Characterization of the Ubiquitin and ISG15 Deconjugase Activity of SARS-CoV-1 and SARS-CoV-2 Papain-Like Protease.
Characterization of the Ubiquitin and ISG15 Deconjugase Activity of SARS-CoV-1 and SARS-CoV-2 Papain-Like Protease.
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SARS-CoV-1 和 SARS-CoV-2 木瓜蛋白酶样蛋白酶的泛素和 ISG15 解缀酶活性的表征。
DOI：
10.1007/978-1-0716-2803-4_11
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Olsen,ShaunK
中科院分区：
文献类型：
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作者：
Nayak,Digant;DosSantosBury,Priscila;Drag,Marcin;Nijkerk,AlfredN;ElOualid,Farid;Olsen,ShaunK
严重急性呼吸综合征冠状病毒1号和2号（SARS-CoV-1和SARS-CoV-2）都编码一种木瓜蛋白酶（PLpro），该蛋白酶在病毒传播中起着至关重要的作用。PLpro通过处理病毒复制所必需的病毒多蛋白，并从宿主的关键免疫信号蛋白中去除小蛋白、泛素和ISG15，从而阻止宿主的先天免疫反应，从而实现这一功能。尽管来自SARS-CoV-1和SARS-CoV-2的PLpro在结构上高度相似（83%的序列同一性），但它们表现出功能变异性。因此，为了进一步阐明PLpro的作用机制和帮助药物发现，需要对其进行生化和动力学表征。本章描述了使用基于活性的探针（ABPs）以及基于荧光的底物在体外评估PLpro活性的一步一步的实验程序。在这里，我们描述了一个循序渐进的实验程序，以评估PLpro的活性在体外使用一套活性探针（ABPs）和荧光底物，以及如何将它们作为快速而灵敏的方法来计算动力学参数。
Both severe acute respiratory syndrome coronavirus 1 and 2 (SARS-CoV-1 and SARS-CoV-2) encode a papain-like protease (PLpro), which plays a vital role in viral propagation. PLpro accomplishes this function by processing the viral polyproteins essential for viral replication and removing the small proteins, ubiquitin and ISG15 from the host’s key immune signaling proteins, thereby preventing the host’s innate immune response. Although PLpro from both SARS-CoV-1 and SARS-CoV-2 are structurally highly similar (83% sequence identity), they exhibit functional variability. Hence, to further elucidate the mechanism and aid in drug discovery efforts, the biochemical and kinetic characterization of PLpro is needed. This chapter describes step-by-step experimental procedures for evaluating PLpro activity in vitro using activity-based probes (ABPs) along with fluorescence-based substrates. Herein we describe a step-by-step experimental procedure to assess the activity of PLpro in vitro using a suite of activity-based probes (ABPs) and fluorescent substrates and how they can be applied as fast and yet sensitive methods to calculate kinetic parameters.