NEDD8-conjugated Cullin4 positive regulates antimicrobial peptides expression in Eriocheir sinensis

The ubiquitin-proteasome system is involved in numerous cellular processes, such as signal transduction, autophagy, cell cycle control, embryogenesis, and regulation of immune response. Neural precursor cell expressed developmentally downregulated 8 (NEDD8) is a ubiquitin-like protein that activates Cullin-RING ligases and modifies substrates via neddylation. However, there is limited information on how neddylation regulates innate immunity in crustaceans. In the present study, we identified the evolutionarily conserved NEDD8 with the ubiquitin homologue domain in the Chinese mitten crab (Eriocheir sinensis), named it EsNEDD8. Then, we analyzed the expression patterns and cellular location of its substrate, EsCullin4. qRT-PCR showed that both EsNEDD8 and EsCullin4 were widely expressed in all the selected tissues, and EsCullin4 was significantly upregulated in hemocytes after bacterial stimulation. Moreover, silencing of EsCullin4 significantly suppressed the expression of antimicrobial peptides (AMPs) in the hemocytes after bacterial stimulation, and inhibition of EsCullin4 neddylation by treatment with the NEDD8-activating enzyme inhibitor MLN4924 significantly inhibited the expression of the AMPs. Thus, the results show that EsNEDD8-modified EsCullin4 could control antimicrobial activities via regulation of AMPs expression in the Chinese mitten crab.