NEMO involves in NF-κB activation by interaction with p65 and promoting its nuclear translocation in large yellow croaker (Larimichthys crocea)

Abstract

NEMO (nuclear factor-κB essential modulator) plays an important role in activating NF-κB signaling pathway, p65 is a pivotal positive-regulator of NF-κB family. However, the role of NEMO in p65-triggered immune activation in teleost is largely unknown. In the present study, the cDNA sequence of LcNEMO was identified from the large yellow croaker (Larimichthys crocea). The predicated LcNEMO protein encoded 565 amino acids, consisting of a N-terminal NEMO domain, followed by two coiled coil (CC) motifs, a CC2-leucine zipper (CC2-LZ) domain, and a C-terminal zinc finger (ZnF) domain. Quantitative PCR showed that the strongest constitutive expression of LcNEMO was detected in blood and the inductive expression of it significantly enhanced after LPS and poly I:C challenge. The effect of LcNEMO on p65, RelB and cRel associated-immune activation detected by dual-luciferase reporter system assay indicated that Lcp65-triggered NF-κB, TNF-α and IL-1β activation could be significantly enhanced by LcNEMO. Furthermore, Co-IP revealed that the protein-protein interaction was existed between LcNEMO and Lcp65. Western blot and confocal microscope observation displayed that Lcp65 nuclear translocation could be promoted by LcNEMO with a dose- and time-dependent manner, which was further verified by RNA interference of LcNEMO expression. Our findings suggest that LcNEMO may be crucial in immune response by promoting p65-mediated immune activation.