Browse
Submit Data
Databases
API
Help

Dataset Information

29 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Development of a DUB-selective fluorogenic substrate.

ABSTRACT: Ubiquitination is a post-translational modification that is involved in a plethora of cellular processes. Target proteins can be specifically modified with a single ubiquitin (Ub) molecule or with complex chains. In recent years, research has focused on deubiquitinating enzymes (DUBs) as potential therapeutic candidates in various diseases. USP16 is an emerging target due to its involvement in mitosis and stem cell self-renewal. Generally, activity-based probes (ABPs) used to study DUBs are based on the ubiquitin scaffold, thus lacking target selectivity. To overcome this issue, we designed a Ub-based activity probe bearing specific mutations to achieve selectivity for USP16, by combining structural modelling and analysis and mutational calculation predictions. We develop a fluorogenic substrate, the first of its kind, that is processed exclusively by USP16, which allows us to monitor USP16 activity in complex samples.

SUBMITTER: Gjonaj L

PROVIDER: S-EPMC6988746 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Development of a DUB-selective fluorogenic substrate.

Gjonaj Lorina L Sapmaz Aysegul A Flierman Dennis D Janssen George M C GMC van Veelen Peter A PA Ovaa Huib H

Chemical science 20190930 44

Ubiquitination is a post-translational modification that is involved in a plethora of cellular processes. Target proteins can be specifically modified with a single ubiquitin (Ub) molecule or with complex chains. In recent years, research has focused on deubiquitinating enzymes (DUBs) as potential therapeutic candidates in various diseases. USP16 is an emerging target due to its involvement in mitosis and stem cell self-renewal. Generally, activity-based probes (ABPs) used to study DUBs are base ...[more]

PMID: 32110315

Similar Datasets

Development of a fluorogenic ADAMTS-7 substrate.

Project description:The extracellular protease ADAMTS-7 has been identified as a potential therapeutic target in atherosclerosis and associated diseases such as coronary artery disease (CAD). However, ADAMTS-7 inhibitors have not been reported so far. Screening of inhibitors has been hindered by the lack of a suitable peptide substrate and, consequently, a convenient activity assay. Here we describe the first fluorescence resonance energy transfer (FRET) substrate for ADAMTS-7, ATS7FP7. ATS7FP7 was used to measure inhibition constants for the endogenous ADAMTS-7 inhibitor, TIMP-4, as well as two hydroxamate-based zinc chelating inhibitors. These inhibition constants match well with IC50 values obtained with our SDS-PAGE assay that uses the N-terminal fragment of latent TGF-β-binding protein 4 (LTBP4S-A) as a substrate. Our novel fluorogenic substrate ATS7FP7 is suitable for high throughput screening of ADAMTS-7 inhibitors, thus accelerating translational studies aiming at inhibition of ADAMTS-7 as a novel treatment for cardiovascular diseases such as atherosclerosis and CAD.

| S-EPMC8494430 | biostudies-literature

Discovery of a sensitive, selective, and tightly binding fluorogenic substrate of bovine plasma amine oxidase.

Project description:We report a novel fluorogenic substrate of bovine plasma amine oxidase (BPAO), namely, (2-(6-(aminomethyl)naphthalen-2-yloxy)ethyl)trimethylammonium (ANETA), which displays extremely tight binding to BPAO (K(m) 183 +/- 14 nM) and yet is metabolized fairly quickly (k(cat) 0.690 +/- 0.010 s(-1)), with the aldehyde turnover product (2-(6-formylnaphthalen-2-yloxy)ethyl)trimethylammonium serving as a real time reporting fluorophore of the enzyme activity. This allowed for the development of a fluorometric noncoupled assay that is 2 orders of magnitude more sensitive than the spectrophotometric benzylamine assay. The discovery of ANETA involved elaboration of the lead compound 6-methoxy-2-naphthalenemethaneamine by structure-based design, which recognized the ancillary cation binding site of BPAO as the most significant structural features controlling binding affinity. Structure-based design further ensured a high level of selectivity: ANETA is a good substrate of BPAO but is not a substrate of either porcine kidney diamine oxidase (pkDAO) or rat liver monoamine oxidase (MAO-B). ANETA represents the first highly sensitive, selective, and tight binding fluorogenic substrate of a copper amine oxidase that is able to respond directly to the enzyme activity in real time.

| S-EPMC2659760 | biostudies-literature

DUB-le vision: snapshots of the proteasome during substrate processing.

Project description:Covalent modification by the small protein ubiquitin can target proteins for destruction by the proteasome, but the ubiquitin signal itself is recycled. Surprisingly, proteasomes contain three different deubiquitinating enzymes (DUBs). Recent work by Zhang and Zou et al. reveals how one of these enzymes, Usp14, regulates, and is regulated by, the proteasome.

| S-EPMC10879984 | biostudies-literature

Proteomics-Based Identification of DUB Substrates Using Selective Inhibitors.

Project description:Deubiquitinating enzymes (DUBs) catalyze the removal of ubiquitin, thereby reversing the activity of E3 ubiquitin ligases and are central to the control of protein abundance and function. Despite the growing interest in DUBs as therapeutic targets, cellular functions for DUBs remain largely unknown and technical challenges often preclude the identification of DUB substrates in a comprehensive manner. Here, we demonstrate that treatment with potent DUB inhibitors coupled to mass spectrometry-based proteomics can identify DUB substrates at a proteome-wide scale. We applied this approach to USP7, a DUB widely investigated as a therapeutic target and identified many known substrates and additional targets. We demonstrate that USP7 substrates are enriched for DNA repair enzymes and E3 ubiquitin ligases. This work provides not only a comprehensive annotation of USP7 substrates, but a general protocol widely applicable to other DUBs, which is critical for translational development of DUB targeted agents.

| S-EPMC7855594 | biostudies-literature

A new fluorogenic substrate for plasmin.

Project description:A new fluorogenic peptide substrate for plasmin, 7-(N-succinoylalanylphenylalanyl-lysylamido)-4-methylcoumarin trifluoroacetate salt, was prepared that can be used in a simple and direct assay. The results obtained by the assay method are linear over a wide range of enzyme concentrations and sensitive enough to detect as little as 10(-5) CTA units of plasmin. By making use of the inhibitor Trasylol and the differences in kinetic constants, plasmin can be specifically assayed even in the presence of the plasminogen activator thrombin, as well as in culture fluids from HeLa cells.

| S-EPMC1161636 | biostudies-other

Synthetic Fluorogenic Peptides Reveal Dynamic Substrate Specificity of Depalmitoylases.

Project description:Palmitoylation is a post-translational modification involving the thioesterification of cysteine residues with a 16-carbon-saturated fatty acid. Little is known about rates of depalmitoylation or the parameters that dictate these rates. Here we report a modular strategy to synthesize quenched fluorogenic substrates for the specific detection of depalmitoylase activity and for mapping the substrate specificity of individual depalmitoylases. We demonstrate that human depalmitoylases APT1 and APT2, and TgPPT1 from the parasite Toxoplasma gondii, have distinct specificities that depend on amino acid residues distal to the palmitoyl cysteine. This information informs the design of optimal and non-optimal substrates as well as isoform-selective substrates to detect the activity of a specific depalmitoylase in complex proteomes. In addition to providing tools for studying depalmitoylases, our findings identify a previously unrecognized mechanism for regulating steady-state levels of distinct palmitoylation sites by sequence-dependent control of depalmitoylation rates.

| S-EPMC6338520 | biostudies-literature

Fluorogenic phospholipid substrate to detect lysophospholipase D/autotaxin activity.

Project description:[reaction: see text] Lysophospholipase D (lysoPLD), also known as autotaxin (ATX), is an important source of the potent mitogen lysophosphatidic acid (LPA). Two fluorogenic substrate analogues for lysoPLD were synthesized in nine steps from (S)-PMB-glycerol. The substrates (FS-2 and FS-3) show significant increases in fluorescence when treated with recombinant ATX and have potential applications in screening for this emerging drug target.

| S-EPMC2528860 | biostudies-literature

Fluorogenic Peptide Substrate for Quantification of Bacterial Enzyme Activities.

Project description:A novel peptide substrate (A G G P L G P P G P G G) was developed for quantifying the activities of bacterial enzymes using a highly sensitive Fluorescence Resonance Energy Transfer (FRET) based assay. The peptide substrate was cleaved by collagenase class I, II, Liberase MTF C/T, collagenase NB1, and thermolysin/neutral protease, which was significantly enhanced in the presence of CaCl2. However, the activities of these enzymes were significantly decreased in the presence of ZnSO4 or ZnCl2. Collagenase I, II, Liberase MTF C/T, thermolysin/neutral protease share similar cleavage sites, L↓G and P↓G. However, collagenase NB1 cleaves the peptide substrate at G↓P and P↓L, in addition to P↓G. The enzyme activity is pH dependent, within a range of 6.8 to 7.5, but was significantly diminished at pH 8.0. Interestingly, the peptide substrate was not cleaved by endogenous pancreatic protease such as trypsin, chymotrypsin, and elastase. In conclusion, the novel peptide substrate is collagenase, thermolysin/neutral protease specific and can be applied to quantify enzyme activities from different microbes. Furthermore, the assay can be used for fine-tuning reaction mixtures of various agents to enhance the overall activity of a cocktail of multiple enzymes and achieve optimal organ/tissue digestion, while protecting the integrity of the target cells.

| S-EPMC5347087 | biostudies-literature

Thiol-selective fluorogenic probes for labeling and release.

Project description:Thiol alkylation is a powerful technique for the labeling of proteins. We report a new class of highly reactive, selective, and fluorogenic probes for thiols in aqueous solution at neutral pH, based on the 7-oxanorbornadiene (OND) framework. The maleate moiety in 7-oxabicyclo[2.2.1]hept-2,5-diene-2,3-dicarboxylic acid esters serves as both a tunable electrophile and an intramolecular quencher of an attached dansyl fluorophore. Thiols have been found to add with high rates (second-order rate constants of 40-200 M(-1) s(-1)) to give adducts that exhibit enhancements of fluorescence intensity up to 180-fold. The resulting adducts are also versatile with respect to cleavage (release) reactions by two mechanisms. First, retro-Diels-Alder fragmentation occurs with half-lives from days to weeks at room temperature, and an epoxide derivative is also reported that is incapable of cycloreversion cleavage. Second, monoamide OND derivatives undergo rapid closure to succinimides upon thiol addition, providing a thiol-triggered mechanism for immediate alcohol release. Peptides and proteins containing free thiol groups were labeled with OND electrophiles with high chemoselectivity. Since the system is so easily assembled from readily accessible modules, various functional groups can be added to OND linkers to allow the attachment of other molecules of interest.

| S-EPMC3166955 | biostudies-literature

Selective fluorogenic chemosensors for distinct classes of nucleases.

Project description:NUCLEASE SENSOR TRIO: Fluorogenic DNA sensors were developed for distinct classes of nucleases: 3'-exonucleases, 5'-exonucleases, and endonucleases. The highly selective sensors, built from very small modified DNA oligomers containing the unnatural fluorescent base pyrene, and employing thymine as a quencher, were found to function in a variety of complex biological media.

| S-EPMC3770146 | 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