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. 2016;1449:395-410.
doi: 10.1007/978-1-4939-3756-1_26.

Monitoring Target Engagement of Deubiquitylating Enzymes Using Activity Probes: Past, Present, and Future

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Free PMC article

Monitoring Target Engagement of Deubiquitylating Enzymes Using Activity Probes: Past, Present, and Future

Jeanine Harrigan et al. Methods Mol Biol. .
Free PMC article

Abstract

Deubiquitylating enzymes or DUBs are a class of enzymes that selectively remove the polypeptide posttranslational modification ubiquitin from a number of substrates. Approximately 100 DUBs exist in human cells and are involved in key regulatory cellular processes, which drive many disease states, making them attractive therapeutic targets. Several aspects of DUB biology have been studied through genetic knock-out or knock-down, genomic, or proteomic studies. However, investigation of enzyme activation and regulation requires additional tools to monitor cellular and physiological dynamics. A comparison between genetic ablation and dominant-negative target validation with pharmacological inhibition often leads to striking discrepancies. Activity probes have been used to profile classes of enzymes, including DUBs, and allow functional and dynamic properties to be assigned to individual proteins. The ability to directly monitor DUB activity within a native biological system is essential for understanding the physiological and pathological role of individual DUBs. We will discuss the evolution of DUB activity probes, from in vitro assay development to their use in monitoring DUB activity in cells and in animal tissues, as well as recent progress and prospects for assessing DUB inhibition in vivo.

Keywords: ABP; Activity-based probe; Acyloxymethyl ketone; DUB; Deubiquitylating enzyme; Hemagglutinin; JAB1/Mov34/Mpr1 Pad1 N-terminal + protease; JAMM; MACHADO-Josephin domain proteas; MJD; MP; Mpr1/Pad1 N-terminal+; OTU; Ovarian tumor protease; PA; Propargy; SENP; Sentrin-specific protease; UCH; USP; Ubiquitin C-terminal hydrolase; Ubiquitin-like protein; Ubiquitin-specific protease; Ubl; VME; VMS; Vinyl methyl ester; Vinyl methyl sulfone.

Figures

Fig. 1
Fig. 1
(a) General structure of an ABP consisting of a reporter (tag), specific molecule (protein), and warhead. (b) General mechanism of action of ABPs. Catalytically competent enzymes react with the electrophilic warhead resulting in a covalently labeled protein. (c) Mechanism of action for labeling DUBs by ubiquitin ABPs
Fig. 2
Fig. 2
Common warheads used for ubiquitin ABPs
Fig. 3
Fig. 3
High-throughput assay design to monitor DUB target engagement using ubiquitin ABPs in cells, animals, or patients tissues: (1) treatment of cells, animals, or patients with DUB inhibitor; (2) generation of protein lysates; (3) incubation of lysates with ubiquitin ABPs; (4) visualization of DUB activity or inhibition

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