Linear Ubiquitination of Hemocyanin Mediated by LUBEL Regulates Innate Immunity in Penaeus vannamei

Penaeus vannamei hemocyanin (PvHMC) exhibits multifunctional roles in immunity, often mediated by various post-translational modifications. While linear ubiquitination catalyzed by LUBAC in mammals regulates immune signaling, its role in crustacean immunity remains unclear. Here, we investigated the regulatory mechanism of PvHMC linear ubiquitination mediated by an E3 ligase PvLUBEL (a HOIP homolog), with emphasis on its role in shrimp immunity defending against diverse pathogens. We detected linear ubiquitination of PvHMC in multiple tissues, including hemocytes and the hepatopancreas. During Vibrio parahaemolyticus infection, the expression of PvLUBEL and the level of PvHMC linear ubiquitination were suppressed, whereas infection by white spot syndrome virus (WSSV) led to their upregulation. Structural analyses revealed that PvLUBEL, which shares a conserved RING-IBR-RING (RBR) domain with mammalian HOIP, serves as the catalytic subunit. Notably, inhibition of PvLUBEL via HOIPIN-1 (a covalent inhibitor) or RNA interference (RNAi) significantly reduced PvHMC linear ubiquitination, thereby increasing pathogen proliferation and decreasing host survival. These findings unveil a novel post-translational regulatory mechanism in which PvLUBEL-mediated linear ubiquitination of PvHMC underpins the shrimp immune response against aquaculture pathogens.