Characterising Plant Deubiquitinases with in vitro Activity-based Labelling and Ubiquitin Chain Disassembly Assays.
Characterising Plant Deubiquitinases with in vitro Activity-based Labelling and Ubiquitin Chain Disassembly Assays.
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DOI：
10.21769/bioprotoc.4015
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发表时间：
2021-05-05
期刊：
影响因子：
0.8
通讯作者：
Spoel SH
中科院分区：
文献类型：
--
作者：
Skelly MJ;Spoel SH
泛素对蛋白质的翻译后修饰是所有真核生物中必不可少的细胞信号传导机制。泛素通过多种具有不同活性和底物特异性的去泛素化酶（DUB）从靶蛋白中去除。了解DUB在体外的功能是揭示其细胞作用的重要第一步。在这里，我们提供了快速分析DUB活性在体外的自杀探针，HA-泛素乙烯基砜（HA-UbVS），泛素链解体测定活性为基础的标记协议。我们以前使用这些方法来分析拟南芥DUB，UBP 6的活性，但原则上，这些方案适用于任何感兴趣的DUB。
Post-translational modification of proteins by ubiquitin is an essential cellular signaling mechanism in all eukaryotes. Ubiquitin is removed from target proteins by a wide range of deubiquitinase (DUB) enzymes with different activities and substrate specificities. Understanding how DUBs function in vitro is a vital first step to uncovering their cellular roles. Here, we provide protocols for the rapid analysis of DUB activity in vitro by activity-based labelling with the suicide probe, HA-ubiquitin vinyl sulfone (HA-UbVS), and ubiquitin chain disassembly assays. We have previously used these methods to analyse the activity of the Arabidopsis thaliana DUB, UBP6, but in principle, these protocols are applicable to any DUB of interest.