Guangzhou Municipal and Guangdong Provincial Key Lab of Protein Modification and Degradation Lab, State Key Lab of Respiratory Disease, School of Basic Medical Sciences, Affiliated Cancer Hospital of Guangzhou Medical University, Guangzhou, China.
Vascular Biology Center, Medical College of Georgia, Augusta University, Augusta, GA, USA.
FASEB J. 2021 Aug;35(8):e21800. doi: 10.1096/fj.202001146RR.
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.
A 型和 B 型血友病是由凝血因子 VIII 或 IX 缺乏引起的先天性出血性疾病，通过下调抗凝血酶来治疗。然而，调节抗凝血酶表达的分子机制仍知之甚少。在这里，我们鉴定出 Cullin 2 和 USP2（泛素特异性肽酶-2）是抗凝血酶表达的新型调节因子，通过调节抗凝血酶的泛素化作用。蛋白酶体抑制剂导致 HepG2 和 SMMC7721 细胞中抗凝血酶及其泛素化形式的积累。值得注意的是，用 MLN4924 抑制 neddylation 抑制了抗凝血酶的泛素化和降解，这通过小干扰 RNA (siRNA) 沉默 neddylation 酶 NAE1、UBA3 和 UBE2M 得到了重现。我们鉴定出 Cullin 2 是抗凝血酶的相互作用伙伴，siRNA 介导的 Cullin 2 敲低减少了抗凝血酶的泛素化并增加了抗凝血酶蛋白。我们进一步发现 USP2 与抗凝血酶相互作用并调节抗凝血酶表达，表明过表达 USP2 抑制抗凝血酶的泛素化和蛋白酶体清除，而药理学抑制或 siRNA 介导的 USP2 敲低下调抗凝血酶。总之，这些结果表明 Cullin 2 E3 泛素连接酶和 USP2 协调调节抗凝血酶的泛素化和降解。因此，靶向 Cullin 2 和 USP2 可能是治疗血友病的一种潜在策略。
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