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The outcome of cell signaling depends not only on signal strength but also on temporal progression. We use Fluorescence Lifetime Imaging of Resonance Energy Transfer (FLIM/FRET) biosensors to investigate intracellular signaling dynamics. We examined the β1 receptor-G αs -cAMP signaling axis using both widefield frequency domain FLIM (fdFLIM) and fast confocal time-correlated single photon counting (TCSPC) setups. Unexpectedly, we observed that fdFLIM revealed transient cAMP responses in HeLa and Cos7 cells, contrasting with sustained responses as detected with TCSPC. Investigation revealed no light-induced effects on cAMP generation or breakdown. Rather, folic acid present in the imaging medium appeared to be the culprit, as its excitation with blue light sensitized degradation of β1 agonists. Our findings highlight the impact of subtle phototoxicity on experimental outcomes, advocating confocal TCSPC for reliable analysis of response kinetics and stressing the need for full disclosure of chemical formulations by scientific vendors., Competing Interests: F.E.O. is a co-founder and shareholder of UbiQ Bio BV., (© 2024 The Authors.)

Both severe acute respiratory syndrome coronavirus 1 and 2 (SARS-CoV-1 and SARS-CoV-2) encode a papain-like protease (PLpro), which plays a vital role in viral propagation. PLpro accomplishes this function by processing the viral polyproteins essential for viral replication and removing the small proteins, ubiquitin and ISG15 from the host's key immune signaling proteins, thereby preventing the host's innate immune response. Although PLpro from both SARS-CoV-1 and SARS-CoV-2 are structurally highly similar (83% sequence identity), they exhibit functional variability. Hence, to further elucidate the mechanism and aid in drug discovery efforts, the biochemical and kinetic characterization of PLpro is needed. This chapter describes step-by-step experimental procedures for evaluating PLpro activity in vitro using activity-based probes (ABPs) along with fluorescence-based substrates. Herein we describe a step-by-step experimental procedure to assess the activity of PLpro in vitro using a suite of activity-based probes (ABPs) and fluorescent substrates and how they can be applied as fast and yet sensitive methods to calculate kinetic parameters., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The E1 enzyme Uba6 initiates signal transduction by activating ubiquitin and the ubiquitin-like protein FAT10 in a two-step process involving sequential catalysis of adenylation and thioester bond formation. To gain mechanistic insights into these processes, we determined the crystal structure of a human Uba6/ubiquitin complex. Two distinct architectures of the complex are observed: one in which Uba6 adopts an open conformation with the active site configured for catalysis of adenylation, and a second drastically different closed conformation in which the adenylation active site is disassembled and reconfigured for catalysis of thioester bond formation. Surprisingly, an inositol hexakisphosphate (InsP6) molecule binds to a previously unidentified allosteric site on Uba6. Our structural, biochemical, and biophysical data indicate that InsP6 allosterically inhibits Uba6 activity by altering interconversion of the open and closed conformations of Uba6 while also enhancing its stability. In addition to revealing the molecular mechanisms of catalysis by Uba6 and allosteric regulation of its activities, our structures provide a framework for developing Uba6-specific inhibitors and raise the possibility of allosteric regulation of other E1s by naturally occurring cellular metabolites., (© 2022. The Author(s).)

Viral papain-like cysteine protease (PLpro, NSP3) is essential for SARS-CoV-2 replication and represents a promising target for the development of antiviral drugs. Here, we used a combinatorial substrate library and performed comprehensive activity profiling of SARS-CoV-2 PLpro. On the scaffold of the best hits from positional scanning, we designed optimal fluorogenic substrates and irreversible inhibitors with a high degree of selectivity for SARS PLpro. We determined crystal structures of two of these inhibitors in complex with SARS-CoV-2 PLpro that reveals their inhibitory mechanisms and provides a molecular basis for the observed substrate specificity profiles. Last, we demonstrate that SARS-CoV-2 PLpro harbors deISGylating activity similar to SARSCoV-1 PLpro but its ability to hydrolyze K48-linked Ub chains is diminished, which our sequence and structure analysis provides a basis for. Together, this work has revealed the molecular rules governing PLpro substrate specificity and provides a framework for development of inhibitors with potential therapeutic value or drug repurposing., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Protein ubiquitination is a key post-translational modification regulating a wide range of biological processes. Ubiquitination involves the covalent attachment of the small protein ubiquitin to a lysine of a protein substrate. In addition to its well-established role in protein degradation, protein ubiquitination plays a role in protein-protein interactions, DNA repair, transcriptional regulation, and other cellular functions. Understanding the mechanisms and functional relevance of ubiquitin as a signaling system requires the generation of antibodies or alternative reagents that specifically detect ubiquitin in a site-specific manner. However, in contrast to other post-translational modifications such as acetylation, phosphorylation, and methylation, the instability and size of ubiquitin-76 amino acids-complicate the preparation of suitable antigens and the generation antibodies detecting such site-specific modifications. As a result, the field of ubiquitin research has limited access to specific antibodies. This severely hampers progress in understanding the regulation and function of site-specific ubiquitination in many areas of biology, specifically in epigenetics and cancer. Therefore, there is a high demand for antibodies recognizing site-specific ubiquitin modifications. Here we describe a strategy for the development of site-specific ubiquitin antibodies. Based on a recently developed antibody against site-specific ubiquitination of histone H2B, we provide detailed protocols for chemical synthesis methods for antigen preparation and discuss considerations for screening and quality control experiments., (Copyright © 2020 van Kruijsbergen, Mulder, Uckelmann, van Welsem, de Widt, Spanjaard, Jacobs, El Oualid, Ovaa and van Leeuwen.)

Ubiquitination is a process in which a protein is modified by the covalent attachment of the C-terminal carboxylic acid of ubiquitin (Ub) to the ε-amine of lysine or N-terminal methionine residue of a substrate protein or another Ub molecule. Each of the seven internal lysine residues and the N-terminal methionine residue of Ub can be linked to the C-terminus of another Ub moiety to form 8 distinct Ub linkages and the resulting differences in linkage types elicit different Ub signaling pathways. Cellular responses are triggered when proteins containing ubiquitin-binding domains (UBDs) recognize and bind to specific polyUb linkage types. To get more insight into the differences between polyUb chains, all of the seven lysine-linked di-ubiquitin molecules (diUbs) were prepared and used as a model to study their structural conformations in solution using NMR spectroscopy. We report the synthesis of diUb molecules, fully 15 N-labeled on the distal (N-terminal) Ub moiety and revealed their structural orientation with respect to the proximal Ub. As expected, the diUb molecules exist in different conformations in solution, with multiple conformations known to exist for K6-, K48-, and K63-linked diUb molecules. These multiple conformations allow structural flexibility in binding with UBDs thereby inducing unique responses. One of the well-known but poorly understood UBD-Ub interaction is the recognition of K6 polyubiquitin by the ubiquitin-associated (UBA) domain of UBXN1 in the BRCA-mediated DNA repair pathway. Using our synthetic 15 N-labeled diUbs, we establish here how a C-terminally extended UBA domain of UBXN1 confers specificity to K6 diUb while the non-extended version of the domain does not show any linkage preference. We show that the two distinct conformations of K6 diUb that exist in solution converge into a single conformation upon binding to this extended form of the UBA domain of the UBXN1 protein. It is likely that more of such extended UBA domains exist in nature and can contribute to linkage-specificity in Ub signaling. The isotopically labeled diUb compounds described here and the use of NMR to study their interactions with relevant partner molecules will help accelerate our understanding of Ub signaling pathways., (Copyright © 2020 Shahul Hameed, van Tilburg, Merkx, Flierman, Wienk, El Oualid, Hofmann, Boelens and Ovaa.)

OTULIN (OTU Deubiquitinase With Linear Linkage Specificity) specifically hydrolyzes methionine1 (Met1)-linked ubiquitin chains conjugated by LUBAC (linear ubiquitin chain assembly complex). Here we report on the mass spectrometric identification of the OTULIN interactor SNX27 (sorting nexin 27), an adaptor of the endosomal retromer complex responsible for protein recycling to the cell surface. The C-terminal PDZ-binding motif (PDZbm) in OTULIN associates with the cargo-binding site in the PDZ domain of SNX27. By solving the structure of the OTU domain in complex with the PDZ domain, we demonstrate that a second interface contributes to the selective, high affinity interaction of OTULIN and SNX27. SNX27 does not affect OTULIN catalytic activity, OTULIN-LUBAC binding or Met1-linked ubiquitin chain homeostasis. However, via association, OTULIN antagonizes SNX27-dependent cargo loading, binding of SNX27 to the VPS26A-retromer subunit and endosome-to-plasma membrane trafficking. Thus, we define an additional, non-catalytic function of OTULIN in the regulation of SNX27-retromer assembly and recycling to the cell surface.

Serine hydroxymethyltransferase 2 (SHMT2) regulates one-carbon transfer reactions that are essential for amino acid and nucleotide metabolism, and uses pyridoxal-5'-phosphate (PLP) as a cofactor. Apo SHMT2 exists as a dimer with unknown functions, whereas PLP binding stabilizes the active tetrameric state. SHMT2 also promotes inflammatory cytokine signalling by interacting with the deubiquitylating BRCC36 isopeptidase complex (BRISC), although it is unclear whether this function relates to metabolism. Here we present the cryo-electron microscopy structure of the human BRISC-SHMT2 complex at a resolution of 3.8 Å. BRISC is a U-shaped dimer of four subunits, and SHMT2 sterically blocks the BRCC36 active site and inhibits deubiquitylase activity. Only the inactive SHMT2 dimer-and not the active PLP-bound tetramer-binds and inhibits BRISC. Mutations in BRISC that disrupt SHMT2 binding impair type I interferon signalling in response to inflammatory stimuli. Intracellular levels of PLP regulate the interaction between BRISC and SHMT2, as well as inflammatory cytokine responses. These data reveal a mechanism in which metabolites regulate deubiquitylase activity and inflammatory signalling.

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.

Promiscuous inhibitors of tyrosine protein kinases, proteases and phosphatases are useful reagents for probing regulatory pathways and stabilizing lysates as well as starting points for the design of more selective agents. Ubiquitination regulates many critical cellular processes, and promiscuous inhibitors of deubiquitinases (DUBs) would be similarly valuable. The currently available promiscuous DUB inhibitors are highly reactive electrophilic compounds that can crosslink proteins. Herein we introduce diarylcarbonate esters as a novel class of promiscuous DUB inhibitors that do not have the liabilities associated with the previously reported compounds. Diarylcarbonates stabilize the high molecular weight ubiquitin pools in cells and lysates. They also elicit cellular phenotypes associated with DUB inhibition, demonstrating their utility in ubiquitin discovery. Diarylcarbonates may also be a useful scaffold for the development of specific DUB inhibitors., (Copyright © 2018. Published by Elsevier Ltd.)

The histone methyltransferase Dot1 is conserved from yeast to human and methylates lysine 79 of histone H3 (H3K79) on the core of the nucleosome. H3K79 methylation by Dot1 affects gene expression and the response to DNA damage, and is enhanced by monoubiquitination of the C-terminus of histone H2B (H2Bub1). To gain more insight into the functions of Dot1, we generated genetic interaction maps of increased-dosage alleles of DOT1. We identified a functional relationship between increased Dot1 dosage and loss of the DUB module of the SAGA co-activator complex, which deubiquitinates H2Bub1 and thereby negatively regulates H3K79 methylation. Increased Dot1 dosage was found to promote H2Bub1 in a dose-dependent manner and this was exacerbated by the loss of SAGA-DUB activity, which also caused a negative genetic interaction. The stimulatory effect on H2B ubiquitination was mediated by the N-terminus of Dot1, independent of methyltransferase activity. Our findings show that Dot1 and H2Bub1 are subject to bi-directional crosstalk and that Dot1 possesses chromatin regulatory functions that are independent of its methyltransferase activity.

Activity-based probes (ABPs) are widely used to monitor the activity of enzyme families in biological systems. Inferring enzyme activity from probe reactivity requires that the probe reacts with the enzyme at its active site; however, probe-labeling sites are rarely verified. Here we present an enhanced chemoproteomic approach to evaluate the activity and probe reactivity of deubiquitinase enzymes, using bioorthogonally tagged ABPs and a sequential on-bead digestion protocol to enhance the identification of probe-labeling sites. We confirm probe labeling of deubiquitinase catalytic Cys residues and reveal unexpected labeling of deubiquitinases on non-catalytic Cys residues and of non-deubiquitinase proteins. In doing so, we identify ZUFSP (ZUP1) as a previously unannotated deubiquitinase with high selectivity toward cleaving K63-linked chains. ZUFSP interacts with and modulates ubiquitination of the replication protein A (RPA) complex. Our reactive-site-centric chemoproteomics method is broadly applicable for identifying the reaction sites of covalent molecules, which may expand our understanding of enzymatic mechanisms.

The methionine 1 (M1)-specific deubiquitinase (DUB) OTULIN acts as a negative regulator of nuclear factor κB signaling and immune homeostasis. By replacing Gly76 in distal ubiquitin (Ub) by dehydroalanine we designed the diubiquitin (diUb) activity-based probe Ub G76Dha -Ub (OTULIN activity-based probe [ABP]) that couples to the catalytic site of OTULIN and thereby captures OTULIN in its active conformation. The OTULIN ABP displays high selectivity for OTULIN and does not label other M1-cleaving DUBs, including CYLD. The only detectable cross-reactivities were the labeling of USP5 (Isopeptidase T) and an ATP-dependent assembly of polyOTULIN ABP chains via Ub-activating E1 enzymes. Both cross-reactivities were abolished by the removal of the C-terminal Gly in the ABP's proximal Ub, yielding the specific OTULIN probe Ub G76Dha -Ub ΔG76 (OTULIN ABPΔG76). Pull-downs demonstrate that substrate-bound OTULIN associates with the linear ubiquitin chain assembly complex (LUBAC). Thus, we present a highly selective ABP for OTULIN that will facilitate studying the cellular function of this essential DUB., (Copyright © 2017 MRC Laboratory of Molecular Biology. Published by Elsevier Ltd.. All rights reserved.)

Ubiquitin (Ub) signaling is a diverse group of processes controlled by covalent attachment of small protein Ub and polyUb chains to a range of cellular protein targets. The best documented Ub signaling pathway is the one that delivers polyUb proteins to the 26S proteasome for degradation. However, studies of molecular interactions involved in this process have been hampered by the transient and hydrophobic nature of these interactions and the lack of tools to study them. Here, we develop Ub-phototrap (Ub PT ), a synthetic Ub variant containing a photoactivatable crosslinking side chain. Enzymatic polymerization into chains of defined lengths and linkage types provided a set of reagents that led to identification of Rpn1 as a third proteasome ubiquitin-associating subunit that coordinates docking of substrate shuttles, unloading of substrates, and anchoring of polyUb conjugates. Our work demonstrates the value of Ub PT , and we expect that its future uses will help define and investigate the ubiquitin interactome., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Post-translational modifications of proteins with ubiquitin (Ub) and ubiquitin-like modifiers (Ubls), orchestrated by a cascade of specialized E1, E2 and E3 enzymes, control a wide range of cellular processes. To monitor catalysis along these complex reaction pathways, we developed a cascading activity-based probe, UbDha. Similarly to the native Ub, upon ATP-dependent activation by the E1, UbDha can travel downstream to the E2 (and subsequently E3) enzymes through sequential trans-thioesterifications. Unlike the native Ub, at each step along the cascade, UbDha has the option to react irreversibly with active site cysteine residues of target enzymes, thus enabling their detection. We show that our cascading probe 'hops' and 'traps' catalytically active Ub-modifying enzymes (but not their substrates) by a mechanism diversifiable to Ubls. Our founder methodology, amenable to structural studies, proteome-wide profiling and monitoring of enzymatic activity in living cells, presents novel and versatile tools to interrogate Ub and Ubl cascades.

The anticancer properties of cruciferous vegetables are well known and attributed to an abundance of isothiocyanates such as benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC). While many potential targets of isothiocyanates have been proposed, a full understanding of the mechanisms underlying their anticancer activity has remained elusive. Here we report that BITC and PEITC effectively inhibit deubiquitinating enzymes (DUB), including the enzymes USP9x and UCH37, which are associated with tumorigenesis, at physiologically relevant concentrations and time scales. USP9x protects the antiapoptotic protein Mcl-1 from degradation, and cells dependent on Mcl-1 were especially sensitive to BITC and PEITC. These isothiocyanates increased Mcl-1 ubiquitination and either isothiocyanate treatment, or RNAi-mediated silencing of USP9x decreased Mcl-1 levels, consistent with the notion that USP9x is a primary target of isothiocyanate activity. These isothiocyanates also increased ubiquitination of the oncogenic fusion protein Bcr-Abl, resulting in degradation under low isothiocyanate concentrations and aggregation under high isothiocyanate concentrations. USP9x inhibition paralleled the decrease in Bcr-Abl levels induced by isothiocyanate treatment, and USP9x silencing was sufficient to decrease Bcr-Abl levels, further suggesting that Bcr-Abl is a USP9x substrate. Overall, our findings suggest that USP9x targeting is critical to the mechanism underpinning the well-established anticancer activity of isothiocyanate. We propose that the isothiocyanate-induced inhibition of DUBs may also explain how isothiocyanates affect inflammatory and DNA repair processes, thus offering a unifying theme in understanding the function and useful application of isothiocyanates to treat cancer as well as a variety of other pathologic conditions., (©2015 American Association for Cancer Research.)

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., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)