The deubiquitinating enzyme BAP1 is an important tumor suppressor that has drawn attention in the clinic since its loss leads to a variety of cancers. BAP1 is activated by ASXL1 to deubiquitinate ...mono-ubiquitinated H2A at K119 in Polycomb gene repression, but the mechanism of this reaction remains poorly defined. Here we show that the BAP1 C-terminal extension is important for H2A deubiquitination by auto-recruiting BAP1 to nucleosomes in a process that does not require the nucleosome acidic patch. This initial encounter-like complex is unproductive and needs to be activated by the DEUBAD domains of ASXL1, ASXL2 or ASXL3 to increase BAP1's affinity for ubiquitin on H2A, to drive the deubiquitination reaction. The reaction is specific for Polycomb modifications of H2A as the complex cannot deubiquitinate the DNA damage-dependent ubiquitination at H2A K13/15. Our results contribute to the molecular understanding of this important tumor suppressor.
Sixteen ovarian tumor (OTU) family deubiquitinases (DUBs) exist in humans, and most members regulate cell-signaling cascades. Several OTU DUBs were reported to be ubiquitin (Ub) chain linkage ...specific, but comprehensive analyses are missing, and the underlying mechanisms of linkage specificity are unclear. Using Ub chains of all eight linkage types, we reveal that most human OTU enzymes are linkage specific, preferring one, two, or a defined subset of linkage types, including unstudied atypical Ub chains. Biochemical analysis and five crystal structures of OTU DUBs with or without Ub substrates reveal four mechanisms of linkage specificity. Additional Ub-binding domains, the ubiquitinated sequence in the substrate, and defined S1’ and S2 Ub-binding sites on the OTU domain enable OTU DUBs to distinguish linkage types. We introduce Ub chain restriction analysis, in which OTU DUBs are used as restriction enzymes to reveal linkage type and the relative abundance of Ub chains on substrates.
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•The 16 human OTU DUBs cleave distinct sets of ubiquitin chain types•Five crystal structures of three human OTU DUBs reveal uncharacterized Ub-binding sites•We reveal four distinct mechanisms of linkage specificity in OTU DUBs•OTU DUBs can be used to identify the linkage types on a ubiquitinated substrate
OTU deubiquitinases use four distinct mechanisms of linkage specificity to hydrolyze ubiquitin, and, due to their specificity, OTU DUBs can be used in ubiquitin chain restriction analysis to characterize the chain types on ubiquitinated proteins.
Deubiquitinating enzymes (DUBs) recognize and cleave linkage-specific polyubiquitin (polyUb) chains, but mechanisms underlying specificity remain elusive in many cases. The severe acute respiratory ...syndrome (SARS) coronavirus papain-like protease (PLpro) is a DUB that cleaves ISG15, a two-domain Ub-like protein, and Lys48-linked polyUb chains, releasing diUbLys48 products. To elucidate this specificity, we report the 2.85 Å crystal structure of SARS PLpro bound to a diUbLys48 activity-based probe. SARS PLpro binds diUbLys48 in an extended conformation via two contact sites, S1 and S2, which are proximal and distal to the active site, respectively. We show that specificity for polyUbLys48 chains is predicated on contacts in the S2 site and enhanced by an S1-S1′ preference for a Lys48 linkage across the active site. In contrast, ISG15 specificity is dominated by contacts in the S1 site. Determinants revealed for polyUbLys48 specificity should prove useful in understanding PLpro deubiquitinating activities in coronavirus infections.
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•A Lys48 linkage-specific diubiquitin activity-based probe selectively labels SARS PLpro•The structure of a diUbLys48∼SARS PLpro complex reveals an extended di-Ub conformation•S2-S1 and S1-S1′ interactions make SARS PLpro specific for K48-linked polyubiquitin•SARS PLpro recognizes Lys48-linked polyUb chains and ISG15 via distinct manners
Békés et al. present a high-resolution crystal structure of a SARS virus PLpro∼diUbLys48 complex that reveals an extended conformation of the Lys48-linked diUb unit and shows the biochemical basis for SARS PLpro’s preference for Lys48-linked polyUb chains.
Changing the subject: An efficient linear solid‐phase peptide synthesis of ubiquitin (Ub) has been developed. This approach allows the incorporation of desired tags and mutations (see picture; blue ...denotes a pseudoproline dipeptide, red a dimethoxybenzyl dipeptide) as well as specific C‐terminal modification and the construction of all diubiquitin conjugates in high yields and purities in a straightforward manner.
USP7 is a highly abundant deubiquitinating enzyme (DUB), involved in cellular processes including DNA damage response and apoptosis. USP7 has an unusual catalytic mechanism, where the low intrinsic ...activity of the catalytic domain (CD) increases when the C-terminal Ubl domains (Ubl45) fold onto the CD, allowing binding of the activating C-terminal tail near the catalytic site. Here we delineate how the target protein promotes the activation of USP7. Using NMR analysis and biochemistry we describe the order of activation steps, showing that ubiquitin binding is an instrumental step in USP7 activation. Using chemically synthesised p53-peptides we also demonstrate how the correct ubiquitinated substrate increases catalytic activity. We then used transient reaction kinetic modelling to define how the USP7 multistep mechanism is driven by target recognition. Our data show how this pleiotropic DUB can gain specificity for its cellular targets.
Deubiquitinating enzymes (DUBs) control vital processes in eukaryotes by hydrolyzing ubiquitin adducts. Their activities are tightly regulated, but the mechanisms remain elusive. In particular, the ...DUB UCH-L5 can be either activated or inhibited by conserved regulatory proteins RPN13 and INO80G, respectively. Here we show how the DEUBAD domain in RPN13 activates UCH-L5 by positioning its C-terminal ULD domain and crossover loop to promote substrate binding and catalysis. The related DEUBAD domain in INO80G inhibits UCH-L5 by exploiting similar structural elements in UCH-L5 to promote a radically different conformation, and employs molecular mimicry to block ubiquitin docking. In this process, large conformational changes create small but highly specific interfaces that mediate activity modulation of UCH-L5 by altering the affinity for substrates. Our results establish how related domains can exploit enzyme conformational plasticity to allosterically regulate DUB activity. These allosteric sites may present novel insights for pharmaceutical intervention in DUB activity.
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•The RPN13 DEUBAD domain activates UCH-L5 by positioning its CL and ULD domain•The INO80G DEUBAD domain inhibits UCH-L5 by blocking ubiquitin binding•The FRF hairpin in the DEUBAD domain of INO80G drives UCH-L5 inhibition•DEUBAD domains regulate UCH-L5 activity by tuning UCH-L5 substrate affinity
Deubiquitinating enzyme UCH-L5 (UCH37) is involved in DNA repair and the proteasome system. Sahtoe et al. uncover how two evolutionarily related DEUBAD domains in RPN13 (ADRM1) and INO80G (NFRKB) can either activate or inhibit UCH-L5. These remarkable regulatory modes exploit flexible structural elements in UCH-L5 to modulate activity.
Ubiquitin chains are important post-translational modifications that control a large number of cellular processes. Chains can be formed via different linkages, which determines the type of signal ...they convey. Deubiquitylating enzymes (DUBs) regulate ubiquitylation status by trimming or removing chains from attached proteins. DUBs can contain several ubiquitin-binding pockets, which confer specificity toward differently linked chains. Most tools for monitoring DUB specificity target binding pockets on opposing sides of the active site; however, some DUBs contain additional pockets. Therefore, reagents targeting additional pockets are essential to fully understand linkage specificity. We report the development of active site-directed probes and fluorogenic substrates, based on non-hydrolyzable diubiquitin, that are equipped with a C-terminal warhead or a fluorogenic activity reporter moiety. We demonstrate that various DUBs in lysates display differential reactivity toward differently linked diubiquitin probes, as exemplified by the proteasome-associated DUB USP14. In addition, OTUD2 and OTUD3 show remarkable linkage-specific reactivity with our diubiquitin-based reagents.
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•Protease-resistant diUb probes bind to DUB S1-S2 sites and react at the proximal end•First kinetic assay showing proximal end cleavage by DUBs using diUb-based substrates•OTUD3 binds K11-linked diUb and OTUD2 binds K11- and K33-linked diUb in S1-S2 pockets•Kinetics suggest different mechanisms for polyUb cleavage by OTUD2 and OTUD3
Deubiquitylating enzymes (DUBs) use multiple ubiquitin-binding pockets to disassemble polyubiquitin chains. Most tools probing DUB specificity target pockets adjacent to the active site. The protease-resistant diUb probes described here monitor linkage specificity of an additional S2 pocket and provide kinetics that explain polyubiquitin chain specificity.
In LC−MS, derivatization is primarily used to improve ionization characteristics, especially for analytes that are not (efficiently) ionized by ESI or APCI such as aldehydes, sugars, and steroids. ...Derivatization strategies are then directed at the incorporation of a group with a permanent charge. A compound class that typically requires derivatization prior to LC−MS is the group of small aliphatic aldehydes that are, for instance, analyzed as the key biomarkers for lipid peroxidation in organisms. Here we report the development of a new tailor-made, highly sensitive, and selective derivatization agent 4-(2-(trimethylammonio)ethoxy)benzenaminium halide (4-APC) for the quantification of aldehydes in biological matrixes with positive ESI-MS/MS without additional extraction procedures. 4-APC possesses an aniline moiety for a fast selective reaction with aliphatic aldehydes as well as a quaternary ammonium group for improved MS sensitivity. The derivatization reaction is a convenient one-pot reaction at a mild pH (5.7) and temperature (10 °C). As a result, an in-vial derivatization can be performed before analysis with an LC−MS/MS system. All aldehydes are derivatized within 30 min to a plateau, except malondialdehyde, which requires 300 min to reach a plateau. All derivatized aldehydes are stable for at least 35 h. Linearity was established between 10 and 500 nM and the limits of detection were in the 3−33 nM range for the aldehyde derivatives. Furthermore, the chosen design of these structures allows tandem MS to be used to monitor the typical losses of 59 and 87 from aldehyde derivatives, thereby enabling screening for aldehydes. Finally, of all aldehydes, pentanal and hexanal were detected at elevated levels in pooled healthy human urine samples.
Active-site directed probes are powerful in studies of enzymatic function. We report an active-site directed probe based on a warhead so far considered unreactive. By replacing the C-terminal ...carboxylate of ubiquitin (Ub) with an alkyne functionality, a selective reaction with the active-site cysteine residue of de-ubiquitinating enzymes was observed. The resulting product was shown to be a quaternary vinyl thioether, as determined by X-ray crystallography. Proteomic analysis of proteins bound to an immobilized Ub alkyne probe confirmed the selectivity toward de-ubiquitinating enzymes. The observed reactivity is not just restricted to propargylated Ub, as highlighted by the selective reaction between caspase-1 (interleukin converting enzyme) and a propargylated peptide derived from IL-1β, a caspase-1 substrate.
•Activity-based probes used to probe DUB inhibitor specificity.•Developments in DUB activity-based probe use.•Determination of linkage preference of DUBs using activity-based probes.•Outlook for ...development in DUB probe design and current challenges.
Protein ubiquitylation is an important regulator of protein function, localization and half-life. It plays a key role in most cellular processes including immune signaling. Deregulation of this process is a major causative factor for many diseases. A major advancement in the identification and characterization of the enzymes that remove ubiquitin, deubiquitylases (DUBs) was made by the development of activity-based probes (ABPs). Recent advances in chemical protein synthesis and ligation methodology has yielded novel reagents for use in ubiquitylation research. We describe recent advances and discuss future directions in reagent development for studying DUBs.
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