Ubiquitin Phosphorylation at Thr12 Modulates the DNA Damage Response.

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.

Ubiquitin ligation to F-box protein targets by SCF-RBR E3-E3 super-assembly.

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 substrates1,2. 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-assemblies3-7. 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.

Structural snapshots along K48-linked ubiquitin chain formation by the HECT E3 UBR5.

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.

Sequence and structural variations determining the recruitment of WNK kinases to the KLHL3 E3 ligase.

The BTB-Kelch protein KLHL3 is a Cullin3-dependent E3 ligase that mediates the ubiquitin-dependent degradation of kinases WNK1-4 to control blood pressure and cell volume. A crystal structure of KLHL3 has defined its binding to an acidic degron motif containing a PXXP sequence that is strictly conserved in WNK1, WNK2 and WNK4. Mutations in the second proline abrograte the interaction causing the hypertension syndrome pseudohypoaldosteronism type II. WNK3 shows a diverged degron motif containing four amino acid substitutions that remove the PXXP motif raising questions as to the mechanism of its binding. To understand this atypical interaction, we determined the crystal structure of the KLHL3 Kelch domain in complex with a WNK3 peptide. The electron density enabled the complete 11-mer WNK-family degron motif to be traced for the first time revealing several conserved features not captured in previous work, including additional salt bridge and hydrogen bond interactions. Overall, the WNK3 peptide adopted a conserved binding pose except for a subtle shift to accommodate bulkier amino acid substitutions at the binding interface. At the centre, the second proline was substituted by WNK3 Thr541, providing a unique phosphorylatable residue among the WNK-family degrons. Fluorescence polarisation and structural modelling experiments revealed that its phosphorylation would abrogate the KLHL3 interaction similarly to hypertension-causing mutations. Together, these data reveal how the KLHL3 Kelch domain can accommodate the binding of multiple WNK isoforms and highlight a potential regulatory mechanism for the recruitment of WNK3.

A Pro-Fluorescent Ubiquitin-Based Probe to Monitor Cysteine-Based E3 Ligase Activity.

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.

Molecular basis of Lys11-polyubiquitin specificity in the deubiquitinase Cezanne.

The post-translational modification of proteins with polyubiquitin regulates virtually all aspects of cell biology. Eight distinct chain linkage types co-exist in polyubiquitin and are independently regulated in cells. This 'ubiquitin code' determines the fate of the modified protein. Deubiquitinating enzymes of the ovarian tumour (OTU) family regulate cellular signalling by targeting distinct linkage types within polyubiquitin, and understanding their mechanisms of linkage specificity gives fundamental insights into the ubiquitin system. Here we reveal how the deubiquitinase Cezanne (also known as OTUD7B) specifically targets Lys11-linked polyubiquitin. Crystal structures of Cezanne alone and in complex with monoubiquitin and Lys11-linked diubiquitin, in combination with hydrogen-deuterium exchange mass spectrometry, enable us to reconstruct the enzymatic cycle in great detail. An intricate mechanism of ubiquitin-assisted conformational changes activates the enzyme, and while all chain types interact with the enzymatic S1 site, only Lys11-linked chains can bind productively across the active site and stimulate catalytic turnover. Our work highlights the plasticity of deubiquitinases and indicates that new conformational states can occur when a true substrate, such as diubiquitin, is bound at the active site.

Exploring the Versatility of the Covalent Thiol-Alkyne Reaction with Substituted Propargyl Warheads: A Deciding Role for the Cysteine Protease.

Terminal unactivated alkynes are nowadays considered the golden standard for cysteine-reactive warheads in activity-based probes (ABPs) targeting cysteine deubiquitinating enzymes (DUBs). In this work, we study the versatility of the thiol-alkyne addition reaction in more depth. Contrary to previous findings with UCHL3, we now show that covalent adduct formation can progress with substituents on the terminal or internal alkyne position. Strikingly, acceptance of alkyne substituents is strictly DUB-specific as this is not conserved among members of the same subfamily. Covalent adduct formation with the catalytic cysteine residue was validated by gel analysis and mass spectrometry of intact ABP-treated USP16CDWT and catalytically inactive mutant USP16CDC205A. Bottom-up mass spectrometric analysis of the covalent adduct with a deuterated propargyl ABP provides mechanistic understanding of the in situ thiol-alkyne reaction, identifying the alkyne rather than an allenic intermediate as the reactive species. Furthermore, kinetic analysis revealed that introduction of (bulky/electron-donating) methyl substituents on the propargyl moiety decreases the rate of covalent adduct formation, thus providing a rational explanation for the commonly lower level of observed covalent adduct compared to unmodified alkynes. Altogether, our work extends the scope of possible propargyl derivatives in cysteine targeting ABPs from unmodified terminal alkynes to internal and substituted alkynes, which we anticipate will have great value in the development of ABPs with improved selectivity profiles.

Targeting TRIM Proteins: A Quest towards Drugging an Emerging Protein Class.

The ubiquitylation machinery regulates several fundamental biological processes from protein homeostasis to a wide variety of cellular signaling pathways. As a consequence, its dysregulation is linked to diseases including cancer, neurodegeneration, and autoimmunity. With this review, we aim to highlight the therapeutic potential of targeting E3 ligases, with a special focus on an emerging class of RING ligases, named tri-partite motif (TRIM) proteins, whose role as targets for drug development is currently gaining pharmaceutical attention. TRIM proteins exert their catalytic activity as scaffolds involved in many protein-protein interactions, whose multidomains and adapter-like nature make their druggability very challenging. Herein, we give an overview of the current understanding of this class of single polypeptide RING E3 ligases and discuss potential targeting options.

Cracking the Ubiquitin Code: The Ubiquitin Toolbox.

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.

A cascading activity-based probe sequentially targets E1-E2-E3 ubiquitin enzymes.

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.

Total Chemical Synthesis of SUMO and SUMO-Based Probes for Profiling the Activity of SUMO-Specific Proteases.

SUMO is a post-translational modifier critical for cell cycle progression and genome stability that plays a role in tumorigenesis, thus rendering SUMO-specific enzymes potential pharmacological targets. However, the systematic generation of tools for the activity profiling of SUMO-specific enzymes has proven challenging. We developed a diversifiable synthetic platform for SUMO-based probes by using a direct linear synthesis method, which permits N- and C-terminal labelling to incorporate dyes and reactive warheads, respectively. In this manner, activity-based probes (ABPs) for SUMO-1, SUMO-2, and SUMO-3-specific proteases were generated and validated in cells using gel-based assays and confocal microscopy. We further expanded our toolbox with the synthesis of a K11-linked diSUMO-2 probe to study the proteolytic cleavage of SUMO chains. Together, these ABPs demonstrate the versatility and specificity of our synthetic SUMO platform for in vitro and in vivo characterization of the SUMO protease family.

Generation of the UFM1 Toolkit for Profiling UFM1-Specific Proteases and Ligases.

Ubiquitin-fold modifier 1 (UFM1) is a reversible post-translational modifier that is covalently attached to target proteins through an enzymatic cascade and removed by designated proteases. Abnormalities in this process, referred to as Ufmylation, have been associated with a variety of human diseases. Given this, the UFM1-specific enzymes represent potential therapeutic targets; however, understanding of their biological function has been hampered by the lack of chemical tools for activity profiling. To address this unmet need, a diversifiable platform for UFM1 activity-based probes (ABPs) utilizing a native chemical ligation (NCL) strategy was developed, enabling the generation of a variety of tools to profile both UFM1 conjugating and deconjugating enzymes. The use of the probes is demonstrated in vitro and in vivo for monitoring UFM1 enzyme reactivity, opening new research avenues.

SARS hCoV papain-like protease is a unique Lys48 linkage-specific di-distributive deubiquitinating enzyme.

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.

A native chemical ligation handle that enables the synthesis of advanced activity-based probes: diubiquitin as a case study.

We present the development of a native chemical ligation handle that also functions as a masked electrophile that can be liberated during synthesis when required. This handle can thus be used for the synthesis of complex activity-based probes. We describe the use of this handle in the generation of linkage-specific activity-based deubiquitylating enzyme probes that contain substrate context and closely mimic the native ubiquitin isopeptide linkage. We have generated activity-based probes based on all seven isopeptide-linked diubiquitin topoisomers and demonstrated their structural integrity and ability to label DUBs in a linkage-specific manner.

Technologies for Direct Detection of Covalent Protein-Drug Adducts.

In the past two decades, drug candidates with a covalent binding mode have gained the interest of medicinal chemists, as several covalent anticancer drugs have successfully reached the clinic. As a covalent binding mode changes the relevant parameters to rank inhibitor potency and investigate structure-activity relationship (SAR), it is important to gather experimental evidence on the existence of a covalent protein-drug adduct. In this work, we review established methods and technologies for the direct detection of a covalent protein-drug adduct, illustrated with examples from (recent) drug development endeavors. These technologies include subjecting covalent drug candidates to mass spectrometric (MS) analysis, protein crystallography, or monitoring intrinsic spectroscopic properties of the ligand upon covalent adduct formation. Alternatively, chemical modification of the covalent ligand is required to detect covalent adducts by NMR analysis or activity-based protein profiling (ABPP). Some techniques are more informative than others and can also elucidate the modified amino acid residue or bond layout. We will discuss the compatibility of these techniques with reversible covalent binding modes and the possibilities to evaluate reversibility or obtain kinetic parameters. Finally, we expand upon current challenges and future applications. Overall, these analytical techniques present an integral part of covalent drug development in this exciting new era of drug discovery.

Plate-Based Screening for DUB Inhibitors.

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.

Chemical Synthesis of Non-hydrolyzable Ubiquitin(-Like) Hybrid Chains.

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.

Expanding Chemical Probe Space: Quality Criteria for Covalent and Degrader Probes.

Within druggable target space, new small-molecule modalities, particularly covalent inhibitors and targeted degraders, have expanded the repertoire of medicinal chemists. Molecules with such modes of action have a large potential not only as drugs but also as chemical probes. Criteria have previously been established to describe the potency, selectivity, and properties of small-molecule probes that are qualified to enable the interrogation and validation of drug targets. These definitions have been tailored to reversibly acting modulators but fall short in their applicability to other modalities. While initial guidelines have been proposed, we delineate here a full set of criteria for the characterization of covalent, irreversible inhibitors as well as heterobifunctional degraders ("proteolysis-targeting chimeras", or PROTACs) and molecular glue degraders. We propose modified potency and selectivity criteria compared to those for reversible inhibitors. We discuss their relevance and highlight examples of suitable probe and pathfinder compounds.

A high-field cellular DNP-supported solid-state NMR approach to study proteins with sub-cellular specificity.

Studying the structural aspects of proteins within sub-cellular compartments is of growing interest. Dynamic nuclear polarization supported solid-state NMR (DNP-ssNMR) is uniquely suited to provide such information, but critically lacks the desired sensitivity and resolution. Here we utilize SNAPol-1, a novel biradical, to conduct DNP-ssNMR at high-magnetic fields (800 MHz/527 GHz) inside HeLa cells and isolated cell nuclei electroporated with [13C,15N] labeled ubiquitin. We report that SNAPol-1 passively diffuses and homogenously distributes within whole cells and cell nuclei providing ubiquitin spectra of high sensitivity and remarkably improved spectral resolution. For cell nuclei, physical enrichment facilitates a further 4-fold decrease in measurement time and provides an exclusive structural view of the nuclear ubiquitin pool. Taken together, these advancements enable atomic interrogation of protein conformational plasticity at atomic resolution and with sub-cellular specificity.

Mutual influence of backbone proline substitution and lipophilic tail character on the biological activity of simplified analogues of caspofungin.

The echinocandins represent the most recent class of antifungal drugs. Previous structure-activity relationship studies on these lipopeptides have relied mainly upon semisynthetic derivatives due to their complex chemical structures. A successful strategy for the rapid enantioselective synthesis of the branched fatty acid chain of caspofungin and analogues was developed to synthesize several simplified analogues of caspofungin. The specific minimum inhibitory activity of each mimic was determined against a panel of Candida strains. This approach gave access to new fully synthetic derived caspofungin mimics with high and selective antifungal activities against Candida strains. In addition, the data suggested an important role of the hydroxy proline residue in the bioactive conformation of the macrocyclic peptide ring structure.