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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.

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.

Post-translational protein modification by ubiquitin (Ub) and Ub-like modifiers is orchestrated by the sequential action of Ub-activating, -conjugating, and -ligating enzymes to regulate a vast array of fundamental biological processes. Unsurprisingly, the dysregulation of the intricate interplay between ubiquitination and deubiquitination gives rise to numerous pathologies, most notably cancer and neurodegenerative diseases. While activity-based probes (ABPs) and assay reagents have been extensively developed and applied for deubiquitinating enzymes, similar tools for the Ub cascade have only recently emerged. Given the recent efforts to develop inhibitors for the Ub system, the urgency for developing ABPs and assay reagents is imminent. In this light, we comprehensively discuss the currently available ABPs with a focus on the newly developed reagents targeting the Ub cascade while illustrating their potential applications., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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. [ABSTRACT FROM AUTHOR]

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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]