研究控制泛素介导的DAF-16/FOXO蛋白周转的机制。
Investigating Mechanisms that Control Ubiquitin-Mediated DAF-16/FOXO Protein Turnover.
作者信息
Heimbucher Thomas, Murphy Coleen T
机构信息
Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, USA.
Paul F. Glenn Laboratories for Aging Research, Princeton University, Princeton, NJ, USA.
出版信息
Methods Mol Biol. 2019;1890:41-49. doi: 10.1007/978-1-4939-8900-3_4.
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.
FOXO家族转录因子的蛋白质周转受泛素-蛋白酶体系统调控。共价连接某些类型泛素链的因子（E3泛素连接酶）与能够去除泛素缀合物的酶（去泛素化酶）之间存在复杂的相互作用，共同调节蛋白酶体对FOXO蛋白的降解。在此，我们描述了将候选E3泛素连接酶和去泛素化酶鉴定为FOXO泛素化状态调节因子的方法。我们的方案可用于在变性条件下从培养细胞中纯化和富集泛素化的FOXO库，这种条件会使细胞去泛素化酶失活，从而保护FOXO蛋白上的泛素缀合物。此外，我们的方法还介绍了如何将泛素化的FOXO蛋白逐步复性，以用作体外去泛素化（DUB）分析的底物。
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