A Novel Strategy to Track Lysine-48 Ubiquitination by Fluorescence Resonance Energy Transfer.
A Novel Strategy to Track Lysine-48 Ubiquitination by Fluorescence Resonance Energy Transfer.
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DOI：
10.1007/978-1-0716-1217-0_7
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Pan ZQ
中科院分区：
文献类型：
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作者：
Wu K;Pan ZQ
赖氨酸-48（K48）连接的泛素（Ub）链对蛋白质的翻译后修饰是选择性蛋白质降解的主要细胞机制，其严重影响生物过程，例如细胞周期检查点。在这一章中，我们描述了一种体外生化方法来检测K48连接的二Ub链的荧光共振能量转移（FRET）。为此，我们详细介绍了相关酶和底物的制备方法，以及高效执行反应的方法。使用这种敏感且高度可重复的形式跟踪K48多聚泛素化提供了高通量筛选的机会，从而鉴定能够改变泛素化以改善人类健康的小分子调节剂。
Posttranslational modification of protein by lysine-48 (K48) linked ubiquitin (Ub) chains is the major cellular mechanism for selective protein degradation that critically impacts biological processes such as cell cycle checkpoints. In this chapter, we describe an in vitro biochemical approach to detect a K48-linked di-Ub chain by fluorescence resonance energy transfer (FRET). To this end, we detail methods for the preparation of the relevant enzymes and substrates, as well as for the execution of the reaction with high efficiency. Tracking K48 polyubiquitination using this sensitive and highly reproducible format provides an opportunity for high-throughput screening that leads to identification of small molecule modulators capable of changing ubiquitination for improving human health.