Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 19 de 19
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Fish Shellfish Immunol ; 141: 109023, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37625735

RESUMO

As a member of Mex3 (muscle excess protein-3) family, Mex3B (Mex-3 RNA binding family member B) is crucial in cell proliferation and migration in mammals. In this study, an ortholog of mammalian Mex3B (denominated CiMex3B, MT276802.1) was cloned and identified in grass carp (Ctenopharyngodon idella). CiMex3B is 1578 bp in length and encodes a polypeptide of 525 amino acids. Consistent with its mammalian counterpart, CiMex3B also contains one C-terminal RING domain and two N-terminal conserved tandem KH domains. CiMex3B up-regulates the expressions of IFN1, ISG15, MX2, as well as the expressions of inflammatory cytokines such as IL6, IL8 and TNFα in response to poly(I:C). A screening test for identifying potential targets indicated that CiMex3B is associated with TLR3 and TRIF. CiMex3B co-localizes with TLR3 in the late endosome, mitochondria and endoplasmic reticulum after poly(I:C) stimulation, whereas they are rarely discovered in the lysosomes. CiMex3B serves as a positive regulator in the phosphorylation of IRF3 and induces IFN1 expression. In addition, two truncation mutants of CiMex3B (1-220 and 221-525) were constructed to better understand the molecular mechanism of CiMex3B-mediated ubiquitination of TLR3. In line with wild-type protein, CiMex3B mutant (1-220) was found mainly in the cytoplasm; however, CiMex3B mutant (221-525) resided in the cytoplasm and the nucleus as well, and it was further confirmed that CiMex3B mutant (221-525) still interacts with TLR3. We also observed that CiMex3B promotes the K63-linked ubiquitination of TLR3, while neither of the truncation mutants (1-220 or 221-525) retains this activity. To sum up, this study revealed that CiMex3B potentiates the K63-linked ubiquitination of TLR3, and then elicits the IRF3-mediated antiviral innate immune responses.


Assuntos
Carpas , Receptor 3 Toll-Like , Animais , Receptor 3 Toll-Like/genética , Carpas/genética , Carpas/metabolismo , Imunidade Inata , Citocinas/genética , Poli I-C/farmacologia , Ubiquitinação , Proteínas de Peixes , Mamíferos/metabolismo
2.
Fish Shellfish Immunol ; 127: 542-548, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35781054

RESUMO

In mammals, DYRK2 increases p53 phosphorylation level by interacting with it and then promotes cell apoptosis. However, the function of fish DYRK2 has not yet been elucidated. In this paper, we cloned and identified the coding sequence (CDS) of a grass carp DYRK2 (CiDYRK2) which is 1773 bp in length and encodes 590 amino acids. SMART predictive analysis showed that CiDYRK2 possesses a serine/threonine kinase domain. Subsequently, we used the dsRNA analog polyinosinic-polycytidylic acid (poly (I:C) and Grass carp reovirus (GCRV) to stimulate grass carp and CIK cells for different times and found that CiDYRK2 mRNA was significantly up-regulated both in fish tissues and cells. To explore the function of CiDYRK2, we carried out overexpression and knockdown experiments of CiDYRK2 in CIK cells. Real-time quantitative PCR (Q-PCR), TdT-mediated dUTP nick end labeling (TUNEL) assay and flow cytometry were used to detect the ratio of BAX/BCL-2 mRNA, the number of TUNEL positive cells, the proportion of Annexin V-positive cells respectively. The results showed that CiDYRK2 significantly up-regulated BAX/Bcl-2 mRNA ratio and increased the number of TUNEL-positive cells, as well as the proportion of Annexin V-positive cells. On the contrary, knock-down of CiDYRK2 significantly down-regulated BAX/Bcl-2 mRNA ratio in the cells. Therefore, CiDYRK2 promoted cell apoptosis. To study the molecular mechanism by which CiDYRK2 promoting cell apoptosis, subcellular localization and immunoprecipitation experiments were used to study the relationship between grass carp DYRK2 and the pro-apoptotic protein p53. The results showed that CiDYRK2 and Cip53 were located and co-localized in the nucleus. Co-immunoprecipitation experiment also showed that CiDYRK2 and Cip53 can bind with each other. We further found that DYRK2 can increase the phosphorylation level of p53. In a word, our results showed that grass carp DYRK2 induces cell apoptosis by increasing the phosphorylation level of p53.


Assuntos
Carpas , Doenças dos Peixes , Infecções por Reoviridae , Reoviridae , Animais , Anexina A5 , Apoptose , Carpas/genética , Carpas/metabolismo , Doenças dos Peixes/genética , Proteínas de Peixes/química , Mamíferos/genética , Mamíferos/metabolismo , Poli I-C/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/genética , Reoviridae/fisiologia , Proteína Supressora de Tumor p53/genética , Proteína X Associada a bcl-2/metabolismo
3.
Front Immunol ; 13: 909315, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35865536

RESUMO

As one of the Mex3 family members, Mex3A is crucial in cell proliferation, migration, and apoptosis in mammals. In this study, a novel gene homologous to mammalian Mex3A (named CiMex3A, MW368974) was cloned and identified in grass carp, which is 1,521 bp in length encoding a putative polypeptide of 506 amino acids. In CIK cells, CiMex3A is upregulated after stimulation with LPS, Z-DNA, and especially with intracellular poly(I:C). CiMex3A overexpression reduces the expressions of IFN1, ISG15, and pro-inflammatory factors IL8 and TNFα; likewise, Mex3A inhibits IRF3 phosphorylation upon treatment with poly(I:C). A screening test to identify potential targets suggested that CiMex3A interacts with RIG-I exclusively. Co-localization analysis showed that Mex3A and RIG-I are simultaneously located in the endoplasmic reticulum, while they rarely appear in the endosome, mitochondria, or lysosome after exposure to poly(I:C). However, RIG-I is mainly located in the early endosome and then transferred to the late endosome following stimulation with poly(I:C). Moreover, we investigated the molecular mechanism underlying CiMex3A-mediated suppression of RIG-I ubiquitination. The results demonstrated that Mex3A truncation mutant (deletion in the RING domain) can still interact physically with RIG-I, but fail to degrade it, suggesting that Mex3A also acts as a RING-type E3 ubiquitin ligase. Taken together, this study showed that grass carp Mex3A can interact with RIG-I in the endoplasmic reticulum following poly(I:C) stimulation, and then Mex3A facilitates the ubiquitination and degradation of RIG-I to inhibit IRF3-mediated innate antiviral immune response.


Assuntos
Carpas , Animais , Carpas/metabolismo , Imunidade Inata , Mamíferos/metabolismo , Poli I-C/metabolismo , Poli I-C/farmacologia , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
4.
Dev Comp Immunol ; 133: 104425, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35452690

RESUMO

PKR plays a significant role in IFN antiviral responses and in mediating apoptosis. Its activity is crucial for cellular antiviral and subsequent recovery. In mammalian cells, Protein Activator of the Interferon-induced Protein Kinase (PACT) and Trans-Activation-Responsive RNA-Binding Protein 2 (TARBP2) have the opposite effect on PKR activity in a dsRNA independent manner. There are some corresponding regulators of PKR in fish, too. In previous studies, we found that grass carp PACT can activate PKR in dsRNA independent manner. In this study, we tried to find out the effect of grass carp TARBP2 on PKR regulation. Grass carp TARBP2 expression is significantly increased at 6h post-poly I:C stimulation in CIK cells and grass carp tissues, indicating that it may play a role in poly I:C-mediated immune response. Then, we found that CiTARBP2 interacts with CiPKR and CiPACT, suggesting that it may regulate PKR activity by direct interaction with PKR or its regulators. Further, poly I:C promotes the phosphorylation of CiTARBP2 and enhances the interaction of CiTARBP2 and CiPKR. Finally, over-expression of CiTARBP2 decreases CiPKR phosphorylation and inhibits PKR-induced apoptosis. Therefore, our study reveals that CiTARBP2 can bind to CiPKR, CiPACT and CiTARBP2. The phosphorylated TARBP2 has stronger affinity to PKR, which results in the decrease of PKR phosphorylation and inhibition of cell apoptosis.


Assuntos
Carpas , Animais , Antivirais , Apoptose , Carpas/metabolismo , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Mamíferos/genética , Fosforilação , Poli I-C/metabolismo , RNA de Cadeia Dupla , Proteínas de Ligação a RNA/genética , eIF-2 Quinase/genética
5.
Dev Comp Immunol ; 129: 104351, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35033573

RESUMO

Subcellular localization analysis implicated that CiPRMT6 was mainly located in the nucleus, with a small part of them located in the cytoplasm. PRMT6, namely protein arginine methyltransferase 6, was first identified and demonstrated to catalyze the methylation of arginine residue on the chromatin histones in mammals. Mammalian PRMT6 usually acts as an arginine methyltransferase in the nucleus, but induces antiviral innate immune response in the cytoplasm. Nowadays, there have been few reports about PRMT6 in teleost. In this study, we investigated the potential molecular mechanisms underlying the interaction of PRMT6 expression and IFN1 response in grass carp. We first cloned and identified a grass carp PRMT6 (named CiPRMT6, MN781672.1), which is 1068bp in length encoding a deduced polypeptide of 355 amino acids. In CIK cell, CiPRMT6 expression was up-regulated upon stimulation with poly (I:C); while overexpression of PRMT6 suppressed the promoter activity of grass carp IFN1 and reduced the phosphorylation of IRF3; however, the amount of PRMT6 mutant (lack of methyltransferase domain) was increased in the cytoplasm. Our results also showed that grass carp PRMT6 and IRF3 (but not TBK1) were co-located and bound to each other in the cytoplasm. The binding of CiPRMT6 to IRF3 impairs the interaction between TBK1 and IRF3, indicating that CiPRMT6 is a negative regulator for IFN1 expression through TBK1-IRF3 signaling pathway in grass carp. In conclusion, we identified that CiPRMT6 negatively regulated IFN1 expression by inhibiting the TBK1-IRF3 interaction as well as IRF3 phosphorylation.


Assuntos
Carpas/metabolismo , Animais , Proteínas de Peixes/genética , Imunidade Inata , Fator Regulador 3 de Interferon , Interferon Tipo I/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Fosforilação , Poli I-C/imunologia , Proteínas Serina-Treonina Quinases , Proteína-Arginina N-Metiltransferases , Transdução de Sinais , Ativação Transcricional , Regulação para Cima
6.
Front Immunol ; 12: 762162, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34880860

RESUMO

Type I interferon and apoptosis elicit multifaceted effects on host defense and various diseases, such as viral infections and cancers. However, the gene/protein network regulating type I interferon and apoptosis has not been elucidated completely. In this study, we selected grass carp (Ctenopharyngodon idella) as an experimental model to investigate the modulation of RNASEK on the secretion of type I interferon and apoptosis. We first cloned two paralogs RNASEK-a and -b in grass carp, defined three exons in each gene, and found the length of both coding regions is 306 bp with 73.27% of protein homology. The protein sequences of the two paralogs are highly conserved across species. Two proteins were mainly localized in early and late endosomes and endoplasmic reticulum. Further, quantitative real-time PCR demonstrated that dsRNA poly I:C and grass carp reovirus upregulated RNASEK-a and -b in grass carp cells and tissues. Overexpression of RNASEK-a and -b individually induced type I interferon expression and the phosphorylation of IRF3/IRF7 shown by Western blot and immunofluorescent staining, increased Bax/Bcl-2 mRNA ratio, DNA fragmentations, TUNEL-positive cells, and the proportion of Annexin V-positive signals in flow cytometry, and activated eIF2α, opposite to that observed when RNASEK-a and -b were knocked down in multiple cell types. Taken together, we claim for the first time that fish paralog proteins RNASEK-a and -b enhance type I interferon secretion and promote apoptosis, which may be involved in the phosphorylation of IRF3/IRF7 and eIF2α, respectively. Our study reveals a previously unrecognized role of RNASEK as a new positive regulator of type I interferon and apoptosis.


Assuntos
Apoptose , Proteínas de Peixes/genética , Interferon Tipo I/metabolismo , Animais , Carpas , Linhagem Celular , Fator Regulador 3 de Interferon/metabolismo , Fator Regulador 7 de Interferon/metabolismo , Interferon Tipo I/genética
7.
Dev Comp Immunol ; 125: 104233, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34403683

RESUMO

Cyclic GMP-AMP synthase (cGAS) is known as a DNA sensor for the initiation of innate immune responses in human and other mammals. However, the knowledge about fish cGAS is limited. In this study, we identified two paralogs of cGAS genes from grass carp (Ctenopharyngodon idellus), namely, CicGASa and CicGASb. Grass carp cGASa and cGASb share some conservative domains with mammalian cGASs; however, cGASb contains a unique transmembrane domain. Grass carp cGASa and cGASb responded to GCRV and poly (dA:dT) infection, but they played opposite roles in the regulation of type I IFN response, i.e. cGASa served as an activator for ISGs and NF-κB in a dose-dependent manner, while cGASb acted as an inhibitor. We found that cGASa and cGASb interacted with STING. Similarly, cGASa is an activator for IRF7, but cGASb inhibited IRF7 expression. Both cGASa and STING can protect cells from GCRV infection. Grass carp cGASb inhibited cGASa-induced type I IFN response by the competitive interaction with STING, suggesting that cGASb may be a negative regulator of cGASa-STING-IRF7 axis.


Assuntos
Carpas/imunologia , Proteínas de Peixes/genética , Nucleotidiltransferases/genética , Isoformas de Proteínas/genética , Infecções por Reoviridae/imunologia , Animais , Proteínas de Peixes/metabolismo , Humanos , Imunidade Inata/genética , Fator Regulador 7 de Interferon/metabolismo , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Nucleotidiltransferases/metabolismo , Reoviridae/fisiologia
8.
Front Immunol ; 12: 808159, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-35046960

RESUMO

Post-translational modifications (PTMs), such as phosphorylation and ubiquitination, etc., have been reported to modulate the activities of IRF3 and IRF7. In this study, we found an acetyltransferase KAT8 in grass carp (CiKAT8, MW286472) that acetylated IRF3/IRF7 and then resulted in inhibition of IFN 1 response. CiKAT8 expression was up-regulated in the cells under poly I:C, B-DNA or Z-DNA stimulation as well as GCRV(strain 873) or SVCV infection. The acetyltransferase domain (MYST domain) of KAT8 promoted the acetylation of IRF3 and IRF7 through the direct interaction with them. So, the domain is essential for KAT8 function. Expectedly, KAT8 without MYST domain (KAT8-△264-487) was granularly aggregated in the nucleus and failed to down-regulate IFN 1 expression. Subcellular localization analysis showed that KAT8 protein was evenly distributed in the nucleus. In addition, we found that KAT8 inhibited the recruitment of IRF3 and IRF7 to ISRE response element. Taken together, our findings revealed that grass carp KAT8 blocked the activities of IRF3 and IRF7 by acetylating them, resulting in a low affinity interaction of ISRE response element with IRF3 and IRF7, and then inhibiting nucleic acids-induced innate immune response.


Assuntos
Proteínas de Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Histona Acetiltransferases/imunologia , Imunidade Inata/imunologia , Fatores Reguladores de Interferon/imunologia , Interferon Tipo I/imunologia , Acetilação , Animais , Carpas
9.
Fish Shellfish Immunol ; 103: 220-228, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32439513

RESUMO

As a NAD+-dependent deacetylase, SIRT1 is widely involved in apoptosis and cellular inflammation via multiple pathways such as p53, NF-кB and STAT. More and more studies have shown that p53 is the first non-histone deacetylation target of SIRT1. SIRT1-p53 axis thus plays an important role in mammalian cells. IRF9 is an important member of interferon regulator factor family and performs an important role in innate immunity against foreign virus invasion. More importantly, human IRF9 can suppress the SIRT1-p53 axis. However, the functions and relationship between IRF9 and SIRT1-p53 axis are rarely studied in fish. To this end, we made a preliminary research on the functions of grass carp (Ctenopharyngodon idella) IRF9, SIRT1 and p53 in apoptosis and innate immunity. Firstly, we cloned and identified the ORF of SIRT1 (named CiSIRT1, MN125614) from C. idella and demonstrated that CiIRF9 promoted apoptosis, while CiSIRT1 inhibited apoptosis by flow cytometry and TUNEL experiments. Next, we found the interaction between CiSIRT1 and Cip53 in vivo by co-immunoprecipitation experiments. Moreover, the colocalization analysis also showed CiSIRT1 and Cip53 were mainly distributed in nucleus. Thirdly, we got a conclusion that CiIRF9 can repress the expression of CiSIRT1, implying that CiIRF9 regulates CiSIRT1-p53 axis. Finally, CiSIRT1 mRNA level was significantly up-regulated and the expression reached the highest level at 24 h post poly (I:C) stimulation in CIK cells. So, CiSIRT1 may exert an important function in innate immunity. Furthermore, we found CiSIRT1 down-regulated the expression of CiIFN1. In summary, CiIRF9 promotes apoptosis and innate immunity by inhibiting SIRT1-p53 axis. These findings will provide a new theoretical basis for the research on teleost innate immunity.


Assuntos
Apoptose/genética , Carpas/imunologia , Proteínas de Peixes/imunologia , Imunidade Inata/genética , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/imunologia , Sirtuína 1/imunologia , Proteína Supressora de Tumor p53/imunologia , Animais , Carpas/genética , Proteínas de Peixes/genética , Regulação da Expressão Gênica/imunologia , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/genética , Sirtuína 1/genética , Proteína Supressora de Tumor p53/genética
10.
Front Immunol ; 11: 597775, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33488591

RESUMO

Accumulating evidence indicates that mammalian NIMA (never in mitosis, gene A)-related kinase 6 (NEK6) plays potential roles during the course of tumorigenesis, but little is known about NEK6 in lower vertebrates. Herein, we reported a mammalian ortholog of NEK6 in grass carp (Ctenopharyngodon idellus) (CiNEK6). Multiple alignment of amino acid sequences and phylogenetic analysis showed that CiNEK6 shares a high level of sequence similarity with its counterparts in birds. CiNEK6 was ubiquitously expressed in all tested tissues, and its expression level was increased under treatment with GCRV (dsRNA virus) or poly I:C (dsRNA analog). Q-PCR and dual-luciferase assays suggested that CiNEK6 overexpression suppressed IFN I activity in CIK cells treated with poly I:C. Knockdown of CiNEK6 resulted in a higher level of IFN I expression in CIK cells treated with poly I:C compared to those which received PBS. Interestingly, analysis of subcellular localization demonstrated that CiNEK6 protein scattered throughout the cytoplasm is gradually congregated together at the edges of karyotheca upon stimulation with poly I:C. Co-IP and co-localization assays suggested that CiNEK6 interacts with CiIRF3 after poly I:C challenge. In poly I:C-treated cells, the phosphorylation of CiIRF3 was increased by CiNEK6 knockdown, but was suppressed by CiNEK6 overexpression, suggesting that CiNEK6 decreases IFN I expression through inhibiting CiIRF3 activity. Cell viability assay, crystal violet staining, and detection of Vp5 also showed that CiNEK6 plays an inhibitory role in IRF3-mediated antiviral responses.


Assuntos
Carpas/genética , Carpas/metabolismo , Fator Regulador 3 de Interferon/metabolismo , Interferon Tipo I/metabolismo , Quinases Relacionadas a NIMA/genética , Quinases Relacionadas a NIMA/metabolismo , RNA de Cadeia Dupla , Sequência de Aminoácidos , Animais , Linhagem Celular , Expressão Gênica , Genes Reporter , Especificidade de Órgãos/genética , Poli I-C , Ligação Proteica , Transporte Proteico
11.
Dev Comp Immunol ; 102: 103475, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31437525

RESUMO

In mammals, interferon regulatory factor 5 (IRF5) can be activated by tumor necrosis factor receptor-associated factor 6 (TRAF6). Upon activation, IRF5 translocates into the nucleus, where it binds to IFN promoter and up-regulates IFN expression. However, there are few reports on the molecular mechanism by which TRAF6 up-regulates IFN expression in fish. In this study, we explored how Grass carp (Ctenopharyngodon idellus) TRAF6 initiated innate immunity by activating IRF5. We found that CiTRAF6, CiIRF5 and CiIFN1 were all significantly up-regulated in LPS-stimulated CIK cells and the expression of CiTRAF6 was earlier than the expressions of CiIRF5 and CiIFN1. These findings suggested that CiIFN1 expression might be induced by CiTRAF6 in fish. CiIFN1 expression, CiIFN1 promoter activity and CO cells viability were all significantly up-regulated in the overexpression experiments, but they were significantly down-regulated in the gene silencing experiments. This indicated that CiTRAF6, along with CiIRF5, regulated CiIFN1 expression. The localization analysis found that both CiTRAF6 and CiIRF5 located in the cytoplasm. Following LPS stimulation, CiIRF5 was observed to translocate to the nucleus. GST-pull down and co-IP experiments revealed that CiTRAF6 interacted with CiIRF5. The colocalization analysis also showed that CiTRAF6 bound with CiIRF5 in the cytoplasm. Overexpression of CiTRAF6 increased the endogenous CiIRF5, promoted its ubiquitination and nuclear translocation. In conclusion, CiTRAF6 bound to CiIRF5 in the cytoplasm, and then activated CiIRF5, resulting in up-regulating the expression of CiIFN1.


Assuntos
Carpas/imunologia , Proteínas de Peixes/metabolismo , Fatores Reguladores de Interferon/metabolismo , Interferon Tipo I/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Animais , Núcleo Celular/metabolismo , Sobrevivência Celular , Células Cultivadas , Proteínas de Peixes/genética , Imunidade Inata , Fatores Reguladores de Interferon/genética , Interferon Tipo I/genética , Lipopolissacarídeos/farmacologia , Ligação Proteica , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Fator 6 Associado a Receptor de TNF/genética , Ativação Transcricional/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacos
12.
Fish Physiol Biochem ; 46(1): 417-428, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31758371

RESUMO

As a member of the Cap 'n' Collar (CNC) family, NRF2 contains a basic leucine zipper (bZip) and can regulate the downstream target gene heme oxygenase 1 (HO-1) in response to oxidative stress. In the present study, a grass carp (Ctenopharyngodon idella) NRF2 ORF was cloned and identified. The largest ORF (1782 bp) encodes a polypeptide of 593 amino acids. The deduced amino acid sequence of grass carp NRF2 (CiNRF2) contains a well-conserved DNA-binding domain (BRLZ domain). Phylogenetic tree analysis revealed that CiNRF2 has a closer evolutionary relationship with other fish counterparts. After CIK (C. idellus kidney) cells were persistently stimulated with tunicamycin (TM), CiNRF2 was significantly upregulated from 12 to 36 h. Then, the expression was dropped at 48 h post-infection. Additionally, when TM or TG (thapsigargin) stimulated CIK cells, overexpression of CiNRF2 in cells downregulated the expression of Bip mRNA, a marker protein of oxidative stress, suggesting that fish NRF2 can alleviate the oxidative stress level induced by TM or TG. To study the protective mechanism of fish NRF2, the DNA sequences of CiNRF2 and CiATF4 (grass carp ATF4) were separately sub-cloned into the expression vectors pEGFP and pCMV-Flag for co-immunoprecipitation and GST pull-down assays. These assays showed that CiNRF2 can combine with CiATF4 through its Neh1 domain. Meanwhile, we cloned grass carp HO-1 promoter sequence and constructed the recombinant plasmid of pGL3-HO-1. Soon afterwards, pGL3-HO-1 was co-transfected into grass carp ovary (CO) cells with pcDNA3.1-CiNRF2 or pcDNA3.1-CiATF4, respectively. The results showed that the luciferase activity of pGL3-HO-1 in the overexpressed CiNRF2 plus CiATF4 cells was significantly increased, along with the increase of cell viability (~ 133%). However, when HO-1 was knocked down in cells, CiNRF2 was unable to perform its function. These results demonstrated that CiNRF2 was effective in protecting grass carp against the oxidative stress induced by TM and increasing cell viability by upregulating HO-1 expression.


Assuntos
Carpas/fisiologia , Proteínas de Peixes/genética , Heme Oxigenase-1/genética , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo/fisiologia , Animais , DNA Complementar , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica , Heme Oxigenase-1/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , RNA Mensageiro
13.
Fish Shellfish Immunol ; 92: 706-711, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31276789

RESUMO

Recently, studies have shown that IκB kinase ß (IKKß), a critical kinase in the nucleus factor kappa-B (NF-κB) pathway, participates in inflammatory responses associated with unfolded protein response (UPR) and plays an important role in ER stress-induced cell death. The unfolded protein response (UPR), which is a regulatory system to restore cellular homeostasis in the endoplasmic reticulum (ER), such as oxidative stress, bacterial infection, and virus invasion. The UPR pathways have been reported to be involved in immune responses in mammals, including the classical NF-κB pathway. However, the molecular mechanism of their crosstalk remains to be elucidated. Previously, we demonstrated that IKKß also has some conserved functions between fish and human, as grass carp (Ctenopharyngodon idella) IKKß (CiIKKß) can activate NF-κB pathway. In this study, we found that CiIKKß level in nucleus was elevated under ER stress and CiIKKß can interact with grass carp X-box-binding protein 1 (CiXBP1S), a key transcription factor in UPR. Consistently, fluorescent histochemical analysis of grass carp kidney (CIK) cells indicated that CiIKKß and CiXBP1S colocalized under ER stress. Furthermore, overexpression of CiIKKß in CIK cells enhanced ER stress tolerance by regulating UPR signaling and resulted in the significant increase of cell viability.


Assuntos
Carpas/genética , Estresse do Retículo Endoplasmático , Proteínas de Peixes/genética , Regulação da Expressão Gênica/imunologia , Quinase I-kappa B/genética , Proteína 1 de Ligação a X-Box/genética , Animais , Carpas/imunologia , Núcleo Celular/genética , Sobrevivência Celular , Proteínas de Peixes/imunologia , Quinase I-kappa B/imunologia , Resposta a Proteínas não Dobradas , Proteína 1 de Ligação a X-Box/imunologia
14.
Front Immunol ; 10: 582, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30984174

RESUMO

PKZ is a fish-specific protein kinase containing Zα domains. PKZ is known to induce apoptosis through phosphorylating eukaryotic initiation factor 2α kinase (eIF2α) in the same way as double-stranded RNA-dependent protein kinase (PKR), but its exact role in detecting pathogens remains to be fully elucidated. Herein, we have found that PKZ acts as a fish-specific DNA sensor by initiating IFN expression through IRF3- or ISGF3-like mediated pathways. The expression pattern of PKZ is similar to those of innate immunity mediators stimulated by poly (dA:dT) and poly (dG:dC). DNA-PKZ interaction can enhance PKZ phosphorylation and dimerization in vitro. These findings indicate that PKZ participates in cytoplasmic DNA-mediated signaling. Subcellular localization assays have also shown that PKZ is located in the cytoplasm, which suggests that PKZ acts as a cytoplasmic PRR. Meanwhile, co-IP assays have shown that PKZ can separately interact with IRF3, STING, ZDHHC1, eIF2α, IRF9, and STAT2. Further investigations have revealed that PKZ can activate IRF3 and STAT2; and that IRF3-dependent and ISGF3-like dependent mediators are critical for PKZ-induced IFN expression. These results demonstrate that PKZ acts as a special DNA pattern-recognition receptor, and that PKZ can trigger immune responses through IRF3-mediated or ISGF3-like mediated pathways in fish.


Assuntos
Proteínas de Peixes/imunologia , Fator Regulador 3 de Interferon/imunologia , Fator Gênico 3 Estimulado por Interferon/imunologia , Proteínas Quinases/imunologia , Animais , Carpas , Células Cultivadas , Feminino , Proteínas de Peixes/genética , Humanos , Imunidade Inata , Rim/citologia , Ovário/citologia , Proteínas Quinases/genética
15.
Fish Shellfish Immunol ; 88: 606-618, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30885743

RESUMO

SAMHD1 is an innate immunity restriction factor that inhibits virus infection through IRF3-mediated antiviral and apoptotic responses. Fish SAMHD1 shares some similar properties with those in mammals. In this study, a SAMHD1 orthologue from grass carp (Ctenopharyngodon idellus) was cloned and characterized. The full-length cDNA of CiSAMHD1 is 2792 bp with an ORF of 1884 bp encoding a polypeptide of 627 amino acids. Multiple alignments showed that SAMHD1 is highly conserved among different species. Phylogenetic tree analysis revealed that CiSAMHD1 shared a high degree of homology with Sinocyclocheilus rhinocerous SAMHD1. Expression analysis indicated that CiSAMHD1 was widely expressed in all tissues tested including the brain, eyes, spleen, gill, intestine, liver, heart and kidney. It was significantly up-regulated in spleen, liver and intestines after treatment with poly I:C. Also, CiSAMHD1 can be induced following stimulation with recombinant IFN in CIK cells. The promoter sequence of CiSAMHD1 was identified to explore the mechanism underlying the transcriptional regulation of CiSAMHD1. The promoter sequence of CiSAMHD1 (1370 bp) consists of IRF1, IRF3, IRF9 and p65 binding elements. Gel mobility shift assay also showed that IRF1, IRF3, IRF9 and p65 prokaryotic proteins can separately interact with CiSAMHD1 promoter. Dual luciferase assay and q-PCR suggested that the promoter of CiSAMHD1 can be activated by the overexpression of CiIRF3 and CiIRF9, but cannot be triggered by CiIRF1 and Cip65. In contrast, knockdown of CiIRF3 or CiIRF9 inhibits the transcription of CiSAMHD1. Intriguingly, CCK assay suggested that CiSAMHD1 decreased cell viability. TUNEL apoptosis assay and Hoechst 33258 staining assay indicated that apoptosis is induced by the overexpression of CiSAMHD1. Crystal violet staining, detection of two GCRV genes (vp3 and vp5) and viral titration showed that CiSAMHD1 can suppress the proliferation of grass carp reovirus (GCRV) in CIK cells.


Assuntos
Apoptose , Carpas/genética , Proteínas de Peixes/genética , Orthoreovirus/fisiologia , Proteína 1 com Domínio SAM e Domínio HD/genética , Replicação Viral , Animais , Proliferação de Células , Clonagem Molecular , DNA Complementar/genética , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica , Imunidade Inata , Fator Regulador 3 de Interferon/genética , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/genética , Regiões Promotoras Genéticas , Infecções por Reoviridae , Proteína 1 com Domínio SAM e Domínio HD/metabolismo
16.
Dev Comp Immunol ; 97: 45-56, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30905684

RESUMO

In mammals, toll-like receptor 3 (TLR3) is capable of recognizing double-stranded RNA and then initiates transcription of IFN-ß. TLR3 can activate the innate immune system by phosphorylating extracellular signal-regulated kinase 1 (ERK1) in the mitogen-activated protein kinase (MAPK) pathway. As a downstream signaling protein of ERK1, ribosomal protein S6 kinase alpha 3 (RSK2) is activated through the "classical" MAPK pathway. So RSK2 plays a critical role in response to innate immune system induced by TRL3. However, the innate immune mechanism of RSK2 remains indistinct in fish. In this study, we cloned and characterized a full length cDNA sequence of RSK2 from Ctenopharyngodon idella (named CiRSK2, MH844551). The full length cDNA of CiRSK2 is 3930 bp with a coding sequence of 2202 bp encoding a polypeptide of 734 amino acids. The expression of CiRSK2 was ubiquitous and significantly up-regulated under the stimulation of poly (I:C) in eight different tissues of C. idella and C. idella kidney cells (CIK). In addition, poly (I:C) stimulation also up-regulated the expression of CiERK1 mRNA in CIK cells and the phosphorylation of CiERK1. We also demonstrated that the activated CiERK1 interacted with CiRSK2 by CO-IP assay and immunofluorescence assay. To further investigate the relationship between CiRSK2 and CiERK1, we performed subcellular localization of CiRSK2 at different periods of CiERK1 stimulation. The result showed that CiERK1 can make CiRSK2 enter the nucleus. Subsequently, we found that CiRSK2 increased the transcriptional level of CiBCL-2 and protein level of CiBCL-2 significantly. Then cell apoptosis was inhibited to a certain extent. Overall, our results suggested that CiRSK2 plays important roles in fish innate immunity and is able to inhibit cell apoptosis by up-regulating CiBCL-2.


Assuntos
Apoptose/genética , Carpas/genética , Proteínas de Peixes/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Animais , Apoptose/efeitos dos fármacos , Carpas/metabolismo , Células Cultivadas , Feminino , Proteínas de Peixes/classificação , Proteínas de Peixes/metabolismo , Perfilação da Expressão Gênica/métodos , Células HEK293 , Humanos , Rim/citologia , Ovário/citologia , Filogenia , Poli I-C/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/classificação , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo
17.
Fish Shellfish Immunol ; 87: 438-448, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30685465

RESUMO

IL-1R-associated kinase 4 (IRAK4), a central TIR signaling mediator in innate immunity, can initiate a cascade of signaling events and lead to induction of inflammatory target gene expression eventually. In the present study, we cloned and characterized an IRAK4 orthologue from grass carp (Ctenopharyngodon idella). The full length cDNA of CiIRAK4 was 2057 bp with an ORF of 1422 bp encoding a polypeptide of 472 amino acids. Multiple alignments showed that IRAK4s were highly conserved among different species. Phylogenetic tree analysis revealed that CiIRAK4 shared high homologous with zebra fish IRAK4. Expression analysis indicated that CiIRAK4 was widely expressed in all tested tissues. It was significantly up-regulated after treatment with poly I:C, especially obvious in liver and spleen. Also, CiIRAK4 could be induced by poly I:C and LPS in CIK cells. Fluorescence microscopy assays showed that CiIRAK4 localized in the cytoplasm. RNAi-mediated knockdown and overexpression assays indicated that CiIRAK4 might have little effect on NF-kappa B p65 translocation from cytoplasm to nucleus, indicating that CiIRAK4 was dispensable for activation of NF-kappa B p65. In addition, IRAK4 promoted IRF5 nuclear translocation, which has nothing to do with the interaction between IRAK4 and IRF5. It suggested that fish IRAK4 kinase regulated IRF5 activity through indirect ways.


Assuntos
Carpas/genética , Carpas/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Quinases Associadas a Receptores de Interleucina-1/genética , Quinases Associadas a Receptores de Interleucina-1/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Quinases Associadas a Receptores de Interleucina-1/química , Lipopolissacarídeos/farmacologia , Filogenia , Poli I-C/farmacologia , Alinhamento de Sequência/veterinária
18.
Fish Shellfish Immunol ; 80: 521-527, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29960062

RESUMO

In mammals, IFN regulatory factor (IRF) 7 is a central regulator of IFN-α expression in response to variable pathogenic infections. There are several pathogenic sensors involved in monitoring pathogen intrusion in mammals. These sensors trigger IRF7-mediated responses through different pathways. TANK-binding kinase 1 (TBK1) is a critical mediator of IRF7 activation upon pathogen infection. In fish, there are many reports on TBK1, IRF3 and IRF7, especially on TBK1-IRF3 signaling pathway. However, it is not very clear how TBK1-IRF7 works in innate immune signaling pathway. In this study, we explored how TBK1 up-regulates IFN, ISG expression, and how TBK1 initiates innate immune response through IRF7 in fish under lipopolysaccharides (LPS) stimulation. After stimulation with LPS, grass carp IRF3 and IRF7 transcriptions were up-regulated, indicating they participate in TLR-mediated antiviral signaling pathway. It is interesting that the response time of grass carp IRF3 to LPS was earlier than that of IRF7. In addition, IRF7 rather than IRF3 acted as a stronger positive regulator of IFN and ISG transcription in Ctenopharyngodon idella kidney cells (CIKs). It is suggested the potential function differentiation between IRF3 and IRF7 upon LPS infection in fish. Dual luciferase assays also showed that overexpression of grass carp IRF7 and TBK1 up-regulated the transcription level of IFN and PKR. However, knockdown of IRF7 inhibits ISG expression, suggesting that grass carp TBK1 regulates the transcription via IRF7. Co-immunoprecipitation and GST pull-down assays proved the binding of grass carp IRF7 to TBK1. Furthermore, grass carp TBK1 can promote the nuclear translocation of IRF7. The results indicated that grass carp TBK1 can bind directly to and activate IRF7.


Assuntos
Carpas/imunologia , Proteínas de Peixes/imunologia , Fator Regulador 7 de Interferon/imunologia , Proteínas Serina-Treonina Quinases/imunologia , Animais , Carpas/genética , Células Cultivadas , Proteínas de Peixes/genética , Células HEK293 , Humanos , Imunidade Inata , Fator Regulador 7 de Interferon/genética , Rim/citologia , Lipopolissacarídeos/farmacologia , Proteínas Serina-Treonina Quinases/genética , Regulação para Cima
19.
Dev Comp Immunol ; 86: 138-146, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29753769

RESUMO

As a host limiting factor, Sterile Alpha Motif and Histidine-Aspartate Domain 1 protein (SAMHD1) is associated with IRF3-mediated antiviral and apoptotic responses in mammals. However, the antiviral mechanism of SAMHD1 remains indistinct in fish. In this study, we found the expression of Ctenopharyngodon idella SAMHD1 (MF326081) was up-regulated after transfection with poly I:C (dsRNA analog), B-DNA or Z-DNA into C. idella kidney cells (CIKs), but these expression profiles had no obvious change when the cells were incubated with these nucleic acids. These data may indicate that CiSAMHD1 participates in the intracellular PRR-mediated signaling pathway rather than extracellular PRR-mediated signaling pathway. Subcellular localization assay suggested that a part of over-expressed CiSAMHD1 were translocated from nuclear to cytoplasm when C. idella ovary cells (COs) were transfected with poly I:C, B-DNA or Z-DNA. Nucleic acid pulldown assays were performed to investigate the reason for nuclear-cytoplasm translocation of CiSAMHD1. The results showed that CiSAMHD1 had a high affinity with B-DNA, Z-DNA and ISD-PS (dsRNA analog). In addition, co-IP assays revealed the interaction of CiSAMHD1 with CiSTING (KF494194). Taken together, all these results suggest that grass carp SAMHD1 performs as an activator for innate immune response through STING-mediated signaling pathway.


Assuntos
Proteínas de Peixes/metabolismo , Interferons/metabolismo , Animais , Carpas/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Imunidade Inata/fisiologia , Rim/metabolismo , Ácidos Nucleicos/metabolismo , Poli I-C/metabolismo , Transporte Proteico/fisiologia , RNA de Cadeia Dupla/metabolismo , Transdução de Sinais/fisiologia , Regulação para Cima/fisiologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...