Grass carp (Ctenopharyngodon idella) DYRK2 modulates cell apoptosis through phosphorylating p53.

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.

Grass carp (Ctenopharyngodon idella) Trans-Activation-Responsive RNA-binding protein 2 (TARBP2) inhibits apoptosis by decreasing PKR phosphorylation.

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.

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.

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) PACT induces cell apoptosis and activates NF-кB via PKR.

As a dsRNA-dependent and interferon-induced protein kinase, PKR is involved in antiviral immune response and apoptosis mediated by various cytokines. In mammalian cells, PKR can also be activated in the absence of dsRNA. A PKR activator, PACT (PKR activating protein), also referred to as RAX (PKR-associated protein X) plays an important role. In recent years, with the increasing recognition of fish interferon system, PKR and PACT have been gradually revealed in fish. However, the function of fish PACT is unclear. In our previous work, we suggested that grass carp (Ctenopharyngodon idella) PACT must be involved in IRF2 and ATF4-mediated stress response pathways. In the present study, we found that the expression of C. idella PACT (CiPACT) and CiPKR were significantly up-regulated under the stimulation of LPS. It indicated that CiPACT and CiPKR may play an important role in response to LPS stimulation. In addition, the response time of CiPACT to LPS is earlier than that of CiPKR. It has also shown that overexpression of CiPACT in CIK cells can significantly enhance the level of p-eIF2α, induces apoptosis and translocation of Cip65 to nucleus from cytoplasm. To further understand the mechanism, we carried out the co-immunoprecipitation assay. It proved that the interaction of CiPACT and CiPKR made the phosphorylation of CiPKR. Overexpression of CiPACT induced the down-regulation of intracellular expression of bcl-2 and up-regulation of bax. However, in CiPKR knocked-down cells the expression of bcl-2 and bax were just the opposite. Therefore, the mechanism of fish PACT induces apoptosis and activates NF-кB is dependent on PKR.