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Ubiquitin (Ub) chain formation by homologous to E6AP C-terminus (HECT)-family E3 ligases regulates vast biology, yet the structural mechanisms remain unknown. We used chemistry and cryo-electron microscopy (cryo-EM) to visualize stable mimics of the intermediates along K48-linked Ub chain formation by the human E3, UBR5. The structural data reveal a ≈ 620 kDa UBR5 dimer as the functional unit, comprising a scaffold with flexibly tethered Ub-associated (UBA) domains, and elaborately arranged HECT domains. Chains are forged by a UBA domain capturing an acceptor Ub, with its K48 lured into the active site by numerous interactions between the acceptor Ub, manifold UBR5 elements and the donor Ub. The cryo-EM reconstructions allow defining conserved HECT domain conformations catalyzing Ub transfer from E2 to E3 and from E3. Our data show how a full-length E3, ubiquitins to be adjoined, E2 and intermediary products guide a feed-forward HECT domain conformational cycle establishing a highly efficient, broadly targeting, K48-linked Ub chain forging machine., (© 2023. The Author(s).)

Protein post-translational modification with ubiquitin (Ub) is a versatile signal regulating almost all aspects of cell biology, and an increasing range of diseases is associated with impaired Ub modification. In this light, the Ub system offers an attractive, yet underexplored route to the development of novel targeted treatments. A promising strategy for small molecule intervention is posed by the final components of the enzymatic ubiquitination cascade, E3 ligases, as they determine the specificity of the protein ubiquitination pathway. Here, we present UbSRhodol, an autoimmolative Ub-based probe, which upon E3 processing liberates the pro-fluorescent dye, amenable to profile the E3 transthiolation activity for recombinant and in cell-extract E3 ligases. UbSRhodol enabled detection of changes in transthiolation efficacy evoked by enzyme key point mutations or conformational changes, and offers an excellent assay reagent amenable to a high-throughput screening setup allowing the identification of small molecules modulating E3 activity., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Hybrid chains are a combination of ubiquitin (Ub) and Ub-like (UbL) proteins, expanding on the finely tuned Ub code. To decipher this intricate code, understanding of its assembly, architecture, as well as specific interactors of these Ub/UbL hybrid chains are important, warranting the development of suitable reagents. Here, we describe the chemical methodology to access linkage specific non-hydrolyzable Ub-NEDD8-based chains endowed with an affinity handle in all possible combinations of K48 hybrid chain dimers between Ub and NEDD8., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The evolutionally conserved and abundant post-translational modifier ubiquitin (Ub) is involved in a vast number of cellular processes. Imbalanced ubiquitination is associated with a range of diseases. Consequently, components of the ubiquitylation machinery, such as deubiquitinating enzymes (DUBs) that control the removal of Ub, are emerging as therapeutic targets. Here, we describe a robust assay suitable for small-molecule inhibitor screening. This assay has the potential to drive the development of small-molecule compounds that can selectively target DUBs., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Post-translational modification with Ubiquitin-like proteins represents a complex signaling language regulating virtually every cellular process. Among these post-translational modifiers is Ubiquitin-fold modifier (UFM1), which is covalently attached to its substrates through the orchestrated action of a dedicated enzymatic cascade. Originally identified to be involved embryonic development, its biological function remains enigmatic. Recent research reveals that UFM1 regulates a variety of cellular events ranging from DNA repair to autophagy and ER stress response implicating its involvement in a variety of diseases. Given the contribution of UFM1 to numerous pathologies, the enzymes of the UFM1 cascade represent attractive targets for pharmacological inhibition. Here we discuss the current understanding of this cryptic post-translational modification especially its contribution to disease as well as expand on the unmet needs of developing chemical and biochemical tools to dissect its role.

E3 ligases are typically classified by hallmark domains such as RING and RBR, which are thought to specify unique catalytic mechanisms of ubiquitin transfer to recruited substrates 1,2 . However, rather than functioning individually, many neddylated cullin-RING E3 ligases (CRLs) and RBR-type E3 ligases in the ARIH family-which together account for nearly half of all ubiquitin ligases in humans-form E3-E3 super-assemblies 3-7 . Here, by studying CRLs in the SKP1-CUL1-F-box (SCF) family, we show how neddylated SCF ligases and ARIH1 (an RBR-type E3 ligase) co-evolved to ubiquitylate diverse substrates presented on various F-box proteins. We developed activity-based chemical probes that enabled cryo-electron microscopy visualization of steps in E3-E3 ubiquitylation, initiating with ubiquitin linked to the E2 enzyme UBE2L3, then transferred to the catalytic cysteine of ARIH1, and culminating in ubiquitin linkage to a substrate bound to the SCF E3 ligase. The E3-E3 mechanism places the ubiquitin-linked active site of ARIH1 adjacent to substrates bound to F-box proteins (for example, substrates with folded structures or limited length) that are incompatible with previously described conventional RING E3-only mechanisms. The versatile E3-E3 super-assembly may therefore underlie widespread ubiquitylation.

The ubiquitin system regulates the DNA damage response (DDR) by modifying histone H2A at Lys15 (H2AK15ub) and triggering downstream signaling events. Here, we find that phosphorylation of ubiquitin at Thr12 (pUbT12) controls the DDR by inhibiting the function of 53BP1, a key factor for DNA double-strand break repair by non-homologous end joining (NHEJ). Detectable as a chromatin modification on H2AK15ub, pUbT12 accumulates in nuclear foci and is increased upon DNA damage. Mutating Thr12 prevents the removal of ubiquitin from H2AK15ub by USP51 deubiquitinating enzyme, leading to a pronounced accumulation of ubiquitinated chromatin. Chromatin modified by pUbT12 is inaccessible to 53BP1 but permissive to the homologous recombination (HR) proteins RNF169, RAD51, and the BRCA1/BARD1 complex. Phosphorylation of ubiquitin at Thr12 in the chromatin context is a new histone mark, H2AK15pUbT12, that regulates the DDR by hampering the activity of 53BP1 at damaged chromosomes., Competing Interests: Declaration of Interests H.O. is a shareholder of UbiQ B.V., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Ubiquitination, a post-translational modification, regulates a vast array of fundamental biological processes with dysregulation of the dedicated enzymes giving rise to pathologies such as cancer and neurodegenerative diseases. Assembly and its ensuing removal of this post-translational modification, determining a large variety of biological functions, is executed by a number of enzymes sequentially activating, conjugating, ligating, as well as deubiquitinating. Considering the vast impact of ubiquitination on regulating cellular homeostasis, understanding the function of these vast enzyme networks merits the development and innovation of tools. Thus, advances in synthetic strategies for generating ubiquitin, permitted the development of a plethora of ubiquitin assay reagents and numerous activity-based probes (ABPs) enable the study of enzymes involved in the complex system of ubiquitination. With ubiquitination playing such a pivotal role in the pathogenesis of a multitude of diseases, the identification of inhibitors for ubiquitin enzymes as well as the development of ABPs and high-throughput assay reagents is of utmost importance. Accordingly, this chapter will review the current state-of-the-art activity-based probes, reporter substrates, and other relevant tools based on Ub as a recognition element while highlighting the need of innovative technologies and unique concepts to study emerging facets of ubiquitin biology.

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.

Many neurodegenerative diseases, such as Huntington's disease, are hallmarked by the formation of intracellular inclusion bodies (IBs) that are decorated with ubiquitin, proteasomes and chaperones. The apparent enrichment of ubiquitin and components involved in protein quality control at IBs suggests local ubiquitin-dependent enzymatic activity. In this study, we examine recruitment of ubiquitin to IBs of polyglutamine-expanded huntingtin fragments (mHtt) by using synthesized TAMRA-labeled ubiquitin moieties. We show that intracellular TAMRA-ubiquitin is dynamic at mHtt IBs and is incorporated into poly-ubiquitin chains of intracellular substrates, such as mHtt, in a conjugation-dependent manner. Furthermore, we report that mHtt IBs recruit catalytically active enzymes involved in (de)-ubiquitination processes based on novel activity-based probes. However, we also find that the overexpression of the GFP-ubiquitin reporter, unlike the endogenous ubiquitin and TAMRA-ubiquitin, becomes irreversibly sequestered as a ring-like structure around the mHtt IBs, suggesting a methodical disadvantage of GFP-tagged ubiquitin. Our data provide supportive evidence for dynamic recruitment of ubiquitin and ubiquitin (de)-conjugating activity at mHtt initiated IBs.

Ubiquitin (Ub) and the Ub-like (Ubl) modifier interferon-stimulated gene 15 (ISG15) participate in the host defence of viral infections. Viruses, including the severe acute respiratory syndrome human coronavirus (SARS hCoV), have co-opted Ub-ISG15 conjugation pathways for their own advantage or have evolved effector proteins to counter pro-inflammatory properties of Ub-ISG15-conjugated host proteins. In the present study, we compare substrate specificities of the papain-like protease (PLpro) from the recently emerged Middle East respiratory syndrome (MERS) hCoV to the related protease from SARS, SARS PLpro. Through biochemical assays, we show that, similar to SARS PLpro, MERS PLpro is both a deubiquitinating (DUB) and a deISGylating enzyme. Further analysis of the intrinsic DUB activity of these viral proteases revealed unique differences between the recognition and cleavage specificities of polyUb chains. First, MERS PLpro shows broad linkage specificity for the cleavage of polyUb chains, whereas SARS PLpro prefers to cleave Lys48-linked polyUb chains. Secondly, MERS PLpro cleaves polyUb chains in a 'mono-distributive' manner (one Ub at a time) and SARS PLpro prefers to cleave Lys48-linked polyUb chains by sensing a di-Ub moiety as a minimal recognition element using a 'di-distributive' cleavage mechanism. The di-distributive cleavage mechanism for SARS PLpro appears to be uncommon among USP (Ub-specific protease)-family DUBs, as related USP family members from humans do not display such a mechanism. We propose that these intrinsic enzymatic differences between SARS and MERS PLpro will help to identify pro-inflammatory substrates of these viral DUBs and can guide in the design of therapeutics to combat infection by coronaviruses.

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Ubiquitin (Ub) and the Ub-like (Ubl) modifier interferonstimulated gene 15 (ISG15) participate in the host defence of viral infections. Viruses, including the severe acute respiratory syndrome human coronavirus (SARS hCoV), have co-opted Ub- ISG15 conjugation pathways for their own advantage or have evolved effector proteins to counter pro-inflammatory properties of Ub-ISG15-conjugated host proteins. In the present study, we compare substrate specificities of the papain-like protease (PLpro) from the recently emerged Middle East respiratory syndrome (MERS) hCoV to the related protease from SARS, SARS PLpro. Through biochemical assays, we show that, similar to SARS PLpro, MERS PLpro is both a deubiquitinating (DUB) and a deISGylating enzyme. Further analysis of the intrinsic DUB activity of these viral proteases revealed unique differences between the recognition and cleavage specificities of polyUb chains. First,MERS PLpro shows broad linkage specificity for the cleavage of polyUb chains, whereas SARS PLpro prefers to cleave Lys48-linked polyUb chains. Secondly, MERS PLpro cleaves polyUb chains in a 'mono-distributive' manner (one Ub at a time) and SARS PLpro prefers to cleave Lys48-linked polyUb chains by sensing a di-Ub moiety as a minimal recognition element using a 'di-distributive' cleavage mechanism. The di-distributive cleavage mechanism for SARS PLpro appears to be uncommon among USP (Ub-specific protease)-family DUBs, as related USP family members from humans do not display such a mechanism. We propose that these intrinsic enzymatic differences between SARS and MERS PLpro will help to identify pro-inflammatory substrates of these viral DUBs and can guide in the design of therapeutics to combat infection by coronaviruses. [ABSTRACT FROM AUTHOR]