A versatile fluorescence polarization-based deubiquitination assay using an isopeptide bond substrate mimetic (IsoMim).

ABSTRACT: Deubiquitinases (DUBs) play a critical role in the regulation of various cellular processes, such as protein homeostasis and signaling, rendering them attractive drug targets. However, the generation of reagents for measuring DUB activity typically involves several steps and is not straightforward. Here, we report the development and characterization of a novel fluorescent polarization assay using an isopeptide bond substrate mimetic (IsoMim) that can be made recombinantly in high yields. The IsoMim assay was able to discern the differential activity of ubiquitin-specific protease family members (USP4, USP15, USP11, and USP2), the ubiquitin C-terminal hydrolase UCHL3, and the Machado-Joseph Domain deubiquitinase JOSD2. A competition assay format of the assay was developed that discerned di

SUBMITTER: Zhang J 

PROVIDER: S-EPMC12273560 | biostudies-literature | 2025 Jun

REPOSITORIES: biostudies-literature