. Subcellular localization revealed that both LcTAK1 and LcTAB1 expressed in the cytoplasm. In addition, LcTAK1 transcripts increased significantly in LCK cells after flagellin, LPS and poly I:C stimulation while LcTAB1 enhanced greatly after LPS and poly I:C challenge. Furthermore, the roles of them in NF-κB activation were investigated by overexpression of LcTAK1 and LcTAB1 in HEK293T cells. Our results revealed that NF-κB luciferase promoter expression could not be induced by overexpression of LcTAK1 or LcTAB1 alone, however, it could be induced by co-expression of LcTAK1 and LcTAB1 together. Moreover, the roles of LcTAK1 and LcTAB1 in immune response analysis showed that NF-κB activation enhanced significantly in co-overexpressed HEK293T cells following LPS and poly I:C stimulation. However, the expression levels of tumor necrosis factor (TNF)-α, Interleukin-6 (IL-6) and IL-8 were induced only after LPS challenge (pâ¯<â¯.05). These findings suggested that the TAK1-TAB1 complex of large yellow croaker might play an important role in pro-inflammatory cytokines and chemokine release after LPS stimulation via inducing NF-κB activation.
Assuntos
Citocinas/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Regulação da Expressão Gênica , Imunidade Inata/genética , Perciformes/genética , Perciformes/imunologia , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Peixes/química , Perfilação da Expressão Gênica/veterinária , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Lipopolissacarídeos/farmacologia , NF-kappa B/metabolismo , FilogeniaRESUMO
Examining the physiological responses of mussels to thermal stress is crucial to evaluate their biogeographic distribution and ability to adapt to a changing climate. In the present study, we investigated the effects of acute cold (8⯰C and 15⯰C) and heat (35⯰C and 42⯰C) stress on the mortality rate, reactive oxygen species (ROS) production, malondialdehyde (MDA) content, mitochondrial membrane potential (MMP) and antioxdative responses in the gill tissue of the green mussel species Perna viridis. Our results showed that cold and heat stress induced a temperature-dependent increase in mortality rate. ROS production increased significantly (pâ¯<â¯0.01) after both cold and heat stress. However, the activities of antioxidant enzymes, including SOD, CAT and GSH-Px, were greatly enhanced only after heat stress. In addition, MDA content and MMP increased significantly under both cold and heat stress. The up-regulation of Hsp70 transcripts was only detected after acute stress at 35⯰C. However, p38-MAPK phosphorylation levels increased after both cold and heat stress. In addition, a moderate activation of caspase-3 was found after mussels were exposed to 8⯰C and 42⯰C stress. Our results suggest that both extreme cold and heat stress could induce ROS production in the gill tissue of P. viridis, which might result in lipid peroxidation and mitochondria dysfunction. Antioxidative enzymes and Hsp70 might be important in the heat stress response of animals, whereas p38-MAPK might be crucial in the acute response to both cold and heat stress. However, caspase-3 activation might be very weak under both cold and heat stress.
Assuntos
Temperatura Baixa , Temperatura Alta , Estresse Oxidativo , Perna (Organismo)/fisiologia , Estresse Fisiológico , Aclimatação/fisiologia , Animais , Antioxidantes/metabolismo , Caspase 3/metabolismo , Catalase/metabolismo , Ativação Enzimática , Brânquias/metabolismo , Glutationa Peroxidase/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Peroxidação de Lipídeos , Malondialdeído/metabolismo , Potencial da Membrana Mitocondrial , Perna (Organismo)/metabolismo , Fosforilação , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
As crucial signaling transducer in Toll-like receptor (TLR) and interleukin (IL)-1 receptor (IL-1R) signaling pathway, IL-1R-associated kinase 4 (IRAK4) mediates downstream signaling cascades and plays important roles in innate and adaptive immune responses. In the present study, an IRAK4 orthologue was characterized from large yellow croaker (Larimichthys crocea), named Lc-IRAK4, with a conservative N-terminal death domain and a C-terminal protein kinase domain. The genome of Lc-IRAK4 is structured into eleven exons and ten introns. Expression analysis indicated that Lc-IRAK4 was widely expressed in tested tissues, with the highest level in liver and weakest in muscle. Additionally, in the spleen, liver tissues and blood, it could be induced by poly I:C and LPS stimulation, but not be induced by Vibrio parahemolyticus infection. Fluorescence microscopy assays revealed that Lc-IRAK4 localized in the cytoplasm in HEK 293T cells. It was also determined that Lc-IRAK4 could interact with MyD88, whereas MyD88-mediated NF-κB activation was significantly impaired when co-transfected the two in HEK 293T cells. These findings collectively indicated that although Lc-IRAK4 was evolutionarily conserved in vertebrates, the exact function especially the signaling transduction mediated by IRAK4 in fish immune response was different from that in mammals, which impaired MyD88-mediated NF-κB activation.
Assuntos
Doenças dos Peixes/genética , Proteínas de Peixes/genética , Quinases Associadas a Receptores de Interleucina-1/genética , Perciformes , Vibrioses/veterinária , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica , Imunidade Inata , Quinases Associadas a Receptores de Interleucina-1/química , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Lipopolissacarídeos/farmacologia , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Filogenia , Poli I-C/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência/veterinária , Vibrio/fisiologia , Vibrioses/genética , Vibrioses/imunologia , Vibrioses/microbiologiaRESUMO
Apoptosis inducing factor (AIF) and cytochrome c (CYC) are two mitochondrial apoptogenic factors. In the present study, the cDNA sequences of AIF (LvAIF) and CYC (LvCYC) were cloned from Pacific white shrimp, Litopenaeus vannamei. The LvAIF was 1664 bp, including a 5'-terminal untranslated region (UTR) of 154 bp, an open reading frame (ORF) of 1323 bp encoding a polypeptide of 440 amino acids (aa) and a 3' UTR of 187 bp. The LvCYC was 582 bp, including a 50 bp 5' UTR, a 315 bp ORF encoding for 104 aa, and a 217 bp 3' UTR. The deduced protein of LvAIF contained a conserved Pyr_redox and AIF_C domain at the N-terminal and the predicted LvCYC included a conservative cytochrome_C domain, respectively. Phylogenetic analysis revealed that LvAIF belonged to AIF1 subfamily and showed a close relationship with AIF1 from vertebrates and LvCYC showed the closest relationship with its counterparts from shrimp Marsupenaeus japonicus. Tissue expression profiles showed that both LvAIF and LvCYC existed in most tissues, with the most predominant expression of LvAIF in intestine, then followed muscle and the weakest expression in gill. The highest expression of LvCYC was detected in muscle, and the weakest expression was in hemocytes. Additionally, after white spot syndrome virus (WSSV) infection, the significant up-regulation of LvAIF, LvCYC and caspase 3 transcripts and the increase of pro-caspase 3 and active-caspase 3 protein were detected at most time points (P < 0.05). However, all of the three genes down-regulated in hemocytes in the early stage after WSSV infection. WSSV proliferation and shrimp mortality showed a time-dependent manner and the production of ROS in hemocytes were significantly increased at 6 and 24 h after infection. Our results showed that the apoptotic genes AIF, CYC and caspase 3 might play crucial roles in hepatopancreas, however, the production of ROS in hemocytes might be important in shrimp defense against WSSV infection.
Assuntos
Fator de Indução de Apoptose/genética , Proteínas de Artrópodes/genética , Citocromos c/genética , Regulação da Expressão Gênica , Imunidade Inata , Penaeidae/genética , Penaeidae/imunologia , Sequência de Aminoácidos , Animais , Fator de Indução de Apoptose/química , Fator de Indução de Apoptose/metabolismo , Proteínas de Artrópodes/química , Proteínas de Artrópodes/metabolismo , Sequência de Bases , Clonagem Molecular , Citocromos c/química , Citocromos c/metabolismo , DNA Complementar/genética , DNA Complementar/metabolismo , Hemócitos/imunologia , Hemócitos/virologia , Hepatopâncreas/imunologia , Hepatopâncreas/virologia , Penaeidae/metabolismo , Penaeidae/virologia , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Vírus da Síndrome da Mancha Branca 1/fisiologiaRESUMO
Interleukin-6 (IL-6) is a multifunctional inflammatory cytokine which exists in multiple tissues and cell lines. In the present study, the full-length cDNA and the genomic sequence of IL-6 (LcIL-6) were cloned from large yellow croaker, Larimichthys crocea. The full-length cDNA of LcIL-6 was 1066 base pairs (bp), containing an open reading frame (ORF) of 678 bp encoding for 225 amino acids, a 5' untranslated region (UTR) of 71 bp and a 3' UTR of 317 bp. The predicted LcIL-6 protein included a 24 amino acids (aa) signal peptide and a conserved IL-6 domain. However, the polypeptide sequence identities between LcIL-6 and its counterparts in mammals and other fish are from 12% to 45%. The genome sequence of LcIL-6 gene was composed of 2126 bp, including five exons and four introns. Phylogenetic analysis revealed that LcIL-6 showed a close relationship with the IL-6 from other bony fish. Quantitative real-time PCR (qRT-PCR) analysis revealed that LcIL-6 mRNA was expressed in most examined tissues, with the most predominant expression in stomach, followed by blood and very weak expression in other tissues. The expression levels of LcIL-6 after challenged with LPS, poly I:C and Vibrio parahaemolyticus were investigated in spleen, head-kidney and liver. LcIL-6 transcripts were induced significantly after immune challenge, with the peak-value of 33.5 times as much as the control in the head-kidney at 3 h after LPS injection (p < 0.05). Overexpression of LcIL-6 enhanced tumor necrosis factor (TNF)-α transcripts significantly (p < 0.05) in L. crocea kidney (LCK) cells. Additionally, recombinant LcIL-6 mature peptide was obtained in the supernatant of Escherichia coli BL21 (DE3). The purified recombinant LcIL-6 fusion protein was also demonstrated to improve the transcriptional expression levels of TNF-α significantly in LCK cells (p < 0.05). However, no significant changes of Mx (myxovirus resistant protein), IL-1ß, janus kinase (JAK)2, signal transducers and activators of transcription (STAT)3 and STAT5 in LCK cells was detected after LcIL-6 overexpression or recombinant LcIL-6 protein stimulation. Our results indicated that LcIL-6 might be important in large yellow croaker immune response and improve the inflammatory response by through activation TNF-α expression.
Assuntos
Proteínas de Peixes/genética , Imunidade Inata , Interleucina-6/genética , Perciformes/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Interleucina-6/química , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Especificidade de Órgãos , Perciformes/imunologia , Perciformes/metabolismo , Filogenia , Poli I-C/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência/veterinária , Vibrio parahaemolyticus/fisiologiaRESUMO
Toll-like receptor 2 (TLR2) plays an important role in innate immune responses. Here we describe the isolation and characterization of the full-length cDNA sequence of toll-like receptor 2 in large yellow croaker Larimichthys crocea (LcTLR2). The LcTLR2 cDNA contains a 5'-terminal untranslated region (5'-UTR) of 135 bp, an open reading frame (ORF) of 2478 bp encoding a polypeptide of 825 amino acid residues and a 3'-UTR of 50 bp. Subcellular localization analysis suggested that the LcTLR2-pEGFP was mainly expressed in cytoplasm. Quantitative real-time reverse transcription PCR (qRT-PCR) analysis revealed a broad expression of LcTLR2 in most examined tissues, with the most predominant expression in blood, followed by spleen, and the weakest expression in stomach. The expression levels of LcTLR2 after injection with Vibrio parahaemolyticus, Lipopolysaccharides (LPS) and poly inosinic:cytidylic (polyI:C) were investigated in spleen, head-kidney and liver. Our results showed that LcTLR2 transcripts increased significantly after all the three immune challenges (p < 0.05). However, compared with polyI:C and LPS, higher expression levels of LcTLR2 were induced in all examined tissues after V. parahaemolyticus stimulation. In addition, the expression levels of LcTLR2 after flagellin, polyI:C, peptidoglycan (PGN) and LPS challenge in LCK were investigated, our findings showed that high LcTLR2 transcripts were induced after flagellin and PGN stimulation, suggesting that LcTLR2 might play a vital role in fish defense against bacterial infection. Furthermore, compared with LPS, flagellin and peptidoglycan might play an important role in LcTLR2 induction in large yellow croaker.
Assuntos
Doenças dos Peixes/genética , Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Perciformes , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Vibrioses/veterinária , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Doenças dos Peixes/microbiologia , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Imunidade Inata/genética , Dados de Sequência Molecular , Especificidade de Órgãos , Filogenia , Reação em Cadeia da Polimerase/veterinária , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência/veterinária , Receptor 2 Toll-Like/química , Vibrioses/genética , Vibrioses/imunologia , Vibrioses/microbiologia , Vibrio parahaemolyticus/fisiologiaRESUMO
Fish is highly affected by many environmental stresses such as temperature and invasive infection. The extracellular signal-regulated kinase (ERK) pathway, part of the mitogen-activated protein kinase (MAPK) family, is found to act as crucial mediators for cell differentiation, proliferation and cell response to various stresses. In the present study, ERK2 (LcERK2) and ERK5 (LcERK2) were cloned and characterized from large yellow croaker, Larimichthys crocea. The full length cDNA sequence of LcERK2 was of 1910 bp, including an ORF of 1110bp encoding a polypeptide of 369 amino acids. The full length cDNA sequence of LcERK5 was of 3720bp, including an ORF of 3375bp encoding a polypeptide of 1124 amino acids. Multiple alignments showed that both LcERK2 and LcERK5 contained highly conserved TEY motif and S_TKc domain in MAPK family and the unique catalytic and active structures of ERK2 and ERK5. Subcellular localization revealed that both LcERK2 and LcERK5 expressed in the cytoplasm and cell nucleus. The expression of LcERK2 and LcERK5 were detected in most tissues of large yellow croaker, with the most predominant expression of LcERK2 in brain and LcERK5 in gill, and the weakest expression of LcERK2 in liver and LcERK5 in intestine, respectively. The expression levels of LcERK2 and LcERK5 after temperature stress and poly I:C and flagellin challenge were investigated in LCK (L. crocea kidney) cells. After temperature stress, significant down-regulations of LcERK2 transcripts were detected after 10 °C stress (p < 0.05) whereas LcERK2 transcripts increased significantly after 35 °C stress (p < 0.05). However, significant down-regulations of LcERK5 expression were detected at most time points after both cold and heat stress (p < 0.05). However, significant up-regulations of LcERK2 and LcERK5 transcripts were found after immune challenge (p < 0.05). Our results showed that LcERK2 transcripts enhanced after heat stress and both LcERK2 and LcERK5 transcripts could be induced by immune challenge. These findings indicated that LcERK2 might be more important in fish response to high temperature stress and both LcERK2 and LcERK5 might play an important role in fish immune response.
Assuntos
Regulação da Expressão Gênica/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 7 Ativada por Mitógeno/genética , Perciformes/genética , Estresse Fisiológico/fisiologia , Temperatura , Animais , Sequência de Bases , Núcleo Celular/metabolismo , Clonagem Molecular , Biologia Computacional , Citoplasma/metabolismo , Primers do DNA/genética , DNA Complementar/genética , Flagelina , Regulação da Expressão Gênica/imunologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Poli I-C , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNARESUMO
The p38 mitogen-activated protein kinase (p38 MAPK) is a multifunctional molecule that is involved in cellular response to various stressful stimuli. In the present study, the full-length cDNA sequence of p38 MAPK (Lcp38 MAPK) was identified from the large yellow croaker Larimichthys crocea, which encoded a polypeptide of 361 amino acid residues. The predicted Lcp38 MAPK protein contained a highly conserved Thr-Gly-Tyr (TGY) motif, a glutamate and aspartate (ED) site, a substrate binding site (Ala-Thr-Arg-Trp < ATRW>), and a serine/threonine kinase catalytic (S_TKc) domain characteristic of the MAPK family. The constitutive expression of Lcp38 MAPK was detected in most of the tissues examined with the strongest expression in intestine. Subcellular localization in LCK cells (kidney cell line from a L. crocea) revealed that Lcp38 MAPK existed in both the cytoplasm and cell nucleus. The expression of Lcp38 MAPK after temperature stress was tested in LCK cells. The results indicated that Lcp38 MAPK transcripts were significantly upregulated under both cold (10 °C) and heat stress (35 °C) (P < 0.05). Furthermore, the phosphorylation levels of p38 MAPK as well the transcriptional levels of heat shock protein 27 (HSP27) and caspase3 in LCK cells were significantly induced under thermal exposure (P < 0.05). However, the cold- and heat induced HSP27 and caspase3 expression was significantly suppressed by SB203580, a specific inhibitor of p38-MAPK (P < 0.05). These findings indicated that Lcp38 MAPK might be involved in the cellular stress response via HSP27 and caspase3 in large yellow croaker.
Assuntos
Perciformes , Proteínas Quinases p38 Ativadas por Mitógeno , Animais , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas de Choque Térmico HSP27/metabolismo , Fosforilação , Temperatura , Perciformes/genética , Perciformes/metabolismoRESUMO
IL-8 plays a crucial role in acute inflammation by recruiting and mediating neutrophils and other cells and in initiating the oxidative burst in neutrophils and inducing wound healing by promoting angiogenesis. In the present study, the full-length cDNA and genome sequence of interleukin-8 (LcIL-8) were cloned from large yellow croaker Larimichthys crocea. The LcIL-8 cDNA sequence was 931 bp, containing a 118-bp 5'-untranslated region (UTR), a 528-bp 3'-UTR and a 285-bp open reading frame (ORF) which encoded 94 amino acids. A putative signal peptide including 20 amino acid residues was found at N-terminal in LcIL-8 protein. And a small cytokine (SCY) domain showing a typical CXC chemokine gene organization was predicted in LcIL-8. The genome sequence of LcIL-8 gene was composed of 1930 nucleotides, including four exons and three introns. Quantitative real-time PCR analysis indicated a broad expression of LcIL-8 in most detected tissues, with the most predominant expression in liver. After injection with LPS, Vibrio parahaemolyticus and poly I:C, LcIL-8 expression levels showed up-regulation in head-kidney and spleen. The peak value was in the spleen with 6 times (at 6 h) greater expression than in the control after LPS injection (p < 0.05). However, LcIL-8 transcripts showed down-regulation in the liver after all the three stimulants injection. Recombinant LcIL-8 mature peptide was produced by Escherichia coli, which enhanced the production of superoxide anion in PCK cells. In addition, 5 single-nucleotide polymorphisms (SNPs) were identified in LcIL-8 gene. The results suggested that LcIL-8 might play an important role in fish's immune response, and the SNPs might be used as potential candidate molecular markers for selection for disease-resistant large yellow croaker.
Assuntos
Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Interleucina-8/genética , Interleucina-8/imunologia , Perciformes/genética , Perciformes/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/análise , DNA Complementar/genética , Escherichia coli/genética , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Perfilação da Expressão Gênica/veterinária , Regulação da Expressão Gênica , Interleucina-8/química , Interleucina-8/metabolismo , Lipopolissacarídeos/administração & dosagem , Dados de Sequência Molecular , Especificidade de Órgãos , Perciformes/metabolismo , Filogenia , Poli I-C/administração & dosagem , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/análise , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência/veterinária , Vibrio parahaemolyticus/imunologiaRESUMO
In a previous study using hemocytes from native and invasive congeners of Mytilus (Mytilus californianus and Mytilus galloprovincialis, respectively) we showed that DNA damage and cell signaling transduction processes related to the cellular stress response and apoptosis were induced by acute temperature stress. The present study extends this work by examining effects of acute heat- and cold stress on total hemocyte counts (THCs) and expression of key regulatory molecules involved in responding to stress: tumor suppressor factor (p53), cell cycle arrest activator (p21), and a DNA base excision repair enzyme (apurinic/apyrimidinic endonuclease (APE)). Hyperthermia (28 °C, 32 °C) led to significant decreases of THCs in both species. The extent of decrease in THC was temperature-, time-, and species-dependent; lower THC values were found in M. californianus, the more cold-adapted species. Western blot analyses of hemocyte extracts with antibodies specific for p53 protein, several site-specific phosphorylation states of p53, p21 protein, and APE indicated that heat- and cold (2 °C) stress induced a time-dependent activation of stress-related proteins in response to DNA damage; these stress-induced changes could govern cell cycle arrest or DNA damage repair. Our results show that the downstream regulatory response to temperature-induced cell damage may play an important role in deciding cellular fate following heat- and cold stress. Compared to M. californianus, the more warm-adapted M. galloprovincialis appears to have a higher temperature tolerance due to a lesser reduction in THC, faster signaling activation and transduction, and stronger DNA repair ability following heat stress.
Assuntos
Hemócitos/fisiologia , Mytilus/fisiologia , Transdução de Sinais/fisiologia , Estresse Fisiológico/fisiologia , Temperatura , Animais , Western Blotting , Pontos de Checagem do Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular/fisiologia , Temperatura Baixa , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Hemócitos/metabolismo , Temperatura Alta , Espécies Introduzidas , Mytilus/citologia , Mytilus/genética , Fosforilação , Especificidade da Espécie , Fatores de Tempo , Proteína Supressora de Tumor p53/metabolismoRESUMO
We investigated the effects of acute heat stress and cold stress on cell viability, lysosome membrane stability, double- and single-stranded DNA breakage, and signaling mechanisms involved in cellular homeostasis and apoptosis in hemocytes of native and invasive mussels, Mytilus californianus and Mytilus galloprovincialis, respectively. Both heat stress (28, 32°C) and cold stress (2, 6°C) led to significant double- and single-stranded breaks in DNA. The type and extent of DNA damage were temperature and time dependent, as was caspase-3 activation, an indicator of apoptosis, which may occur in response to DNA damage. Hemocyte viability and lysosomal membrane stability decreased significantly under heat stress. Western blot analyses of hemocyte extracts with antibodies for proteins associated with cell signaling and stress responses [including members of the phospho-specific mitogen-activated protein kinase (MAPK) family c-JUN NH(2)-terminal kinase (JNK) and p38-MAPK, and apoptosis executor caspase-3] revealed that heat and cold stress induced a time-dependent activation of JNK, p38-MAPK and caspase-3 and that these signaling and stress responses differed between species. The thermal limits for activation of cell signaling processes linked to the repair of stress-induced damage may help determine cellular thermal tolerance limits. Our results show similarities in responses to cold and heat stress and suggest causal linkages between levels of DNA damage at both extremes of temperature and downstream regulatory responses, including induction of apoptosis. Compared with M. californianus, M. galloprovincialis might have a wider temperature tolerance due to a lower amount of single- and double-stranded DNA damage, faster signaling activation and transduction, and stronger repair ability against temperature stress.
Assuntos
Hemócitos/citologia , Mytilus/fisiologia , Animais , Apoptose , Dano ao DNA , Hemócitos/metabolismo , Transdução de Sinais , Estresse Fisiológico , TemperaturaRESUMO
The interferon regulatory factor (IRF)3 and IRF7 are considered to play essential roles in innate immune system's antiviral responses. In this report, the full-length cDNA and genomic structure and immune response characterizations of IRF3 and IRF7 were investigated in large yellow croaker, Larimichthys crocea. The full-length cDNA of L. crocea (Lc)IRF3 was of 2204 bp, including a 5'-terminal untranslated region (UTR) of 41 bp, a 3'-terminal UTR of 774 bp and an open reading frame (ORF) of 1389 bp encoding a polypeptide of 462 amino acids residues. The full-length cDNA of LcIRF7 was of 1979 bp, including a 5'-terminal UTR of 47 bp, a 3'-terminal UTR of 636 bp and an ORF of 1296 bp encoding a polypeptide of 431 amino acids. The putative amino acid sequence of both LcIRF3 and LcIRF7 contained a typical IRF domain at the N-terminal and an IRF3 domain at the C-terminal. Furthermore, we obtained 4517 nucleotides (nt) LcIRF3 genome sequence based on the full-length cDNA, which contained 11 exons and 10 introns. The full-length genome sequence of LcIRF7 was of 3991 nucleotides, including 9 exons and 8 introns. Quantitative real-time reverse transcription PCR analysis revealed a broad expression of LcIRF3 and LcIRF7 with the most predominant expression of LcIRF3 and LcIRF7 in the liver and in the gill, respectively. The expression levels of LcIRF3 and LcIRF7 after challenged with LPS, poly I:C and Vibrio parahaemolyticus were tested in blood, spleen and liver. The results showed that the highest relative expression of LcIRF3 was in the liver at 24 h after poly I:C injection with 90 times greater than that of the non-injection group (p < 0.05). Moreover, LcIRF3 transcription increased significantly at most time point in blood and spleen tissue after poly I:C stimulation compared with that of the control group. After LPS injection, the peak value of LcIRF7 was in the liver with 207 times (at 3 h) as much as that in the control group (p < 0.05). In addition, LcIRF7 expression was significantly induced by poly I:C injection in spleen. Both LcIRF3 and LcIRF7 transcripts did not show significant change after V. parahaemolyticus stimulation. These results indicated that IRF3 and IRF7 might play an important role in large yellow croaker's defense against viral and bacterial infection.
Assuntos
Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/imunologia , Perciformes/genética , Perciformes/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Fator Regulador 3 de Interferon/química , Fator Regulador 7 de Interferon/química , Dados de Sequência Molecular , Poli I-C/farmacologia , Alinhamento de Sequência , Vibrio/fisiologiaRESUMO
Toll like receptor 5 (TLR5) plays a crucial role in the innate immune response by recognizing bacterial flagellin proteins. In the present study, the genomic and 5'-flanking sequences of LcTLR5M (membrane) and LcTLR5S (soluble) were cloned from the large yellow croaker, Larimichthys crocea. Then, their promoter activities were determined in human embryonic kidney (HEK293T) cells. LcTLR5M contained 4 exons and 3 introns, and LcTLR5S contained 2 exons and 1 intron. Bioinformatic prediction of transcription factor binding sites (TFBSs) indicated that the promoter structures were different between LcTLR5M and LcTLR5S. A dual luciferase assay showed that the deletion mutant -471 to +189 of LcTLR5M promoter possessed the greatest activity with 36 times activity of the control (P < 0.05). For LcTLR5S, two deletion mutants, -1687 to +106 and - 720 to +106, showed high promoter activity. Furthermore, site-directed mutation demonstrated that a -392 to -391 AT/GG substitution in Oct-1 binding site, and a -104 to -103 GG/TT and a -98 to -97 CC/AA substitution in the NF-κB binding site of TLR5S caused a significant decline of luciferase activity (P < 0.05). Moreover, the co-transfection of an NF-κB/p65 over-expression plasmid with wild type TLR5S (-720 to +106) resulted in an extremely significant increase of promoter activity by more than 9 times compared with the NF-kB mutant (P < 0.01). Our findings suggest that the genomic organization and promoter structure may differ between LcTLR5M and LcTLR5S. Oct1 and NF-κB binding sites might be cis-regulatory elements in the LcTLR5S promoter. NF-κB/p65 plays an important role in LcTLR5S promoter activation through binding with the NF-κB binding site.
Assuntos
NF-kappa B , Perciformes , Animais , Sítios de Ligação , Proteínas de Peixes/genética , Células HEK293 , Humanos , Imunidade Inata , NF-kappa B/genética , NF-kappa B/metabolismo , Perciformes/genética , Perciformes/metabolismoRESUMO
Calmodulin (CaM) plays an important role in calcium-dependent signal transduction pathways. In the present study, two alternative splicing isoforms of CaM (named LvCaM-A and LvCaM-B) cDNA were cloned from the Pacific white shrimp, Litopenaeus vannamei. LvCaM-A was of 1101 bp, including a 5'-terminal untranslated region (UTR) of 70 bp, a 3'-terminal UTR of 581 bp and an open reading frame (ORF) of 450 bp encoding a polypeptide of 149 amino acids with a calculated molecular weight (Mw) of 17 kDa and pI of 4.41. LvCaM-B was 689 bp, including a same 5'-UTR of 70 bp, a 3'-terminal UTR of 109 bp and an ORF of 510 bp encoding a polypeptide of 169 amino acids with a calculated Mw of 19 kDa and pI of 4.36. Both LvCaM-A and LvCaM-B contained 4 conservative EF-hand motifs. Quantitative real-time reverse transcription PCR analysis revealed LvCaM-A to be expressed in most shrimp tissues, with the predominant expression in nerve and the weakest expression in heart. However, LvCaM-B expression level was much weaker than those of LvCaM-A in all the tested tissues with main expression in hepatopancreas. The expression of LvCaM-A and LvCaM-B after challenge with Vibrio parahaemolyticus and WSSV were tested in hemocytes, hepatopancreas and nerve. The results indicated that LvCaM-A and LvCaM-B transcripts could be significantly induced in hemocytes and hepatopancreas respectively by injection with V. parahaemolyticus. The highest expression of LvCaM-A was in the hemocytes with 2.3 times (at 48 h) greater expression than in the control (p < 0.05). However, sharp down-regulation of both LvCaM-A and LvCaM-B were detected in nerve after Vibrio- and WSSV injection (p < 0.05). These results suggested that CaM might play an important role in shrimp's defense against pathogenic infection.
Assuntos
Calmodulina/genética , Calmodulina/imunologia , Penaeidae/genética , Penaeidae/imunologia , Vibrio parahaemolyticus , Vírus da Síndrome da Mancha Branca 1 , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar , Hemócitos/imunologia , Hemócitos/microbiologia , Hemócitos/virologia , Hepatopâncreas/imunologia , Hepatopâncreas/microbiologia , Hepatopâncreas/virologia , Dados de Sequência Molecular , Penaeidae/microbiologia , Penaeidae/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de SequênciaRESUMO
Toll-like receptor 3 (TLR3) plays an important role in innate immune responses. In this report, the full-length cDNA sequence and genomic structure of Pseudosciaena crocea TLR3 (PcTLR3) were identified and characterized. The full-length cDNA of PcTLR3 was of 3384 bp, including a 5'-terminal untranslated region (UTR) of 65 bp, a 3'-terminal UTR of 589 bp and an open reading frame (ORF) of 2730 bp encoding a polypeptide of 909 amino acid residues. The full-length genome sequence of PcTLR3 was composed of 5721 nucleotides, including five exons and four introns. The putative PcTLR3 protein contained a signal peptide sequence, 16 leucine-rich repeat (LRR) motifs, a transmembrane region and a Toll/interleukin-1 receptor (TIR) domain. Quantitative real-time reverse transcription PCR analysis revealed a broad expression of PcTLR3 in most tissues, with the predominant expression in liver, then intestine, and the weakest expression in blood cells. The expression of PcTLR3 after injection with poly inosinic:cytidylic (I:C) and Vibrio parahemolyticus was tested in spleen, blood cells and liver. The results indicated that PcTLR3 transcripts could be induced in the three tissues by injection with poly I:C. The highest expression was in the blood cells with 43.5 times (at 6h) greater expression than in the control (p<0.05). In addition, after V. parahemolyticus challenge, a moderate up-regulation and down-regulation of PcTLR3 was found in blood cells and liver, respectively. Our results suggested that PcTLR3 might play an important role in fish's defense against both viral and bacterial infection.