Lindner Holger A, Lytvyn Viktoria, Qi Hongtao, Lachance Paule, Ziomek Edmund, Ménard Robert
Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montreal, Que., Canada H4P 2R2.
Arch Biochem Biophys. 2007 Oct 1;466(1):8-14. doi: 10.1016/j.abb.2007.07.006. Epub 2007 Jul 14.
The severe acute respiratory syndrome coronavirus papain-like protease (SARS-CoV PLpro) carries out N-terminal processing of the viral replicase polyprotein, and also exhibits Lys48-linked polyubiquitin chain debranching and ISG15 precursor processing activities in vitro. Here, we used SDS-PAGE and fluorescence-based assays to demonstrate that ISG15 derivatives are the preferred substrates for the deubiquitinating activity of the PLpro. With k(cat)/K(M) of 602,000 M(-1)s(-1), PLpro hydrolyzes ISG15-AMC 30- and 60-fold more efficiently than Ub-AMC and Nedd8-AMC, respectively. Data obtained with truncated ISG15 and hybrid Ub/ISG15 substrates indicate that both the N- and C-terminal Ub-like domains of ISG15 contribute to this preference. The enzyme also displays a preference for debranching Lys48- over Lys63-linked polyubiquitin chains. Our results demonstrate that SARS-CoV PLpro can differentiate between ubiquitin-like modifiers sharing a common C-terminal sequence, and that the debranching activity of the PLpro is linkage type selective. The potential structural basis for the demonstrated specificity of SARS-CoV PLpro is discussed.
严重急性呼吸综合征冠状病毒木瓜样蛋白酶（SARS-CoV PLpro）负责病毒复制酶多聚蛋白的N端加工，并且在体外还表现出K48连接的多聚泛素链去分支和ISG15前体加工活性。在此，我们使用SDS-PAGE和基于荧光的分析方法来证明ISG15衍生物是PLpro去泛素化活性的优选底物。PLpro水解ISG15-AMC的效率分别比Ub-AMC和Nedd8-AMC高30倍和60倍，其k(cat)/K(M)为602,000 M(-1)s(-1)。用截短的ISG15和杂交Ub/ISG15底物获得的数据表明，ISG15的N端和C端类泛素结构域均促成了这种偏好。该酶还表现出对K48连接的多聚泛素链去分支的偏好高于K63连接的多聚泛素链。我们的结果表明，SARS-CoV PLpro可以区分具有共同C端序列共享的类泛素修饰物，并且PLpro的去分支活性具有连接类型选择性。本文讨论了所证明的SARS-CoV PLpro特异性的潜在结构基础。
