Identification and functional characterization of NEMO in Crassostrea gigas reveals its crucial role in the NF-κB activation

Abstract NEMO (NF-κB essential modulator) is one of the important regulatory subunits of the IκB kinase (IκK) complex that controls the activation of the NF-κB signaling pathway. Here, we have identified the homolog of NEMO from the pacific oyster Crassostrea gigas. Cg NEMO harbors the conserved the IκK binding region, NEMO ubiquitin binding domain and Zinc finger domain. In terms of tissue distribution, Cg NEMO is expressed in various tissues with an observed highest expression in the hemocytes. Furthermore, infection by two related Vibrio strains significantly increased Cg NEMO expression in the hemocytes. Cell culture based luciferase reporter assays showed that Cg NEMO activates the NF-κB reporter in a dose-pendent manner. Moreover, Cg NEMO was also found to counter the IkB-dependent inhibitory effect on NF-κB activation, providing a plausible mechanism of NF-κB activation by Cg NEMO. Meanwhile, site-directed mutagenesis demonstrated that the putative ubiquitination site K535 is required for the activation of NF-κB, implying that ubiquitination of NEMO may be involved in regulating its activity. Finally, RNAi mediated knockdown of Cg NEMO in vivo significantly compromised the bacterial induction of key cytokines TNF-α and IL-17, strongly suggesting a role for Cg NEMO in acute immune defense in oyster. In conclusion, this study provides new insights into our understanding about the evolution of NEMO mediated NF-κB activation and the induction of cytokine. Our findings may provide valuable information about diseases control and management in oyster aquaculture.