Characterization of a PIAS4 Homologue from Zebrafish: Insights into Its Conserved Negative Regulatory Mechanism in the TRIF, MAVS, and IFN Signaling Pathways during Vertebrate Evolution

Abstract

Members of the protein inhibitor of activated STAT (PIAS) family are key regulators of various human and mammalian signaling pathways, but data on their occurrence and functions in ancient vertebrates are limited. This study characterizes for the first time to our knowledge a PIAS4 homologue (PIAS4a) from zebrafish. Structurally, this zebrafish PIAS4a (zfPIAS4a) shares a number of conserved functional domains with mammalian PIAS4 proteins, including the scaffold attachment factor A/B/acinus/PIAS box, PINIT, and RING-finger–like zinc-binding domains and a highly acidic domain in the C-terminal region. Subcellular localization analysis shows that zfPIAS4a is a nuclear-localized protein and that the C terminus of the molecule harbors strict nuclear localization signals. Functionally, zfPIAS4a expression can be dramatically induced by the stimulation of polyinosinic-polycytidylic acid and zebrafish IFNϕ1. It acts as a critical negative regulator of the TIR domain-containing adapter inducing IFN-β, mitochondrial antiviral signaling (MAVS), and IFN signaling pathways, and it is the first PIAS protein that plays a role in the MAVS-mediated pathway to be identified. The structure and functionality of PIAS4 seem highly conserved from zebrafish to mammals, making zebrafish an attractive model for screens designed to uncover genes involved in IFN- and inflammatory cytokine-induced signaling pathways. This study provides preliminary evidence that the PIAS regulatory mechanism already existed in fish during vertebrate evolution. It presents valuable clues for improving the understanding of not only the negative regulation of cytokine signaling in fish but also the evolutionary history of the PIAS family from fish to mammals as a whole.

Fish MAVS is involved in RLR pathway-mediated IFN response

“…Recently MAVS homologous genes have been identified in several fish species, including zebrafish D. rerio [21,28], Japanese flounder P. olivaceus [26], grass carp C. idella [37], common carp Cyprinus carpio [38], and pufferfish T. nigroviridis [27]. These studies have shown that fish MAVS not only activates IFN promoter but also induces the transcription of IFN and some ISGs [21,26,28,39], and that overexpression of fish MAVS confers protection on cultured fish cells against DNA and RNA virus infection [21,26,28]. However, whether fish utilize a conserved signaling pathway involving MAVSmediated IFN response remains elusive.…”

Spring viraemia of carp virus: recent advances

“…In contrast, variant 1c, which lacks a C-terminal transmembrane domain, has no influence on either IRF3 or NFk B signalling pathways (Lad et al, 2008). MAVS orthologues have been identified in zebrafish (Xiong et al, 2012), salmon (Lauksund et al, 2009), Japanese flounder (Simora et al, 2010), spotted green pufferfish (Xiang et al, 2011) and grass carp (Wan et al, 2013). Although alternative splicing variants of MAVS have been reported in zebrafish and salmon (Biacchesi et al, 2009), their functions and mechanisms of interacting with other signalling proteins remain poorly understood.…”

Characterization of SIGIRR/IL-1R8 Homolog from Zebrafish Provides New Insights into Its Inhibitory Role in Hepatic Inflammation

“…The encoding sequences of DrMyD88, DrTLR3, and DrTLR22 were subcloned into pCMV-Tag2B (Invitrogen) or pcDNA6/myc-HisB. The plasmids of pEGFP-TRIF and pCMV-TRIF were previously constructed in our laboratory (33). The NF-kB luciferase construct was purchased from Clontech, and the pRL-TK (Renilla luciferase reporter plasmid) vector was obtained from Promega.…”

“…The empty plasmid was added to ensure that all samples received equal amounts of DNA. At 24 h after microinjection, embryos were collected for luciferase activity detection as previously described (32,33). For IFN production assay, plasmids in various combinations (pEGFP-TRIF alone, pEGFP-TRIF plus DrMO, and pEGFP-TRIF plus DrMO and DrSIGIRR mRNA) were microinjected into one-cell stage embryos.…”

“…At 24 h after microinjection, the embryos with strong and GFP expression were used for further analysis. Real-time PCR was performed to measure the mRNA levels of IFN regulatory factor (IRF)3 and IFN1 as described earlier (33,34).…”