Cullin 2-RBX1 E3 ligase and USP2 regulate antithrombin ubiquitination and stability

Hemophilia A and B are congenital bleeding disorders caused by a deficiency in pro-coagulant factor VIII or IX that is treated by downregulation of antithrombin. However, the molecular mechanisms that regulate antithrombin expression remain poorly understood. Here, we identified Cullin 2 and USP2 (ubiquitin-specific peptidase-2) as novel regulators of antithrombin expression that act by modulating antithrombin ubiquitination. Inhibition of the proteasome caused accumulation of antithrombin and its ubiquitinated forms in HepG2 and SMMC7721 cells. Notably, inhibition of neddylation with MLN4924 suppressed both ubiquitination and degradation of antithrombin, which is recapitulated by silencing of the neddylation enzymes, NAE1, UBA3, and UBE2M, with small interfering RNA (siRNA). We identified Cullin 2 as the interaction partner of antithrombin, and siRNA-mediated Cullin 2 knockdown reduced antithrombin ubiquitination and increased antithrombin protein. We further found that USP2 interacted with antithrombin and regulated antithrombin expression, showing that overexpression of USP2 inhibits the ubiquitination and proteasomal clearance of antithrombin, whereas pharmacological inhibition or siRNA-mediated knockdown of USP2 downregulates antithrombin. Collectively, these results suggest that Cullin 2 E3 ubiquitin ligase and USP2 coordinately regulate antithrombin ubiquitination and degradation. Thus, targeting Cullin 2 and USP2 could be a potential strategy for treatment of hemophilia.