Development and Optimization of an IQF USP5 Activity Assay

Development and optimization of a fluorescence based deubiquitinase (DUB) activity assay for USP5 with internally quenched fluorophore pair (IQF) substrates: di-ubiquitin lysine linked K48 and K63 (Ub2K48, Ub2K63). The inhibitory capability of USP5 zinc-finger ubiquitin binding domain (ZnF-UBD) ligands was investigated.本研究开发并优化了一种面向泛素特异性蛋白酶5（USP5）的荧光法去泛素化酶（deubiquitinase, DUB）活性检测方法，所用底物为带有内部淬灭荧光基团对（internally quenched fluorophore pair, IQF）的K48与K63位赖氨酸连接型双泛素（Ub2K48、Ub2K63）。此外，本研究还考察了USP5锌指泛素结合结构域（zinc-finger ubiquitin binding domain, ZnF-UBD）配体的抑制活性。