Arsenault Heather E, Benanti Jennifer A
Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA, USA.
Methods Mol Biol. 2023;2591:237-253. doi: 10.1007/978-1-0716-2803-4_14.
A significant hurdle to understanding the functions of deubiquitinases (DUBs) is the identification of their in vivo substrates. Substrate identification can be difficult for two reasons. First, many proteins that are degraded by the ubiquitin-proteasome system are expressed at relatively low levels in the cell, and second, redundancy between DUBs complicates loss of function screening approaches. Here, we describe a systematic overexpression approach that takes advantage of genome-wide resources available in S. cerevisiae to overcome these challenges and identify DUB substrates in cells.
理解去泛素化酶（DUBs）功能的一个重大障碍是确定其体内底物。底物鉴定可能因两个原因而困难。首先，许多被泛素-蛋白酶体系统降解的蛋白质在细胞中表达水平相对较低，其次，DUBs之间的冗余使功能丧失筛选方法变得复杂。在这里，我们描述了一种系统性过表达方法，该方法利用酿酒酵母中可用的全基因组资源来克服这些挑战并鉴定细胞中的DUB底物。
