Meng L, Mohan R, Kwok B H, Elofsson M, Sin N, Crews C M
Department of Molecular, Cellular and Developmental Biology, Yale University, 219 Prospect Street, New Haven, CT 06520-8103, USA.
Proc Natl Acad Sci U S A. 1999 Aug 31;96(18):10403-8. doi: 10.1073/pnas.96.18.10403.
The proteasome regulates cellular processes as diverse as cell cycle progression and NF-kappaB activation. In this study, we show that the potent antitumor natural product epoxomicin specifically targets the proteasome. Utilizing biotinylated-epoxomicin as a molecular probe, we demonstrate that epoxomicin covalently binds to the LMP7, X, MECL1, and Z catalytic subunits of the proteasome. Enzymatic analyses with purified bovine erythrocyte proteasome reveal that epoxomicin potently inhibits primarily the chymotrypsin-like activity. The trypsin-like and peptidyl-glutamyl peptide hydrolyzing catalytic activities also are inhibited at 100- and 1,000-fold slower rates, respectively. In contrast to peptide aldehyde proteasome inhibitors, epoxomicin does not inhibit nonproteasomal proteases such trypsin, chymotrypsin, papain, calpain, and cathepsin B at concentrations of up to 50 microM. In addition, epoxomicin is a more potent inhibitor of the chymotrypsin-like activity than lactacystin and the peptide vinyl sulfone NLVS. Epoxomicin also effectively inhibits NF-kappaB activation in vitro and potently blocks in vivo inflammation in the murine ear edema assay. These results thus define epoxomicin as a novel proteasome inhibitor that likely will prove useful in exploring the role of the proteasome in various in vivo and in vitro systems.
蛋白酶体调节多种细胞过程，如细胞周期进程和核因子κB激活。在本研究中，我们表明强效抗肿瘤天然产物环氧霉素特异性靶向蛋白酶体。利用生物素化环氧霉素作为分子探针，我们证明环氧霉素与蛋白酶体的LMP7、X、MECL1和Z催化亚基共价结合。用纯化的牛红细胞蛋白酶体进行的酶分析表明，环氧霉素主要强效抑制类胰凝乳蛋白酶活性。类胰蛋白酶和肽基 - 谷氨酰肽水解催化活性也分别以慢100倍和1000倍的速率受到抑制。与肽醛类蛋白酶体抑制剂不同，环氧霉素在浓度高达50微摩尔时不抑制诸如胰蛋白酶、胰凝乳蛋白酶、木瓜蛋白酶、钙蛋白酶和组织蛋白酶B等非蛋白酶体蛋白酶。此外，环氧霉素比乳胞素和肽乙烯砜NLVS对类胰凝乳蛋白酶活性的抑制作用更强。环氧霉素在体外也有效抑制核因子κB激活，并在小鼠耳部水肿试验中有效阻断体内炎症。因此，这些结果将环氧霉素定义为一种新型蛋白酶体抑制剂，可能在探索蛋白酶体在各种体内和体外系统中的作用方面证明是有用的。
