Biochemical properties of PD-L1 ubiquitination by CRL3SPOP, ARIH1 and NEDD4 family ubiquitin ligases

As a key immune checkpoint ligand, PD-L1 represents a critical target in cancer immunotherapy. While multiple E3 ubiquitin ligases including CRL3SPOP, ARIH1, and NEDD4 have been implicated in PD-L1 degradation, their precise enzymatic mechanisms remain unclear. Through in vitro reconstitution with purified components, we systematically compared the enzymatic activities of CRL3SPOP, ARIH1, and NEDD4 ligases on PD-L1's cytoplasmic domain. ARIH1, rather than CRL3SPOP, could independently ubiquitinate PD-L1. We demonstrated a unique mechanism that ARIH1 acts as a substrate receptor to cooperate with CRLs to catalyze PD-L1 ubiquitination. We also biochemically validated the E3 ligase activity of the NEDD4 family E3s towards PD-L1. Using liposomes in the enzymatic assays, we illustrated that the phosphorylation enhanced PD-L1 ubiquitination by disrupting its membrane association. Our study provides previously neglected biochemical insights into PD-L1 ubiquitination, which advances our understanding of the molecular details of PD-L1 regulation mechanism.