A protein inhibitor of activated STAT (CgPIAS) negatively regulates the expression of ISGs by inhibiting STAT activation in oyster Crassostrea gigas

Abstract

The protein inhibitor of activated STAT (PIAS) family proteins act as the important negative regulators in janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway, which can be also involved in regulating the expression of interferon-stimulated genes (ISGs). In the present study, a PIAS homologue (designated as CgPIAS) was identified from oyster Crassostrea gigas. The open reading frame (ORF) of CgPIAS cDNA was of 1887 bp encoding a peptide of 628 amino acid residues. The CgPIAS protein contains a conserved scaffold attachment factor A/B/acinus/PIAS (SAP) domain, a Pro-Ile-Asn-Ile-Thr (PINIT) motif, a RING-finger-like zinc-binding domain (RLD) and two SUMO-interaction Motifs (SIMs). The deduced amino acid sequence of CgPIAS shared 74.58–81.36% similarity with other PIAS family members in the RLD domain. The mRNA transcripts of CgPIAS were detected in all the tested tissues with highest level in haemocytes (32.98-fold of mantles, p < 0.001). After poly (I:C) and recombinant Interferon-like protein (rCgIFNLP) stimulation, the mRNA expression of CgPIAS in haemocytes significantly up-regulated to the highest level at 48 h (7.38-fold, p < 0.001) and at 24 h (13.08-fold, p < 0.01), respectively. Moreover, the nuclear translocation of CgPIAS was observed in haemocytes after poly (I:C) stimulation. Biolayer Interferometry (BLI) assay revealed that the recombinant protein CgPIAS-RLD could interact with the recombinant protein CgSTAT in vitro with the KD value of 3.88 × 10−8 M. In the CgPIAS-RNAi oysters, the green signals of CgSTAT protein in nucleus of haemocytes increased compared with that in NC-RNAi group, and the mRNA expression of myxovirus resistance (CgMx1), oligoadenylate synthase-like proteins (CgOASL), CgViperin and IFN-induced protein 44-like (CgIFI44L-1) in haemocytes significantly increased at 12 h after poly (I:C) stimulation, which were 2.39-fold (p < 0.05), 2.18-fold (p < 0.001), 1.74-fold (p < 0.05), and 2.89-fold (p < 0.01) of that in control group, respectively. The above results indicated that CgPIAS negatively regulated the ISG expression by inhibiting STAT activation in oyster C. gigas.