Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

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.

SUBMITTER: Cho J

PROVIDER: S-EPMC7460613 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Assay Systems for Profiling Deubiquitinating Activity.

Cho Jinhong J Park Jinyoung J Kim Eunice EunKyeong EE Song Eun Joo EJ

International journal of molecular sciences 20200806 16

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 ...[more]

PMID: 32781716

Similar Datasets

Use of focused ultrasonication in activity-based profiling of deubiquitinating enzymes in tissue.

Project description:To develop a reproducible tissue lysis method that retains enzyme function for activity-based protein profiling, we compared four different methods to obtain protein extracts from bovine lung tissue: focused ultrasonication, standard sonication, mortar & pestle method, and homogenization combined with standard sonication. Focused ultrasonication and mortar & pestle methods were sufficiently effective for activity-based profiling of deubiquitinases in tissue, and focused ultrasonication also had the fastest processing time. We used focused-ultrasonicator for subsequent activity-based proteomic analysis of deubiquitinases to test the compatibility of this method in sample preparation for activity-based chemical proteomics.

| S-EPMC5094370 | biostudies-literature

Regulation of USP28 deubiquitinating activity by SUMO conjugation.

Project description:USP28 (ubiquitin-specific protease 28) is a deubiquitinating enzyme that has been implicated in the DNA damage response, the regulation of Myc signaling, and cancer progression. The half-life stability of major regulators of critical cellular pathways depends on the activities of specific ubiquitin E3 ligases that target them for proteosomal degradation and deubiquitinating enzymes that promote their stabilization. One function of the post-translational small ubiquitin modifier (SUMO) is the regulation of enzymatic activity of protein targets. In this work, we demonstrate that the SUMO modification of the N-terminal domain of USP28 negatively regulates its deubiquitinating activity, revealing a role for the N-terminal region as a regulatory module in the control of USP28 activity. Despite the presence of ubiquitin-binding domains in the N-terminal domain, its truncation does not impair deubiquitinating activity on diubiquitin or polyubiquitin chain substrates. In contrast to other characterized USP deubiquitinases, our results indicate that USP28 has a chain preference activity for Lys(11), Lys(48), and Lys(63) diubiquitin linkages.

| S-EPMC4263883 | biostudies-literature

Deubiquitinating enzyme amino acid profiling reveals a class of ubiquitin esterases.

Project description:The reversibility of ubiquitination by the action of deubiquitinating enzymes (DUBs) serves as an important regulatory layer within the ubiquitin system. Approximately 100 DUBs are encoded by the human genome, and many have been implicated with pathologies, including neurodegeneration and cancer. Non-lysine ubiquitination is chemically distinct, and its physiological importance is emerging. Here, we couple chemically and chemoenzymatically synthesized ubiquitinated lysine and threonine model substrates to a mass spectrometry-based DUB assay. Using this platform, we profile two-thirds of known catalytically active DUBs for threonine esterase and lysine isopeptidase activity and find that most DUBs demonstrate dual selectivity. However, with two anomalous exceptions, the ovarian tumor domain DUB class demonstrates specific (iso)peptidase activity. Strikingly, we find the Machado-Joseph disease (MJD) class to be unappreciated non-lysine DUBs with highly specific ubiquitin esterase activity rivaling the efficiency of the most active isopeptidases. Esterase activity is dependent on the canonical catalytic triad, but proximal hydrophobic residues appear to be general determinants of non-lysine activity. Our findings also suggest that ubiquitin esters have appreciable cellular stability and that non-lysine ubiquitination is an integral component of the ubiquitin system. Its regulatory sophistication is likely to rival that of canonical ubiquitination.

| S-EPMC7848729 | biostudies-literature

Photocaged Cell-Permeable Ubiquitin Probe for Temporal Profiling of Deubiquitinating Enzymes.

Project description:Photocaged cell-permeable ubiquitin probe holds promise in profiling the activity of cellular deubiquitinating enzymes (DUBs) with the much needed temporal control. Here we report a new photocaged cell-permeable ubiquitin probe that undergoes photoactivation upon 365 nm UV treatment and enables intracellular deubiquitinating enzyme profiling. We used a semisynthetic approach to generate modular ubiquitin-based probe containing a tetrazole-derived warhead at the C-terminus of ubiquitin and employed a cyclic polyarginine cell-penetrating peptide (cR10) conjugated to the N-terminus of ubiquitin via a disulfide linkage to deliver the probe into live cells. Upon 365 nm UV irradiation, the tetrazole group is converted to a nitrilimine intermediate in situ, which reacts with nearby nucleophilic cysteine residue from the DUB active site. The new photocaged cell-permeable probe showed good reactivity toward purified DUBs, including USP2, UCHL1, and UCHL3, upon photoirradiation. The Ub-tetrazole probe was also assessed in HeLa cell lysate and showed robust labeling only upon photoactivation. We further carried out protein profiling in intact HeLa cells using the new photocaged cell-permeable ubiquitin probe and identified DUBs captured by the probe using label-free quantitative (LFQ) mass spectrometry. Importantly, the photocaged cell-permeable ubiquitin probe captured DUBs specifically in respective G1/S and G2/M phases in synchronized HeLa cells. Moreover, using this probe DUBs were profiled at different time points following the release of HeLa cells from G1/S phase. Our results showed that photocaged cell-permeable probe represents a valuable new tool for achieving a better understanding of the cellular functions of DUBs.

| S-EPMC8462974 | biostudies-literature

A high-throughput, multiplexed assay for superfamily-wide profiling of enzyme activity.

Project description:The selectivity of an enzyme inhibitor is a key determinant of its usefulness as a tool compound or its safety as a drug. Yet selectivity is never assessed comprehensively in the early stages of the drug discovery process, and only rarely in the later stages, because technical limitations prohibit doing otherwise. Here, we report EnPlex, an efficient, high-throughput method for simultaneously assessing inhibitor potency and specificity, and pilot its application to 96 serine hydrolases. EnPlex analysis of widely used serine hydrolase inhibitors revealed numerous previously unrecognized off-target interactions, some of which may help to explain previously confounding adverse effects. In addition, EnPlex screening of a hydrolase-directed library of boronic acid- and nitrile-containing compounds provided structure-activity relationships in both potency and selectivity dimensions from which lead candidates could be more effectively prioritized. Follow-up of a series of dipeptidyl peptidase 4 inhibitors showed that EnPlex indeed predicted efficacy and safety in animal models. These results demonstrate the feasibility and value of high-throughput, superfamily-wide selectivity profiling and suggest that such profiling can be incorporated into the earliest stages of drug discovery.

| S-EPMC5953424 | biostudies-literature

Development of a Multiplexed Activity-Based Protein Profiling Assay to Evaluate Activity of Endocannabinoid Hydrolase Inhibitors.

Project description:Endocannabinoids, an important class of signaling lipids involved in health and disease, are predominantly synthesized and metabolized by enzymes of the serine hydrolase superfamily. Activity-based protein profiling (ABPP) using fluorescent probes, such as fluorophosphonate (FP)-TAMRA and β-lactone-based MB064, enables drug discovery activities for serine hydrolases. FP-TAMRA and MB064 have distinct, albeit partially overlapping, target profiles but cannot be used in conjunction due to overlapping excitation/emission spectra. We therefore synthesized a novel FP-probe with a green BODIPY as a fluorescent tag and studied its labeling profile in mouse proteomes. Surprisingly, we found that the reporter tag plays an important role in the binding potency and selectivity of the probe. A multiplexed ABPP assay was developed in which a probe cocktail of FP-BODIPY and MB064 visualized most endocannabinoid serine hydrolases in mouse brain proteomes in a single experiment. The multiplexed ABPP assay was employed to profile endocannabinoid hydrolase inhibitor activity and selectivity in the mouse brain.

| S-EPMC6154214 | biostudies-literature

Distinct USP25 and USP28 Oligomerization States Regulate Deubiquitinating Activity.

Project description:The evolutionarily related deubiquitinating enzymes (DUBs) USP25 and USP28 comprise an identical overall domain architecture but are functionally non-redundant: USP28 stabilizes c-MYC and other nuclear proteins, and USP25 regulates inflammatory TRAF signaling. We here compare molecular features of USP25 and USP28. Active enzymes form distinctively shaped dimers, with a dimerizing insertion spatially separating independently active catalytic domains. In USP25, but not USP28, two dimers can form an autoinhibited tetramer, where a USP25-specific, conserved insertion sequence blocks ubiquitin binding. In full-length enzymes, a C-terminal domain with a previously unknown fold has no impact on oligomerization, but N-terminal regions affect the dimer-tetramer equilibrium in vitro. We confirm oligomeric states of USP25 and USP28 in cells and show that modulating oligomerization affects substrate stabilization in accordance with in vitro activity data. Our work highlights how regions outside of the catalytic domain enable a conceptually intriguing interplay of DUB oligomerization and activity.

| S-EPMC6509359 | biostudies-literature

DUB-1, a deubiquitinating enzyme with growth-suppressing activity.

Project description:Cytokines regulate cell growth by inducing the expression of specific target genes. Using the differential display method, we have cloned a cytokine-inducible immediate early gene, DUB-1 (for deubiquitinating enzyme). DUB-1 is related to members of the UBP superfamily of deubiquitinating enzymes, which includes the oncoprotein Tre-2. A glutathione S-transferase-DUB-1 fusion protein cleaved ubiquitin from a ubiquitin-beta-galactosidase protein. When a conserved cysteine residue of DUB-1, required for ubiquitin-specific thiol protease activity, was mutated to serine (C60S), deubiquitinating activity was abolished. Continuous expression of DUB-1 from a steroid-inducible promoter induced growth arrest in the G1 phase of the cell cycle. Cells arrested by DUB-1 expression remained viable and resumed proliferation upon steroid withdrawal. Our results suggest that DUB-1 regulates cellular growth by modulating either the ubiquitin-dependent proteolysis or the ubiquitination state of an unknown growth regulatory factor(s).

| S-EPMC39596 | biostudies-other

E2 ubiquitin-conjugating enzymes regulate the deubiquitinating activity of OTUB1.

Project description:OTUB1 is a Lys48-specific deubiquitinating enzyme that forms a complex in vivo with E2 ubiquitin (Ub)-conjugating enzymes including UBC13 and UBCH5. OTUB1 binds E2~Ub thioester intermediates and prevents ubiquitin transfer, thereby noncatalytically inhibiting accumulation of polyubiquitin. We report here that a second role of OTUB1-E2 interactions is to stimulate OTUB1 cleavage of Lys48 polyubiquitin. This stimulation is regulated by the ratio of charged to uncharged E2 and by the concentration of Lys48-linked polyubiquitin and free ubiquitin. Structural and biochemical studies of human and worm OTUB1 and UBCH5B show that the E2 enzyme stimulates binding of the Lys48 polyubiquitin substrate by stabilizing folding of the OTUB1 N-terminal ubiquitin-binding helix. Our results suggest that OTUB1-E2 complexes in the cell are poised to regulate polyubiquitin chain elongation or degradation in response to changing levels of E2 charging and available free ubiquitin.

| S-EPMC3941643 | biostudies-literature

Ubiquitination directly enhances activity of the deubiquitinating enzyme ataxin-3.

Project description:Deubiquitinating enzymes (DUBs) control the ubiquitination status of proteins in various cellular pathways. Regulation of the activity of DUBs, which is critically important to cellular homoeostasis, can be achieved at the level of gene expression, protein complex formation, or degradation. Here, we report that ubiquitination also directly regulates the activity of a DUB, ataxin-3, a polyglutamine disease protein implicated in protein quality control pathways. Ubiquitination enhances ubiquitin (Ub) chain cleavage by ataxin-3, but does not alter its preference for K63-linked Ub chains. In cells, ubiquitination of endogenous ataxin-3 increases when the proteasome is inhibited, when excess Ub is present, or when the unfolded protein response is induced, suggesting that the cellular functions of ataxin-3 in protein quality control are modulated through ubiquitination. Ataxin-3 is the first reported DUB in which ubiquitination directly regulates catalytic activity. We propose a new function for protein ubiquitination in regulating the activity of certain DUBs and perhaps other enzymes.

| S-EPMC2646149 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data