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.

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.

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.

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 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.

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.

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.

A Comprehensive Guide for Assessing Covalent Inhibition in Enzymatic Assays Illustrated with Kinetic Simulations.

Covalent inhibition has become more accepted in the past two decades, as illustrated by the clinical approval of several irreversible inhibitors designed to covalently modify their target. Elucidation of the structure-activity relationship and potency of such inhibitors requires a detailed kinetic evaluation. Here, we elucidate the relationship between the experimental read-out and the underlying inhibitor binding kinetics. Interactive kinetic simulation scripts are employed to highlight the effects of in vitro enzyme activity assay conditions and inhibitor binding mode, thereby showcasing which assumptions and corrections are crucial. Four stepwise protocols to assess the biochemical potency of (ir)reversible covalent enzyme inhibitors targeting a nucleophilic active site residue are included, with accompanying data analysis tailored to the covalent binding mode. Together, this will serve as a guide to make an educated decision regarding the most suitable method to assess covalent inhibition potency. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol I: Progress curve analysis of substrate association competition Basic Data Analysis Protocol 1A: Two-step irreversible covalent inhibition Basic Data Analysis Protocol 1B: One-step irreversible covalent inhibition Basic Data Analysis Protocol 1C: Two-step reversible covalent inhibition Basic Data Analysis Protocol 1D: Two-step irreversible covalent inhibition with substrate depletion Basic Protocol II: Incubation time-dependent potency IC50 (t) Basic Data Analysis Protocol 2: Two-step irreversible covalent inhibition Basic Protocol III: Preincubation time-dependent inhibition without dilution Basic Data Analysis Protocol 3: Preincubation time-dependent inhibition without dilution Basic Data Analysis Protocol 3Ai: Two-step irreversible covalent inhibition Alternative Data Analysis Protocol 3Aii: Two-step irreversible covalent inhibition Basic Data Analysis Protocol 3Bi: One-step irreversible covalent inhibition Alternative Data Analysis Protocol 3Bii: One-step irreversible covalent inhibition Basic Data Analysis Protocol 3C: Two-step reversible covalent inhibition Basic Protocol IV: Preincubation time-dependent inhibition with dilution/competition Basic Data Analysis Protocol 4: Preincubation time-dependent inhibition with dilution Basic Data Analysis Protocol 4Ai: Two-step irreversible covalent inhibition Alternative Data Analysis Protocol 4Aii: Two-step irreversible covalent inhibition Basic Data Analysis Protocol 4Bi: One-step irreversible covalent inhibition Alternative Data Analysis Protocol 4Bii: One-step irreversible covalent inhibition.

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.

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.

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.

Highly Specialized Ubiquitin-Like Modifications: Shedding Light into the UFM1 Enigma.

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.

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.

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.

Strategy for Development of Site-Specific Ubiquitin Antibodies.

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.

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.

Profiling DUBs and Ubl-specific proteases with activity-based probes.

Protein (poly-)ubiquitination is a posttranslational modification that plays a key role in almost all cellular processes. It involves the installment of either single ubiquitin (Ub) moieties or one of eight different polyUb linkage types, each giving a distinct cellular outcome. Deubiquitinating enzymes (DUBs) reverse Ub signaling by disassembly of one or multiple poly-Ub chain types and their malfunction is often associated with human disease. The Ub system displays significant crosstalk with structurally homologous ubiquitin-like proteins (Ubls), including SUMO, Nedd8, and ISG15. This can be seen with the existence of heterogeneous chains made from Ub-Ubl mixtures as well as the proteolytic cross reactivity displayed by several DUBs toward other Ubl systems. In addition, numerous pathogens have been found to encode Ub(l)-ligases and deconjugating enzymes in order to facilitate infection and fight the host immune response. Studying the activity of DUBs and Ubl-specific proteases, both human as well as pathogen-derived, gives fundamental insights into their physiological roles. Activity-based probes (ABPs) have proven to be valuable tools to achieve this, as they report on enzyme activities by making a (often irreversible) covalent complex, rather than on their relative abundance. In this chapter, we explain the potential of ABPs to assess substrate preferences, structural features, and activity of Ub and Ubl deconjugating enzymes. We further demonstrate the practical use of ABPs to (1) characterize the activity of viral proteases toward Ub and Ubls and (2) to gain more insight in the structural determinants of substrate preference of DUBs.

Kinetic analysis of multistep USP7 mechanism shows critical role for target protein in activity.

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.

SUMOylation and the HSF1-Regulated Chaperone Network Converge to Promote Proteostasis in Response to Heat Shock.

The role of stress-induced increases in SUMO2/3 conjugation during the heat shock response (HSR) has remained enigmatic. We investigated SUMO signal transduction at the proteomic and functional level during the HSR in cells depleted of proteostasis network components via chronic heat shock factor 1 inhibition. In the recovery phase post heat shock, high SUMO2/3 conjugation was prolonged in cells lacking sufficient chaperones. Similar results were obtained upon inhibiting HSP90, indicating that increased chaperone activity during the HSR is critical for recovery to normal SUMO2/3 levels post-heat shock. Proteasome inhibition likewise prolonged SUMO2/3 conjugation, indicating that stress-induced SUMO2/3 targets are subsequently degraded by the ubiquitin-proteasome system. Functionally, we suggest that SUMOylation can enhance the solubility of target proteins upon heat shock, a phenomenon that we experimentally observed in vitro. Collectively, our results implicate SUMO2/3 as a rapid response factor that coordinates proteome degradation and assists the maintenance of proteostasis upon proteotoxic stress.