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

<i>Penaeus vannamei</i> hemocyanin (<i>Pv</i>HMC) 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 <i>Pv</i>HMC linear ubiquitination mediated by an E3 ligase <i>Pv</i>LUBEL (a HOIP homolog), with emphasis on its role in shrimp immunity defending against diverse pathogens. We detected linear ubiquitination of <i>Pv</i>HMC in multiple tissues, including hemocytes and the hepatopancreas. During <i>Vibrio parahaemolyticus</i> infection, the expression of <i>Pv</i>LUBEL and the level of <i>Pv</i>HMC linear ubiquitination were suppressed, whereas infection by white spot syndrome virus (WSSV) led to their upregulation. Structural analyses revealed that <i>Pv</i>LUBEL, which shares a conserved RING-IBR-RING (RBR) domain with mammalian HOIP, serves as the catalytic subunit. Notably, inhibition of <i>Pv</i>LUBEL via HOIPIN-1 (a covalent inhibitor) or RNA interference (RNAi) significantly reduced <i>Pv</i>HMC linear ubiquitination, thereby increasing pathogen proliferation and decreasing host survival. These findings unveil a novel post-translational regulatory mechanism in which <i>Pv</i>LUBEL-mediated linear ubiquitination of <i>Pv</i>HMC underpins the shrimp immune response against aquaculture pathogens.