Investigating Mechanisms that Control Ubiquitin-Mediated DAF-16/FOXO Protein Turnover.
Investigating Mechanisms that Control Ubiquitin-Mediated DAF-16/FOXO Protein Turnover.
复制标题
研究控制泛素介导的 DAF-16/FOXO 蛋白质周转的机制。
DOI：
10.1007/978-1-4939-8900-3_4
复制
发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Murphy,ColeenT
中科院分区：
文献类型：
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作者：
Heimbucher,Thomas;Murphy,ColeenT
FOXO家族转录因子的蛋白质周转受泛素-蛋白酶体系统调节。共价连接某些类型泛素链的因子（E3-泛素连接酶）和能够去除泛素缀合物的酶（去泛素化酶）的复杂相互作用调节蛋白酶体对FOXO蛋白的降解。在这里，我们描述的方法来表征候选E3-泛素连接酶和去泛素化酶的FOXO泛素化状态的监管机构。我们的协议可以用来纯化和丰富的泛素化的FOXO池从培养的细胞在变性条件下，失活细胞去泛素化酶，从而保护FOXO蛋白上的泛素缀合物。此外，我们的方法描述了泛素化的FOXO蛋白如何以逐步的方式复性，以作为体外去泛素化（DUB）测定的底物。
Protein turnover of FOXO family transcription factors is regulated by the ubiquitin-proteasome system. A complex interplay of factors that covalently attach certain types of ubiquitin chains (E3-ubiquitin ligases), and enzymes that are able to remove ubiquitin conjugates (deubiquitylases), regulate the degradation of FOXO proteins by the proteasome. Here, we describe methods to characterize candidate E3-ubiquitin ligases and deubiquitylases as regulators of the FOXO ubiquitylation status. Our protocol can be utilized to purify and enrich a ubiquitylated FOXO pool from cultured cells under denaturing conditions, which inactivates cellular deubiquitylases and thereby protects ubiquitin conjugates on FOXO proteins. In addition, our method describes how ubiquitylated FOXO proteins can be renatured in a stepwise fashion to serve as substrates for in vitro deubiquitylation (DUB) assays.