The papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme
The papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme
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DOI：
10.1128/jvi.79.24.15199-15208.2005
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发表时间：
2005-12-01
影响因子：
5.4
通讯作者：
Ménard, R
中科院分区：
文献类型：
--
作者：
Lindner, HA;Fotouhi-Ardakani, N;Ménard, R
严重急性呼吸综合征冠状病毒木瓜蛋白酶样蛋白酶（SARS-CoV PLpro）参与病毒多蛋白的加工，从而有助于病毒复制复合物的生物合成。结构生物信息学已经揭示了SARS-CoV PLpro与疱疹病毒相关的泛素特异性蛋白酶（HAUSP）的关系，HAUSP是一种泛素特异性蛋白酶，表明除了其在多蛋白加工中的功能外，还具有潜在的去泛素化活性（T. Sulea，H. A. Lindner，E. O. Purisima和R.梅纳尔，J. 79：4550-4551，2005）。为了证实这一预测，我们从大肠杆菌中过表达并纯化了SARS-CoV PLpro（氨基酸[aa] 1507至1858）。纯化的酶水解泛素-7-氨基-4-甲基香豆素（Ub-AMC），一种通用的去泛素化酶底物，催化效率为13，100 M（-1）s（-1），比小的合成肽底物Z-LRGG-AMC的效率高220倍，后者包含泛素的C-末端4个残基。此外，SARS-CoV PLpro被特异性去泛素化酶抑制剂泛素醛抑制，抑制常数为210 nM。纯化的SARS-CoV PLpro分解长度为2至7（Ub 2 -7）或4（Ub 4）单位的分支聚泛素链，其涉及异肽键切割。SARS-CoV PLpro加工活性也针对融合到泛素样修饰物ISG 15的C末端的蛋白质进行检测，无论是在体外使用纯化的酶还是在HeLa细胞中通过与SARS-CoV PLpro（aa 1198至2009）共表达。这些结果清楚地表明SARS-CoV PLpro是一种去泛素化酶，从而证实了我们早期的预测。冠状病毒木瓜蛋白酶样蛋白酶的这种意想不到的活性表明了一种新的病毒策略，可以调节宿主细胞的泛素化机制。
The severe acute respiratory syndrome coronavirus papain-like protease (SARS-CoV PLpro) is involved in the processing of the viral polyprotein and, thereby, contributes to the biogenesis of the virus replication complex. Structural bioinformatics has revealed a relationship for the SARS-CoV PLpro to herpesvirus-associated ubiquitin-specific protease (HAUSP), a ubiquitin-specific protease, indicating potential deubiquitinating activity in addition to its function in polyprotein processing (T. Sulea, H. A. Lindner, E. O. Purisima, and R. Menard, J. Virol. 79:4550-4551, 2005). In order to confirm this prediction, we overexpressed and purified SARS-CoV PLpro (amino acids [aa] 1507 to 1858) from Escherichia coli. The purified enzyme hydrolyzed ubiquitin-7-amino-4-methylcoumarin (Ub-AMC), a general deubiquitinating enzyme substrate, with a catalytic efficiency of 13,100 M(-1)s(-1), 220-fold more efficiently than the small synthetic peptide substrate Z-LRGG-AMC, which incorporates the C-terminal four residues of ubiquitin. In addition, SARS-CoV PLpro was inhibited by the specific deubiquitinating enzyme inhibitor ubiquitin aldehyde, with an inhibition constant of 210 nM. The purified SARS-CoV PLpro disassembles branched polyubiquitin chains with lengths of two to seven (Ub2-7) or four (Ub4) units, which involves isopeptide bond cleavage. SARS-CoV PLpro processing activity was also detected against a protein fused to the C terminus of the ubiquitin-like modifier ISG15, both in vitro using the purified enzyme and in HeLa cells by coexpression with SARS-CoV PLpro (aa 1198 to 2009). These results clearly establish that SARS-CoV PLpro is a deubiquitinating enzyme, thereby confirming our earlier prediction. This unexpected activity for a coronavirus papain-like protease suggests a novel viral strategy to modulate the host cell ubiquitination machinery to its advantage.