Activity-Based Ubiquitin Probes Capture the Sulfenylated State of Deubiquitinases
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo Sapiens
SUBMITTER: 
Jing Huang
PROVIDER: PXD067721 | iProX | Tue Aug 26 00:00:00 BST 2025
REPOSITORIES: iProX
Similar Datasets
Project description:Ubiquitination is emerging as an important post-translational modification (PTM) for numerous cellular functions including protein degradation, DNA damage repair and tolerance, and cell cycle progression. Compared with other small-molecule modifiers found in phosphorylation, acetylation and glycosylation, ubiquitin is a small protein modifier that exists as either a single ubiquitin or a polyubiquitin chain. Furthermore, the polyubiquitin chains are formed via various linkages imparting an additional layer of specificity in cellular signaling. In order to adequately study ubiquitin signaling and particularly deubiquitination, a number of ubiquitin activity-based probes (ABPs) were developed and utilized in understanding the deubiquitinase (DUBs) function. Here, we focus on the current state of the DUB ABP development and their application in understanding DUB function and specificity for polyubiquitin chains and ubiquitinated proteins.
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Project description:Post-translational modification of proteins by Ubiquitin (Ub) and Ubiquitin-like proteins (Ubls) can be reversed by deconjugating enzymes, which have been implicated in different pathways and associated with various human diseases. To understand the activity and dynamics of deconjugating enzymes, multiple synthetic and semi-synthetic Ub/Ubl probes have been developed, and some of them have been applied to screen inhibitors of deconjugating enzymes. Since these Ub/Ubl probes are generally not cell-permeable, different strategies have been developed to deliver Ub/Ubl probes to live cells. However, till now no Ub/Ubl probes can be expressed in live cells to directly report on the activities of deconjugating enzymes in the most relevant cellular environment. Here, we genetically encoded cross-linkable Ub/Ubl probes in live E. coli and HEK293T cells. These probes can cross-link with deconjugating enzymes in vitro and in vivo, and the extent of cross-linking is dependent on the activities of deconjugating enzymes.
2022-09-23 | PXD036955 |
Project description:Activity-based protein profiling is an invaluable technique for studying enzyme biology and facilitating the development of therapeutics. Ubiquitin E3 ligases (E3s) are one of the largest enzyme families and regulate a host of (patho)physiological processes. The largest subtype are the RING E3s of which there are >600 members. RING E3s have adaptor-like activity that can be subject to diverse regulatory mechanisms and have become attractive drug targets. Activity-based probes (ABPs) for measuring RING E3 activity do not exist. Here we re-engineer ubiquitin-charged E2 conjugating enzymes to produce photocrosslinking ABPs. We demonstrate activity-dependent profiling of two divergent cancer-associated RING E3s, RNF4 and c-Cbl, in response to their native activation signals. We also demonstrate profiling of endogenous RING E3 ligase activation in response to epidermal growth factor (EGF) stimulation. These photocrosslinking ABPs should advance E3 ligase research and the development of selective modulators against this important class of enzymes.
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Project description:Advancement in our knowledge of deubiquitinases (DUBs) and their biological functions requires biochemical tools permitting interrogation of DUB activities under physiologically relevant conditions. Activity-based DUB probes (DUB ABPs) have been widely used in investigating the function and activity of DUBs. However, most ubiquitin (Ub)-based DUB ABPs are not cell-permeable, limiting their utility to purified proteins and cell lysates. Lysis of cells usually leads to dilution of the cytoplasm and disruption of the normal cellular organization, which may alter the activity of many DUBs and DUB complexes. Here, we report a new class of cell-permeable DUB ABPs that enable intracellular DUB profiling. We used a semisynthetic approach to generate modular ubiquitin-based DUB probes containing a reactive warhead for covalent trapping of DUBs with a catalytic cysteine. We employed cell-penetrating peptides (CPPs), particualrly cyclic polyarginine (cR10), to deliver the DUB ABPs into cells, as confirmed using live-cell fluorescence microscopy and DUB ABPs containing a fluorophore at the C-terminus of Ub. In comparison to TAT, enhanced intacellular delivery was observed through conjugation of a cyclic polyarginine (cR10) to the N-terminus of ubiquitin via a disulfide linkage. Using the new cell-permeable DUB ABPs, we carried out DUB profiling in intact HeLa cells, and identified active DUBs using immunocapture and label-free quantitative mass spectrometry. Additionally, we demonstrated that the cell-permeable DUB ABPs can be used in assessing the inhibition of DUBs by small-molecule inhibitors in intact cells. Our results indicate that cell-permeable DUB ABPs hold great promise in providing a better understanding of the cellular functions of DUBs and advancing drug discovery efforts targeting human DUBs.
| S-EPMC8011864 | biostudies-literature
Project description:Ubiquitination is an essential eukaryotic post-translational modification that regulates various cellular processes. The removal of ubiquitin from its target protein is catalyzed by deubiquitinating enzymes (DUBs). Although it was proposed that many DUBs specifically interact and recognize ubiquitinated proteins as substrates, more direct evidence is needed to support this notion. Here we report protein-targeting activity-based DUB probes that allowed the identification of DUBs recognizing monoubiquitinated proliferating cell nuclear antigen (PCNA) in Saccharomyces cerevisiae. This new class of DUB probes contain a Michael acceptor as a warhead between ubiquitin and the target protein PCNA through a linkage that mimics the native isopeptide bond. We selected two known and biologically relevant ubiquitination sites on PCNA to generate the DUB probes. This allowed us to interrogate the site-specific deubiquitination of a target protein by DUBs. DUBs were profiled in yeast cell lysates using the two Ub-PCNA DUB probes in conjunction with two control probes that contain a noncleavable linkage but no warhead. We identified yeast DUBs through pulldown coupled with quantitative mass spectrometry analysis of the pulled down proteins. Our results showed that specific yeast DUBs recognize monoubiquitinated PCNA and corroborated previous genetic study. We also identified DUBs as potential new deubiquitinase of PCNA. Remarkably, identified DUBs clearly distinguish the different modification sites on PCNA, thus supporting a high level of DUB specificity beyond the target protein identity.
| S-EPMC6194582 | biostudies-literature
Project description: Not available
| S-EPMC3021723 | biostudies-literature
Project description:Protein modification by ubiquitin (Ub) and ubiquitin-like modifiers (Ubl) requires the action of activating (E1), conjugating (E2), and ligating (E3) enzymes and is a key step in the specific destruction of proteins. Deubiquitinating enzymes (DUBs) deconjugate substrates modified with Ub/Ubl's and recycle Ub inside the cell. Genome mining based on sequence homology to proteins with known function has assigned many enzymes to this pathway without confirmation of either conjugating or DUB activity. Function-dependent methodologies are still the most useful for rapid identification or assessment of biological activity of expressed proteins from cells. Activity-based protein profiling uses chemical probes that are active-site-directed for the classification of protein activities in complex mixtures. Here we show that the design and use of an expanded set of Ub-based electrophilic probes allowed us to recover and identify members of each enzyme class in the ubiquitin-proteasome system, including E3 ligases and DUBs with previously unverified activity. We show that epitope-tagged Ub-electrophilic probes can be used as activity-based probes for E3 ligase identification by in vitro labeling and activity studies of purified enzymes identified from complex mixtures in cell lysate. Furthermore, the reactivity of our probe with the HECT domain of the E3 Ub ligase ARF-BP1 suggests that multiple cysteines may be in the vicinity of the E2-binding site and are capable of the transfer of Ub to self or to a substrate protein.
| S-EPMC2693349 | biostudies-literature
Project description:The nucleophilic thiol-alkyne addition reaction can form a covalent thiovinyl bond with cysteine reactivity specific,as the classical standards for cysteine-reactive warheads in activity-based probes (ABPs).Utilizing this classical reactivity property, we designed two potentially reactive unnatural amino acids containing alkyne moiety in its side chain, named as 4-propargyloxy-L-phenylalanine (pPR) and enrichable 4-propynyloxy-L-phenylalanine (epPR),for capturing deubiquitinases or ubiquitin-like protein specific proteases in the ubiquitin (Ub) and ubiquitin-like (Ubl) modification.The technology was extended to polyubiquitin chains through Sortase A mediated transpeptidation using reduced N6-((2-azidoacetyl)glycyl)-L-lysine (AzGGK) to triglycine tail (GGK),constructing ubiquitin conjuates,such as di diubiquitin (Diub). Rare codon recoding techonology (RCR) can be used to incorporation unnatural amino acid to mammalian cell, utilizing the rare codons to introduce multiple unnatural amino acids into proteins, creating multireactive and high-efficiency crosslinking probes. Here,we genetically encoded and sortase-mediated transpeptidation to develope crosslinkable and enrichable Ub/Ubl/Diub ABPs in E.coli and HEK293T cells.Meanwhile,the alkyne cysteine specificity of the covalent binding to the deubiquitinases allows for the preliminary screening of pan and irreversible inhibitors.Since most chemical Ub/Ubl/Diub ABPs are generally not cell-permeable, we performed functional complementation studies using genetically encoded biosynthetic Ub/Ubl/Diub ABPs. Meanwhile, the insertion of unnatural amino acids at multiple sites of UFM1 multi-reactive crosslinking probe was realized to be expressed in living cells by using the RCR technology, providing a novel methodology for comprehensive mapping of UFM1 interaction networks.
2025-05-14 | PXD063756 |
Project description:HA antibody beads were used to pulldown HA proteins in a cell lysate. The HA protein is an activty based probe (consisting of a small protein and a warhead) and was spiked into the cell lysate. 35, 156, 149 and 61 are different probes. Experiments were performed with and without pretreatment of the cell lysate with IAA an alkylating agent which should reduce the binding between the probe and its targets in cell lysate (cysteine based proteases).
2022-11-29 | MSV000090802 | MassIVE
Project description:Epitope-tagged active-site-directed probes are widely used to visualize the activity of deubiquitinases (DUBs) in cell extracts, to investigate the specificity and potency of small-molecule DUB inhibitors, and to isolate and identify DUBs by mass spectrometry. With DUBs arising as novel potential drug targets, probes are required that can be produced in sufficient amounts and to meet the specific needs of a given experiment. The established method for the generation of DUB probes makes use of labor-intensive intein-based methods that have inherent limitations concerning the incorporation of unnatural amino acids and the amount of material that can be obtained. Here, we describe the total chemical synthesis of active-site-directed probes and their application to activity-based profiling and identification of functional DUBs. This synthetic methodology allowed the easy incorporation of desired tags for specific applications, for example, fluorescent reporters, handles for immunoprecipitation or affinity pull-down, and cleavable linkers. Additionally, the synthetic method can be scaled up to provide significant amounts of probe. Fluorescent ubiquitin probes allowed faster, in-gel detection of active DUBs, as compared to (immuno)blotting procedures. A biotinylated probe holding a photocleavable linker enabled the affinity pull-down and subsequent mild, photorelease of DUBs. Also, DUB activity levels were monitored in response to overexpression or knockdown, and to inhibition by small molecules. Furthermore, fluorescent probes revealed differential DUB activity profiles in a panel of lung and prostate cancer cells.
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