β-arrestin interacts with TRAF6 to negatively regulate the NF-κB pathway in triangle sail mussel Hyriopsis cumingii

As members of arrestins family, β-arrestins are widely expressed in monocytes, macrophages, neutrophils and other immune cells. They can regulate the immune response of bodies through various ways. In the present study, a β-arrestin homolog named Hcβ-arrestin was cloned and identified from Hyriopsis cumingii. Predicted Hcβ-arrestin protein contained a conserved arrestin domain, which could be further divided into arrestin-N (39-192aa) and arrestin-C (211-365aa). Amino acid sequence alignment showed that it had the highest identity with Mytilus galloprovincialis and Mytilus edulis counterpart, which was 89.02% and 87.68%, respectively. Furthermore, real-time quantitative PCR analysis showed that the Hcβ-arrestin gene was widely expressed in the detected tissues and with the highest expression in hepatopancreas. The transcription of Hcβ-arrestin in hepatopancreas and gill of mussels was significantly up-regulated after stimulation with peptidoglycan, lipopolysaccharide (LPS) and polyinosinic polycytidylic acid. Knockdown of Hcβ-arrestin gene significantly increased the expression of some antibacterial effector genes, such as lysozyme, LPS-binding protein/bactericidal permeability increasing protein and theromacin in hepatopancreas and gills of LPS stimulated mussels, but only had little effect on TLR pathway genes. In addition, GST pull-down assay confirmed that Hcβ-arrestin can bind to HcTRAF6 protein in vitro. Dual luciferase reporter assay showed that the co-expression of HcTRAF6 and Hcβ-arrestin inhibited the activation of NF-κB reporter by HcTRAF6. These findings indicated that Hcβ-arrestins could interact with HcTRAF6 to negatively regulate the NF-κB pathway in H. cumingii.