Lawson T G, Gronros D L, Werner J A, Wey A C, DiGeorge A M, Lockhart J L, Wilson J W, Wintrode P L
Department of Chemistry, Bates College, Lewiston, Maine 04240.
J Biol Chem. 1994 Nov 11;269(45):28429-35.
The encephalomyocarditis virus 3C protease has been shown to be rapidly degraded in infected cells and in vitro in rabbit reticulocyte lysate. The in vitro degradation, at least, is accomplished by a virus-independent, ATP-dependent proteolytic system. Here we identify this proteolytic system as the ubiquitin-mediated system. Incubation of the 3C protease in rabbit reticulocyte or cultured mouse cell lysate preparations, alone or in the presence of added ubiquitin or methylated ubiquitin, resulted in the generation of new higher molecular weight species. These new products were shown to be 3C protease-ubiquitin conjugates by their ability to bind antibodies against both the 3C protease and ubiquitin. Supplemental ubiquitin also stimulated the degradation of the 3C protease in these preparations. Large 3C protease-polyubiquitin conjugates were observed to accumulate in reticulocyte lysate in the presence of adenosine 5'-O-(3-thiotriphosphate), an inhibitor of the 26 S multicatalytic protease. This, combined with the fact that the proteolytic activity could be removed from the lysate by sedimentation, implicates the multicatalytic protease in the degradation of the 3C protease-ubiquitin conjugates. It was also found that the slow rate of degradation of a model polyprotein, which resembles the stable viral 3CD diprotein produced in vivo, is likely due to the fact that the polyprotein is a poor substrate for the ubiquitin-conjugating system.
脑心肌炎病毒3C蛋白酶已被证明在受感染细胞以及兔网织红细胞裂解液的体外环境中会迅速降解。至少在体外，这种降解是由一种不依赖病毒、依赖ATP的蛋白水解系统完成的。在此，我们确定这种蛋白水解系统就是泛素介导的系统。在兔网织红细胞或培养的小鼠细胞裂解液制剂中孵育3C蛋白酶，无论是单独孵育还是在添加泛素或甲基化泛素的情况下，都会产生新的高分子量产物。这些新产物通过其结合抗3C蛋白酶和泛素抗体的能力，被证明是3C蛋白酶-泛素缀合物。补充泛素也会刺激这些制剂中3C蛋白酶的降解。在存在26S多催化蛋白酶抑制剂5'-O-(3-硫代三磷酸腺苷)的情况下，观察到大型3C蛋白酶-多聚泛素缀合物在网织红细胞裂解液中积累。这一点，再加上蛋白水解活性可通过沉淀从裂解液中去除这一事实，表明多催化蛋白酶参与了3C蛋白酶-泛素缀合物的降解。还发现，一种模拟体内产生的稳定病毒3CD双蛋白的模型多蛋白降解速度缓慢，这可能是因为该多蛋白是泛素缀合系统的不良底物。
