Assay Systems for Profiling Deubiquitinating Activity

Abstract

Deubiquitinating enzymes regulate various cellular processes, particularly protein degradation, localization, and protein–protein interactions. The dysregulation of deubiquitinating enzyme (DUB) activity has been linked to several diseases; however, the function of many DUBs has not been identified. Therefore, the development of methods to assess DUB activity is important to identify novel DUBs, characterize DUB selectivity, and profile dynamic DUB substrates. Here, we review various methods of evaluating DUB activity using cell lysates or purified DUBs, as well as the types of probes used in these methods. In addition, we introduce some techniques that can deliver DUB probes into the cells and cell-permeable activity-based probes to directly visualize and quantify DUB activity in live cells. This review could contribute to the development of DUB inhibitors by providing important information on the characteristics and applications of various probes used to evaluate and detect DUB activity in vitro and in vivo.

An expanded lexicon for the ubiquitin code

“…PTM ‘codes’ depend not only on writers but also on ‘eraser’ DUBs that remove modifications 30 . Previously, it was not readily possible to systematically assay DUB activity towards different bond types because the technology was not robust enough to generate comparable probes with the C terminus of ubiquitin linked to various amino acids 31 . This challenge was overcome by taking advantage of the ability of the RCR domain of MYCBP2 to forge ester bonds 32 .…”

USP13: Multiple Functions and Target Inhibition

“…To date, seven structurally distinct DUB families have been described, including ubiquitin-specific proteases (USPs), ovarian tumor proteases (OTUs), ubiquitin C-terminal hydrolase (UCHs), Machado–Josephin domain-containing proteases (MJDs), motifs interacting with the ubiquitin-containing novel DUB family (MINDYs), JAB1, MPN, MOV34 family (JAMMs), and zinc finger containing Ub peptidase 1 (ZUP1) ( Kwasna et al, 2018 ; Cho et al, 2020 ; Wang and Wang, 2021 ). JAMMs are zinc metallopeptidases, while the other six DUB families are cysteine peptidases.…”

Characterising Plant Deubiquitinases with in vitro Activity-based Labelling and Ubiquitin Chain Disassembly Assays

“…To understand DUB function, their activity can be analysed by various in vitro methods to determine substrate specificity and regulation of activity ( Cho et al , 2020 ). These methods include activity-based protein profiling using synthetic ubiquitin suicide probes that irreversibly label DUB active sites ( Borodovsky et al , 2002 ; Gong et al , 2018 ; Schauer et al , 2020 ), as well as assays in which purified ubiquitin chains are incubated with DUBs and chain disassembly is monitored by western blotting or gel staining.…”