The ubiquitin- and proteasome-dependent degradation of COX-2 is regulated by the COP9 signalosome and differentially influenced by coxibs
The ubiquitin- and proteasome-dependent degradation of COX-2 is regulated by the COP9 signalosome and differentially influenced by coxibs
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DOI：
10.1007/s00109-007-0197-y
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发表时间：
2007-09-01
影响因子：
4.7
通讯作者：
Dubiel, Wolfgang
中科院分区：
文献类型：
--
作者：
Neuss, Heiko;Huang, Xiaohua;Dubiel, Wolfgang
环氧合酶-2（考克斯-2）在炎症和肿瘤组织中被诱导。它产生刺激肿瘤血管生成和肿瘤生长的洋地黄素。因此，破坏和/或特异性抑制考克斯-2应该是未来肿瘤治疗的一个重要方面。近年来，特异性的考克斯-2抑制剂coxibs的临床应用受到了质疑，因为长期应用会引起严重的肾脏和心血管并发症。人们对确切的潜在机制知之甚少，也没有解释不同昔布的不同作用。以前已经证明考克斯-2被泛素（Ub）蛋白酶体系统（UPS）降解。然而，泛素化是如何完成和调节的尚不清楚。UPS的一个重要调节因子是COP 9信号体（CSN），它控制许多蛋白质的稳定性。在这里，我们表明，考克斯-2在HeLa细胞裂解液和HeLa细胞中的蛋白酶体依赖性降解刺激姜黄素，CSN相关激酶的抑制剂。这些数据表明CSN在考克斯-2降解中的功能。此外，帕瑞昔布可显著加速考克斯-2的蛋白水解，而塞来昔布或罗非昔布则无此作用。通过密度梯度离心和免疫沉淀，我们证明了考克斯-2与CSN的物理相互作用。此外，考克斯-2与由CSN、cullin-RING Ub连接酶和26 S蛋白酶体组成的大复合物相关。用Flag-COX-2的下拉实验揭示了cullin 1和cullin 4作为大的超复合物的组分。Cullin 1和4是Ub连接酶的支架蛋白，推测其泛素化考克斯-2。用帕瑞昔布处理HeLa细胞导致内源性考克斯-2的加速降解，伴随着考克斯-2-Ub缀合物的增加。在HeLa细胞中，帕瑞昔布转化为选择性考克斯-2抑制剂伐地昔布。加入伐地考昔还刺激HeLa细胞中的考克斯-2降解。因此，我们得出结论，伐地昔布特异性地与考克斯-2相互作用，并诱导一种可用于泛素化和降解的构象。
The cyclooxygenase-2 (COX-2) enzyme is induced upon inflammation and in neoplastic tissues. It produces prostaglandins that stimulate tumor angiogenesis and tumor growth. Therefore, destruction and/or specific inhibition of COX-2 should be an important aspect of future tumor therapy. Recently, clinical application of specific COX-2 inhibitors called coxibs became doubtfully because they produce serious renal and cardiovascular complications under long term application. The exact underlying mechanisms are poorly understood and the different effects of diverse coxibs are not explained. It has been demonstrated before that COX-2 is degraded by the ubiquitin (Ub) proteasome system (UPS). However, how ubiquitination is accomplished and regulated was unclear. An important regulator of the UPS is the COP9 signalosome (CSN), which controls the stability of many proteins. Here we show that the proteasome-dependent degradation of COX-2 in HeLa cell lysate and in HeLa cells was stimulated by curcumin, an inhibitor of CSN-associated kinases. These data suggest a function of the CSN in the degradation of COX-2. In addition, proteolysis of COX-2 was significantly accelerated by parecoxib, but not by celecoxib or rofecoxib. By density gradient centrifugation and immunoprecipitation we demonstrate that COX-2 physically interacts with the CSN. Moreover, COX-2 is associated with large complexes consisting of the CSN, cullin-RING Ub ligases and the 26S proteasome. Pulldown experiments with Flag-COX-2 revealed cullin 1 and cullin 4 as components of the large super-complexes. Cullin 1 and 4 are scaffolding proteins of Ub ligases that presumably ubiquitinate COX-2. Treatment of HeLa cells with parecoxib results in an accelerated degradation of endogenous COX-2 accompanied by an increase of COX-2-Ub conjugates. In HeLa cells parecoxib is converted to the selective COX-2 inhibitor valdecoxib. Addition of valdecoxib also stimulates COX-2 degradation in HeLa cells. We therefore conclude that valdecoxib specifically interacts with COX-2 and induces a conformation accessible for ubiquitination and degradation.