Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 3E1, Canada; Donnelly Centre for Cellular and Biomolecular Research, Banting and Best Department of Medical Research, University of Toronto, Toronto, ON, M5S3E1, Canada.
Donnelly Centre for Cellular and Biomolecular Research, Banting and Best Department of Medical Research, University of Toronto, Toronto, ON, M5S3E1, Canada.
J Mol Biol. 2019 Mar 15;431(6):1160-1171. doi: 10.1016/j.jmb.2019.02.007. Epub 2019 Feb 11.
We applied a yeast-two-hybrid (Y2H) analysis to screen for ubiquitin variant (UbV) inhibitors of a human deubiquitinase (DUB), ubiquitin-specific protease 2 (USP2). The Y2H screen used USP2 as the bait and a prey library consisting of UbVs randomized at four specific positions, which were known to interact with USP2 from phage display analysis. The screen yielded numerous UbVs that bound to USP2 both as a Y2H interaction in vivo and as purified proteins in vitro. The Y2H-derived UbVs inhibited the catalytic activity of USP2 in vitro with nanomolar-range potencies, and they bound and inhibited USP2 in human cells. Mutational and structural analysis showed that potent and selective inhibition could be achieved by just two substitutions in a UbV, which exhibited improved hydrophobic and hydrophilic contacts compared to the wild-type ubiquitin interaction with USP2. Our results establish Y2H as an effective platform for the development of UbV inhibitors of DUBs in vivo, providing an alternative strategy for the analysis of DUBs that are recalcitrant to phage display and other in vitro methods.
我们应用酵母双杂交（Y2H）分析筛选了人泛素特异性蛋白酶 2（USP2）的泛素变体（UbV）抑制剂。Y2H 筛选以 USP2 为诱饵，以由四个特定位置随机化的 UbV 组成的前体文库为诱饵，这些位置是从噬菌体展示分析中已知与 USP2 相互作用的。筛选产生了许多 UbV，它们既可以在体内作为 Y2H 相互作用，也可以作为体外纯化蛋白与 USP2 结合。Y2H 衍生的 UbV 以纳摩尔范围的效力在体外抑制 USP2 的催化活性，并且它们在人细胞中结合并抑制 USP2。突变和结构分析表明，UbV 中的两个取代即可实现有效且选择性的抑制，与野生型泛素与 USP2 的相互作用相比，UbV 显示出改善的疏水性和亲水性接触。我们的结果确立了 Y2H 作为体内 DUBs 的 UbV 抑制剂开发的有效平台，为分析对噬菌体展示和其他体外方法有抗性的 DUBs 提供了另一种策略。
