Grass carp (Ctenopharyngodon idella) NIK up-regulates the expression of IL-8 by activating the NF-κB canonical pathway.

NIK (NF-κB inducing kinase) belongs to the mitogen-activated protein kinase family, which activates NF-κB and plays a vital role in immunology, inflammation, apoptosis, and a series of pathological responses. In NF-κB noncanonical pathway, NIK and IKKα have been often studied in mammals and zebrafish. However, few have explored the relationship between NIK and other subunits of the IKK complex. As a classic kinase in the NF-κB canonical pathway, IKKß has never been researched with NIK in fish. In this paper, the full-length cDNA sequence of grass carp (Ctenopharyngodon idella) NIK (CiNIK) was first cloned and identified. The expression level of CiNIK in grass carp cells was increased under GCRV stimuli. Under the stimulation of GCRV, poly (I:C), and LPS, the expression of NIK in various tissues of grass carp was also increased. This suggests that CiNIK responds to viral stimuli. To study the relationship between CiNIK and CiIKKß, we co-transfected CiNIK-FLAG and CiIKKB-GFP into grass carp cells in coimmunoprecipitation and immunofluorescence experiments. The results revealed that CiNIK interacts with CiIKKß. Besides, the degree of autophosphorylation of CiNIK was enhanced under poly (I:C) stimulation. CiIKKß was phosphorylated by CiNIK and then activated the activity of p65. The activity change of p65 indicates that NF-κB downstream inflammatory genes will be functioning. CiNIK or CiIKKß up-regulated the expression of IL-8. It got higher when CiNIK and CiIKKß coexisted. This paper revealed that NF-κB canonical pathway and noncanonical pathway are not completely separated in generating benefits.

Grass carp (Ctenopharyngodon idella) deacetylase SIRT1 targets p53 to suppress apoptosis in a KAT8 dependent or independent manner.

Sirtuin1 (SIRT1) is known as a deacetylase to control various physiological processes. In mammals, SIRT1 inhibits apoptotic process, but the detailed mechanism is not very clear. Here, our study revealed that grass carp (Ctenopharyngodon idella) SIRT1 (CiSIRT1, MN125614.1) inhibits apoptosis through targeting p53 in a KAT8-dependent or a KAT8-independent manner. In CIK cells, CiSIRT1 over-expression results in significant decrease of some apoptotic gene expressions, including Bax/Bcl2, caspase3 and caspase9, whereas CiKAT8 or Cip53 facilitates the induction of apoptosis. Because CiSIRT1 separately interacted with CiKAT8 and Cip53, we speculated that CiSIRT1 blocked apoptosis may be by virtue of KAT8-p53 axis or directly by p53. In a KAT8-dependent manner, CiSIRT1 interacted with CiKAT8, then reduced the acetylation of CiKAT8 and subsequently promoted its degradation. Then, CiKAT8 acetylated p53 and induced p53-mediated apoptosis. MYST domain of CiKAT8 was critical in this pathway. In a KAT8-independent manner, CiSIRT1 also inhibited p53-induced apoptosis by directly deacetylating p53 and promoting the degradation of p53. Generally, these findings uncovered two pathways in which CiSIRT1 decreases the acetylation of p53 via a KAT8-dependent or a KAT8-independent manner.

Grass carp (Ctenopharyngodon idella) Mex3B positively regulates innate immunity by promoting the K63-linked ubiquitination of TLR3.

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.

Grass Carp Mex3A Promotes Ubiquitination and Degradation of RIG-I to Inhibit Innate Immune Response.

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.

Grass carp PRMT6 negatively regulates innate immunity by inhibiting the TBK1/IRF3 binding and cutting down IRF3 phosphorylation level.

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.

Cloning and functional analysis of SNX8 from grass carp (Ctenopharyngodon idellus).

Sorting nexin 8 (SNX8), a member of sorting nexin protein family, plays important roles in endocytosis, endosomal sorting, and innate immune response. To date, a few homologs of SNX8 have been found in fish except in mammals. In this study, a teleost SNX8 cDNA was identified from grass carp (Ctenopharyngodon idellus). CiSNX8 was up-regulated significantly after infection with poly I:C or GCRV. We found that SNX8 was mainly distributed in the endoplasmic reticulum (ER) in CIK cells. Further analysis indicated that CiSNX8 might negatively regulate RLR signaling pathway that is quite distinct from mammalian SNX8. In addition, CiSNX8 could interact with MAVS, STING, TBK1, IRF3 and IRF7. Either wild type CiSNX8 or mutants of N-terminal PX domain (aa 1-245) and C-terminal BAR domain (aa 256-519) could associate with STING. These results suggested that fish SNX8 participated in innate immune response through different molecular mechanisms.

Ethoprophos induces cardiac toxicity in zebrafish embryos.

Ethoprophos is an effective and widely pesticide that used in controlling nemathelminth and soil insect. However, ethoprophos has been frequently detected in environment and freshwater. The potential toxicity to aquatic organisms is still not be explored. In this study, zebrafish embryo model was used to evaluated the toxicity of ethoprophos during cardiovascular developmental process of zebrafish. Zebrafish embryos were separately exposed to 10 mg/L, 20 mg/L, 30 mg/L, 40 mg/L and 50 mg/L of ethoprophos exposure at 96 h post-fertilization (hpf), which induced cardiac defects, such as low heart rate, pericardium edema and long SV-BA distance, but had no influence to vascular development. Mechanistically, the expression of cardiac-related genes were abnormal. Moreover, ethoprophos exposure significantly increased oxidative stress in zebrafish embryos by inhibiting the production of antioxidant enzyme (SOD) and activating reactive oxygen species. Expectedly, some apoptosis genes were induced and the apoptotic cardiomyocytes were detected by acridine orange staining. In addition, ethoprophos exposure also inhibited the expression of genes in wnt signaling pathway, such as ß-catenin, Axin2, GSK3ß and Sox9b. BML284, an activator of wnt signaling pathway, can rescue the cardiotoxic effect of embryos. These results indicated that oxidative stress and blocking wnt signaling pathway were molecular basis of ethoprophos-induced injure in zebrafish. Generally, our study showed that ethoprophos exposure led to severe cardiotoxicity to zebrafish embryo.

Grass carp (Ctenopharyngodon idellus) PIAS1 inhibits innate immune response via interacting with STAT1.

Protein inhibitor of activated signal transducer and activator of transcription (PIAS) family protein involved in gene transcriptional regulation acts as negative regulator in Janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling pathway. But until now, the roles of PIAS in fish are not clear. In this study, we identified the two mammalian PIAS1 orthologs from Ctenopharyngodon idellus, namely CiPIAS1a and CiPIAS1b, respectively. They can respond to the stimulation from Polyribocytidylic acid (Poly I:C), Grass Carp Reovirus (GCRV) and Lipopolysaccharides (LPS) respectively, so we suggested that they could participate in interferon (IFN)-mediated antiviral and antibacterial immune response. The subcellular localization and nuclear cytoplasm extraction showed that CiPIAS1a and CiPIAS1b were mainly distributed in the nucleus. In addition, Co-IP showed that they separately inhibited the phosphorylation of STAT1 via interacting with it, which leads to the reduction of IFN1 expression.

Grass Carp (Ctenopharyngodon idella) KAT8 Inhibits IFN 1 Response Through Acetylating IRF3/IRF7.

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.

Ctenopharyngodon idellus DDX41 initiates IFN I and ISG15 expression in response to GCRV infection.

As a member of DExD/H-box helicase family, DDX41 (DEAD box polypeptide 41) acts as an intracellular DNA sensor that induces type I IFN expression in mammals. Fish DDX41 shares some similar properties with the mammalian counterparts. In this study, a DDX41 orthologous gene from grass carp (Ctenopharyngodon idellus) (CiDDX41) was cloned and characterized. The ORF of CiDDX41 encodes a polypeptide of 614 amino acids. Multiple alignments showed that DDX41 is highly conserved among different species. Phylogenetic tree analysis revealed that CiDDX41 shares a high degree of homology with Sinocyclocheilus rhinocerous DDX41. CiDDX41 is highly expressed in kidney, intestines, liver and spleen. Their expressions are up-regulated more obviously after the treatment with GCRV. Over-expression of CiDDX41 in CIK cells increases the transcription level of grass carp IFN I and ISG15. On the contrary, knockdown of CiDDX41 inhibits the IFN I and ISG15 transcription. Moreover, a part of CiDDX41 translocates from the nuclear to cytoplasm to interact with grass carp STING (CiSTING). In CIK cells, overexpression of CiDDX41 and CiSTING can promote the phosphorylation and nuclear-cytoplasm translocation of grass carp IRF7 (CiIRF7) and then acutely up-regulate the IFN I and ISG15 expression. However, the knockdown of CiDDX41 inhibits the phosphorylation IRF7. Taken together, all these results above suggested that CiDDX41 performs as an activator for innate immune through STING-IRF7 mediated signaling pathway.

The immunotoxicity and neurobehavioral toxicity of zebrafish induced by famoxadone-cymoxanil.

As a new protective and therapeutic fungicide, studies on famoxadone-cymoxanil are rare, and its toxicity to aquatic organisms has not been reported. In the present study, zabrafish embryos were exposed to several concentrations of famoxadone-cymoxanil at 10 hpf. Then, the changes of their shape, heart rate, development and function of innate and adaptive immune cells, oxidative stress, apoptosis, the expression of apoptosis-related genes and immune-related genes, the locomotor behavior were observed and detected in acute toxicity of famoxadone-cymoxanil. Our studies showed that, after exposure to famoxadone-cymoxanil, zebrafish embryos had decreased heart rate, shortened body length, swollen yolk sac. Secondly, the number of innate and adaptive immune cells was significantly reduced; and neutrophil migration and retention at the injury area were inhibited, indicating the developmental toxicity and immunotoxicity of famoxadone-cymoxanil on the zebrafish. We also found that the oxidative stress related indicators of embryos were changed significantly, and apoptosis were substantially increased. Further investigation of changes of some key genes in TLR signaling including TLR4, MYD88 and NF-κB p65 revealed that the mRNA expression of these genes was up-regulated. Meanwhile, the mRNA expression of some proinflammatory cytokines such as TNF-α, IFN-γ, IL6 and IL-1ß was also up-regulated. In addition, the activity, the total distance, time and average speed were decreased along with the increase of exposure concentration. The absolute turn angle, sinuosity and the enzymatic activity of acetylcholinesterase (AChE) were also increased. These results suggested that famoxadone-cymoxanil can induce developmental toxicity, immunotoxicity and neurobehavioral toxicity in zebrafish larvae.

Grass Carp (Ctenopharyngodon idellus) NIMA-Related Kinase 6 Blocks dsRNA-Induced IFN I Response by Targeting IRF3.

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.

5-Butyl-amino-6-(4-fluoro-phen-yl)-7-oxo-1-p-tolyl-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-3-carbonitrile.

In the title compound, C(23)H(21)FN(6)O, the dihedral angle between the fluoro-phenyl and pyrimidinone rings is 75.9 (1)°, and the dihedral angle between the methyl-phenyl and pyrazole rings is 40.3 (1)°. In the crystal structure, weak C-H⋯π(arene) and C-N⋯π(arene) inter-actions and intermolecular C-H⋯N and N-H⋯O hydrogen-bonding inter-actions are present.