快速高效检测植物体内蛋白泛素化修饰研究方法
收稿日期: 2019-08-12
录用日期: 2019-09-24
网络出版日期: 2019-10-09
基金资助
山东大学齐鲁青年学科建设经费(No.11200087963080)
An Quick and Efficient Assay for In Vivo Protein Ubiquitination
Received date: 2019-08-12
Accepted date: 2019-09-24
Online published: 2019-10-09
UPS参与植物中绝大多数的信号转导通路。其中, 一些激素的受体本身就是E3泛素连接酶, 如茉莉酸(JA)受体COI1和生长素(auxin)受体TIR1都是F-box蛋白, 它们通过特异性介导相应转录抑制子的泛素化降解来传递激素信号, 但对于整个UPS体系而言, 由于技术的限制, 迄今为止仅见少量泛素连接酶与特异性底物间生化机制的报道。用大肠杆菌(Escherichia coli)表达蛋白实施泛素连接酶泛素化修饰底物的体外实验是验证泛素连接酶/底物对的常用方法, 但由于体外实验缺乏某些蛋白必需的转录后修饰, 导致实验结果有时存在假阴性。利用农杆菌注射烟草(Nicotiana benthamiana)瞬时表达蛋白的方法, 建立高效的植物体内检测蛋白泛素化系统, 可以快速检测蛋白泛素化, 包括检测泛素连接酶和底物的特异性相互作用、底物蛋白的自身泛素化、泛素连接酶对底物降解的促进作用、26S蛋白酶体抑制剂MG132对底物降解的抑制作用以及用植物内源表达蛋白进行体外泛素化反应。
刘利静,赵庆臻,谢旗,于菲菲 . 快速高效检测植物体内蛋白泛素化修饰研究方法[J]. 植物学报, 2019 , 54(6) : 753 -763 . DOI: 10.11983/CBB19153
The UPS (ubiquitination/proteasome system) plays a vital role in nearly all plant signaling processes, for example, jasmonic acid receptor COI1 (coronotine insensitive protein 1) and auxin receptor TIR1 (transport inhibitor response 1) are F-box type E3s ligases, and they promote the ubiquitination then degradation of specific transcriptional repressors through 26S proteasome to activate hormone signaling. However, for the whole UPS system, only a few E3 ligase/substrate pairs’ interactions have been demonstrated due to technical limitations in plants. Generally, E3 ligase and substrate are expressed in Escherichia coli for ubiquitination assay, which may lack post-translational modifications that are important for protein function, then gives false negative result. We describe a quick and efficient assay for detecting E3-mediated protein ubiquitination in vivo by means of agroinfiltration for transient expression of relevant genes in tobacco (Nicotiana benthamiana) leaves. In detail, this method can detect the specific interaction between E3 ligase and substrate, substrate ubiquitination, the effect of E3 ligase on degradation of its substrate, inhibition of substrate degradation by 26S proteasome inhibitor MG132, and in vitro ubiquitination assay with endogenous plant proteins.
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