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A General in Vitro Assay for Studying Enzymatic Activities of the Ubiquitin System

Yukun Zuo, Boon Keat Chong, Kun Jiang,Daniel Finley,David Klenerman,Yu Ye

Biochemistry(2020)SCI 3区

Univ Cambridge | Harvard Med Sch

Cited 3|Views16
Abstract
The ubiquitin (Ub) system regulates a wide range of cellular signaling pathways. Several hundred E1, E2 and E3 enzymes are together responsible for protein ubiquitination, thereby controlling cellular activities. Due to the numerous enzymes and processes involved, studies on ubiquitination activities have been challenging. We here report a novel FRET-based assay to study the in vitro kinetics of ubiquitination. FRET is established between binding of fluorophore-labeled Ub to eGFP-tagged ZnUBP, a domain that exclusively binds unconjugated Ub. We name this assay the Free Ub Sensor System (FUSS). Using Uba1, UbcH5 and CHIP as model E1, E2 and E3 enzymes, respectively, we demonstrate that ubiquitination results in decreasing FRET efficiency, from which reaction rates can be determined. Further treatment with USP21, a deubiquitinase, leads to increased FRET efficiency, confirming the reversibility of the assay. We subsequently use this assay to show that increasing the concentration of CHIP or UbcH5 but not Uba1 enhances ubiquitination rates, and develop a novel machine learning approach to model ubiquitination. The overall ubiquitination activity is also increased upon incubation with tau, a substrate of CHIP. Our data together demonstrate the versatile applications of a novel ubiquitination assay that does not require labeling of E1, E2, E3 or substrates, and is thus likely compatible with any E1-E2-E3 combinations.
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Deubiquitinating Enzymes
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要点】:本文提出了一种基于FRET技术的新型体外实验方法——Free Ub Sensor System(FUSS),用于研究泛素系统的酶活性和泛素化过程,并成功应用于模型酶和泛素化底物的研究,同时创新性地引入了机器学习模型来预测泛素化速率。

方法】:作者通过构建一种FRET传感器系统,将荧光标记的泛素与eGFP标记的ZnUBP结合,当泛素与ZnUBP结合时,FRET效率降低,从而可以检测泛素化反应的动力学。

实验】:使用Uba1、UbcH5和CHIP作为模型E1、E2和E3酶,通过FUSS系统研究了泛素化反应的速率,并用USP21去泛素化酶验证了实验的可逆性。实验结果表明,增加CHIP或UbcH5的浓度能提高泛素化速率,而增加Uba1的浓度则没有这种效果。此外,tau蛋白作为CHIP的底物,其泛素化活性在共孵育后也增强。实验使用了模型酶和tau作为数据集,未提及具体数据集名称。