Distinct USP25 and USP28 Oligomerization States Regulate Deubiquitinating Activity.

The evolutionarily related deubiquitinating enzymes (DUBs) USP25 and USP28 comprise an identical overall domain architecture but are functionally non-redundant: USP28 stabilizes c-MYC and other nuclear proteins, and USP25 regulates inflammatory TRAF signaling. We here compare molecular features of USP25 and USP28. Active enzymes form distinctively shaped dimers, with a dimerizing insertion spatially separating independently active catalytic domains. In USP25, but not USP28, two dimers can form an autoinhibited tetramer, where a USP25-specific, conserved insertion sequence blocks ubiquitin binding. In full-length enzymes, a C-terminal domain with a previously unknown fold has no impact on oligomerization, but N-terminal regions affect the dimer-tetramer equilibrium in vitro. We confirm oligomeric states of USP25 and USP28 in cells and show that modulating oligomerization affects substrate stabilization in accordance with in vitro activity data. Our work highlights how regions outside of the catalytic domain enable a conceptually intriguing interplay of DUB oligomerization and activity.

Molecular basis for ubiquitin/Fubi cross-reactivity in USP16 and USP36.

Ubiquitin and ubiquitin-like proteins typically use distinct machineries to facilitate diverse functions. The immunosuppressive ubiquitin-like protein Fubi is synthesized as an N-terminal fusion to a ribosomal protein (Fubi-S30). Its proteolytic maturation by the nucleolar deubiquitinase USP36 is strictly required for translationally competent ribosomes. What endows USP36 with this activity, how Fubi is recognized and whether other Fubi proteases exist are unclear. Here, we report a chemical tool kit that facilitated the discovery of dual ubiquitin/Fubi cleavage activity in USP16 in addition to USP36 by chemoproteomics. Crystal structures of USP36 complexed with Fubi and ubiquitin uncover its substrate recognition mechanism and explain how other deubiquitinases are restricted from Fubi. Furthermore, we introduce Fubi C-terminal hydrolase measurements and reveal a synergistic role of USP16 in Fubi-S30 maturation. Our data highlight how ubiquitin/Fubi specificity is achieved in a subset of human deubiquitinases and open the door to a systematic investigation of the Fubi system.

N-Cyanopiperazines as Specific Covalent Inhibitors of the Deubiquitinating Enzyme UCHL1.

Cyanamides have emerged as privileged scaffolds in covalent inhibitors of deubiquitinating enzymes (DUBs). However, many compounds with a cyanopyrrolidine warhead show cross-reactivity toward small subsets of DUBs or toward the protein deglycase PARK7/DJ-1, hampering their use for the selective perturbation of a single DUB in living cells. Here, we disclose N'-alkyl,N-cyanopiperazines as structures for covalent enzyme inhibition with exceptional specificity for the DUB UCHL1 among 55 human deubiquitinases and with effective target engagement in cells. Notably, transitioning from 5-membered pyrrolidines to 6-membered heterocycles eliminated PARK7 binding and introduced context-dependent reversibility of the isothiourea linkage to the catalytic cysteine of UCHL1. Compound potency and specificity were analysed by a range of biochemical assays and with a crystal structure of a cyanopiperazine in covalent complex with UCHL1. The structure revealed a compound-induced conformational restriction of the cross-over loop, which underlies the observed inhibitory potencies. Through the rationalization of specificities of different cyanamides, we introduce a framework for the investigation of protein reactivity of bioactive nitriles of this compound class. Our results represent an encouraging case study for the refining of electrophilic compounds into chemical probes, emphasizing the potential to engineer specificity through subtle chemical modifications around the warhead.

Native Semisynthesis of Isopeptide-Linked Substrates for Specificity Analysis of Deubiquitinases and Ubl Proteases.

Post-translational modifications with ubiquitin (Ub) and ubiquitin-like proteins (Ubls) are regulated by isopeptidases termed deubiquitinases (DUBs) and Ubl proteases. Here, we describe a mild chemical method for the preparation of fluorescence polarization substrates for these enzymes that is based on the activation of C-terminal Ub/Ubl hydrazides to acyl azides and their subsequent functionalization to isopeptides. The procedure is complemented by native purification routes and thus circumvents the previous need for desulfurization and refolding. Its broad applicability was demonstrated by the generation of fully cleavable substrates for Ub, SUMO1, SUMO2, NEDD8, ISG15, and Fubi. We employed these reagents for the investigation of substrate specificities of human UCHL3, USPL1, USP2, USP7, USP16, USP18, and USP36. Pronounced selectivity of USPL1 for SUMO2/3 over SUMO1 was observed, which we rationalize with crystal structures and biochemical assays, revealing a SUMO paralogue specificity mechanism distinct from SENP family deSUMOylases. Moreover, we investigated the recently identified Fubi proteases USP16 and USP36 and found both to act as bona fide deFubiylases, harboring catalytic activity against isopeptide-linked Fubi. Surprisingly, we also noticed the activity of both enzymes toward ISG15, previously not identified in chemoproteomics, which makes USP16 and USP36 the first human DUBs with specific isopeptidase activity toward three distinct modifiers. The methods described here for the preparation of isopeptide-linked, fully folded substrates will aid in the characterization of further DUBs/Ubl proteases. More broadly, our findings highlight possible limitations associated with fluorogenic substrates and Ubl activity-based probes and stress the importance of isopeptide-containing reagents for validating isopeptidase activities and quantifying substrate specificities.

Molecular basis of USP7 inhibition by selective small-molecule inhibitors.

Ubiquitination controls the stability of most cellular proteins, and its deregulation contributes to human diseases including cancer. Deubiquitinases remove ubiquitin from proteins, and their inhibition can induce the degradation of selected proteins, potentially including otherwise 'undruggable' targets. For example, the inhibition of ubiquitin-specific protease 7 (USP7) results in the degradation of the oncogenic E3 ligase MDM2, and leads to re-activation of the tumour suppressor p53 in various cancers. Here we report that two compounds, FT671 and FT827, inhibit USP7 with high affinity and specificity in vitro and within human cells. Co-crystal structures reveal that both compounds target a dynamic pocket near the catalytic centre of the auto-inhibited apo form of USP7, which differs from other USP deubiquitinases. Consistent with USP7 target engagement in cells, FT671 destabilizes USP7 substrates including MDM2, increases levels of p53, and results in the transcription of p53 target genes, induction of the tumour suppressor p21, and inhibition of tumour growth in mice.

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.

Ubiquitin Ser65 phosphorylation affects ubiquitin structure, chain assembly and hydrolysis.

The protein kinase PINK1 was recently shown to phosphorylate ubiquitin (Ub) on Ser65, and phosphoUb activates the E3 ligase Parkin allosterically. Here, we show that PINK1 can phosphorylate every Ub in Ub chains. Moreover, Ser65 phosphorylation alters Ub structure, generating two conformations in solution. A crystal structure of the major conformation resembles Ub but has altered surface properties. NMR reveals a second phosphoUb conformation in which ß5-strand slippage retracts the C-terminal tail by two residues into the Ub core. We further show that phosphoUb has no effect on E1-mediated E2 charging but can affect discharging of E2 enzymes to form polyUb chains. Notably, UBE2R1- (CDC34), UBE2N/UBE2V1- (UBC13/UEV1A), TRAF6- and HOIP-mediated chain assembly is inhibited by phosphoUb. While Lys63-linked poly-phosphoUb is recognized by the TAB2 NZF Ub binding domain (UBD), 10 out of 12 deubiquitinases (DUBs), including USP8, USP15 and USP30, are impaired in hydrolyzing phosphoUb chains. Hence, Ub phosphorylation has repercussions for ubiquitination and deubiquitination cascades beyond Parkin activation and may provide an independent layer of regulation in the Ub system.

Selective Activation of Human Caseinolytic Protease†P (ClpP).

Caseinolytic protease P (ClpP) is the proteolytic component of the ClpXP protein degradation complex. Eukaryotic ClpP was recently found to act within the mitochondria-specific unfolded protein response (UPRmt ). However, its detailed function and dedicated regulation remain largely unexplored. A small molecule (D9) acts as a potent and species-selective activator of human ClpP (hClpP) by mimicking the natural chaperone ClpX. Structure-activity relationship studies highlight the importance of a halogenated benzyl motif within D9 that interacts with a unique aromatic amino acid network in hClpP. Mutational and structural studies suggest that this YYW motif tightly controls hClpP activity and regulates substrate turnover by interaction with cognate ligands. This signature motif is unique to ClpP from higher organisms and does not exist in tested bacterial homologues, allowing a species-selective analysis. Thus, D9 is a versatile tool to analyze mechanistic features of hClpP.

Deuteration of Hyperpolarized 13 C-Labeled Zymonic Acid Enables Sensitivity-Enhanced Dynamic MRI of pH.

Aberrant pH is characteristic of many pathologies such as ischemia, inflammation or cancer. Therefore, a non-invasive and spatially resolved pH determination is valuable for disease diagnosis, characterization of response to treatment and the design of pH-sensitive drug-delivery systems. We recently introduced hyperpolarized [1,5-13 C2 ]zymonic acid (ZA) as a novel MRI probe of extracellular pH utilizing dissolution dynamic polarization (DNP) for a more than 10000-fold signal enhancement of the MRI signal. Here we present a strategy to enhance the sensitivity of this approach by deuteration of ZA yielding [1,5-13 C2 , 3,6,6,6-D4 ]zymonic acid (ZAd ), which prolongs the liquid state spin lattice relaxation time (T1 ) by up to 39 % in vitro. Measurements with ZA and ZAd on subcutaneous MAT B III adenocarcinoma in rats show that deuteration increases the signal-to-noise ratio (SNR) by up to 46 % in vivo. Furthermore, we demonstrate a proof of concept for real-time imaging of dynamic pH changes in vitro using ZAd , potentially allowing for the characterization of rapid acidification/basification processes in vivo.

Structural and functional insights into caseinolytic proteases reveal an unprecedented regulation principle of their catalytic triad.

Caseinolytic proteases (ClpPs) are large oligomeric protein complexes that contribute to cell homeostasis as well as virulence regulation in bacteria. Although most organisms possess a single ClpP protein, some organisms encode two or more ClpP isoforms. Here, we elucidated the crystal structures of ClpP1 and ClpP2 from pathogenic Listeria monocytogenes and observe an unprecedented regulation principle by the catalytic triad. Whereas L. monocytogenes (Lm)ClpP2 is both structurally and functionally similar to previously studied tetradecameric ClpP proteins from Escherichia coli and Staphylococcus aureus, heptameric LmClpP1 features an asparagine in its catalytic triad. Mutation of this asparagine to aspartate increased the reactivity of the active site and led to the assembly of a tetradecameric complex. We analyzed the heterooligomeric complex of LmClpP1 and LmClpP2 via coexpression and subsequent labeling studies with natural product-derived probes. Notably, the LmClpP1 peptidase activity is stimulated 75-fold in the complex providing insights into heterooligomerization as a regulatory mechanism. Collectively, our data point toward different preferences for substrates and inhibitors of the two ClpP enzymes and highlight their structural and functional characteristics.

Disruption of oligomerization and dehydroalanine formation as mechanisms for ClpP protease inhibition.

Over 100 protease inhibitors are currently used in the clinics, and most of them use blockage of the active site for their mode of inhibition. Among the protease drug targets are several enzymes for which the correct multimeric assembly is crucial to their activity, such as the proteasome and the HIV protease. Here, we present a novel mechanism of protease inhibition that relies on active-site-directed small molecules that disassemble the protease complex. We show the applicability of this mechanism within the ClpP protease family, whose members are tetradecameric serine proteases and serve as regulators of several cellular processes, including homeostasis and virulence. Compound binding to ClpP in a substoichiometric fashion triggers the formation of completely inactive heptamers. Moreover, we report the selective ß-sultam-induced dehydroalanine formation of the active site serine. This reaction proceeds through sulfonylation and subsequent elimination, thereby obliterating the catalytic charge relay system. The identity of the dehydroalanine was confirmed by mass spectrometry and crystallography. Activity-based protein profiling experiments suggest the formation of a dehydroalanine moiety in living S. aureus cells upon ß-sultam treatment. Collectively, these findings extend our view on multicomponent protease inhibition that until now has mainly relied on blockage of the active site or occupation of a regulatory allosteric site.

Selective inhibition of the immunoproteasome by ligand-induced crosslinking of the active site.

The concept of proteasome inhibition ranks among the latest achievements in the treatment of blood cancer and represents a promising strategy for modulating autoimmune diseases. In this study, we describe peptidic sulfonyl fluoride inhibitors that selectively block the catalytic ß5 subunit of the immunoproteasome by inducing only marginal cytotoxic effects. Structural and mass spectrometric analyses revealed a novel reaction mechanism involving polarity inversion and irreversible crosslinking of the proteasomal active site. We thus identified the sulfonyl fluoride headgroup for the development and optimization of immunoproteasome selective compounds and their possible application in autoimmune disorders.

Variety in the USP deubiquitinase catalytic mechanism.

The ubiquitin-specific protease (USP) family of deubiquitinases (DUBs) controls cellular ubiquitin-dependent signaling events. This generates therapeutic potential, with active-site inhibitors in preclinical and clinical studies. Understanding of the USP active site is primarily guided by USP7 data, where the catalytic triad consists of cysteine, histidine, and a third residue (third critical residue), which polarizes the histidine through a hydrogen bond. A conserved aspartate (fourth critical residue) is directly adjacent to this third critical residue. Although both critical residues accommodate catalysis in USP2, these residues have not been comprehensively investigated in other USPs. Here, we quantitatively investigate their roles in five USPs. Although USP7 relies on the third critical residue for catalysis, this residue is dispensable in USP1, USP15, USP40, and USP48, where the fourth critical residue is vital instead. Furthermore, these residues vary in importance for nucleophilic attack. The diverging catalytic mechanisms of USP1 and USP7 are independent of substrate and retained in cells for USP1. This unexpected variety of catalytic mechanisms in this well-conserved protein family may generate opportunities for selective targeting of individual USPs.

Insights into structural network responsible for oligomerization and activity of bacterial virulence regulator caseinolytic protease P (ClpP) protein.

The barrel-shaped caseinolytic protease P (ClpP) is a main virulence regulator in the bacterial pathogen Staphylococcus aureus (SaClpP). It consists of two heptameric rings forming a homotetradecamer with an inner chamber that houses the 14 active sites. We recently showed that SaClpP is able to adopt a compressed, inactive conformation. We present here the 2.3 Å resolution structure of SaClpP in its closed, active conformation as well as the structure of the S98A mutant. Comprehensive mutational analysis aiming at destabilizing one or the other or both conformations was able to pinpoint key residues involved in this catalytic switch and in the heptamer-heptamer interaction. By probing the active site serine with a covalently modifying ß-lactone probe, we could show that the tetradecameric organization is essential for a proper formation of the active site. Structural data suggest that a highly conserved hydrogen-bonding network links oligomerization to activity. A comparison of ClpP structures from different organisms provides suggestive evidence for the presence of a universal mechanism regulating ClpP activity in which binding of one subunit to the corresponding subunit on the other ring interface is necessary for the functional assembly of the catalytic triad and thus for protease function. This mechanism ensures controlled access to the active sites of a highly unspecific protease.

Electrophilic natural products and their biological targets.

The study of biologically active natural products has resulted in seminal contributions to our understanding of living systems. In the case of electrophilic natural products, the covalent nature of their interaction has largely facilitated the identification of their biological binding partners. In this review, we provide a comprehensive compilation of electrophilic natural products from all major chemical classes together with their biological targets. Covering Michael acceptor systems, ring-strained compounds and other electrophiles, such as esters or carbamates, we highlight representative and instructive examples for over 20 electrophilic moieties. The fruitful cooperation of natural product chemistry, medicinal chemistry and chemical biology has produced a collection of well-studied examples for how electrophilic natural products exert their biological functions that range from antibiotic to antitumor effects. Special emphasis is put on the elucidation of their respective biological targets via activity-based protein profiling, which together with the recent advancements in mass spectrometry has been crucial to the success of the field. The wealth of naturally occurring electrophilic moieties and their chemical complexity enables binding of a large variety of biological targets, such as enzymes of all classes, nonenzymatic proteins, DNA and other cellular compounds. With approximately 30,000 genes in the human genome but only 266 confirmed protein drug targets, the study of biologically active, electrophilic natural products has the potential to provide insights into fundamental biological processes and to greatly aid the discovery of new drug targets.

Structural basis for specific inhibition of the deubiquitinase UCHL1.

Ubiquitination regulates protein homeostasis and is tightly controlled by deubiquitinases (DUBs). Loss of the DUB UCHL1 leads to neurodegeneration, and its dysregulation promotes cancer metastasis and invasiveness. Small molecule probes for UCHL1 and DUBs in general could help investigate their function, yet specific inhibitors and structural information are rare. Here we report the potent and non-toxic chemogenomic pair of activity-based probes GK13S and GK16S for UCHL1. Biochemical characterization of GK13S demonstrates its stereoselective inhibition of cellular UCHL1. The crystal structure of UCHL1 in complex with GK13S shows the enzyme locked in a hybrid conformation of apo and Ubiquitin-bound states, which underlies its UCHL1-specificity within the UCH DUB family. Phenocopying a reported inactivating mutation of UCHL1 in mice, GK13S, but not GK16S, leads to reduced levels of monoubiquitin in a human glioblastoma cell line. Collectively, we introduce a set of structurally characterized, chemogenomic probes suitable for the cellular investigation of UCHL1.

USP28 deletion and small-molecule inhibition destabilizes c-MYC and elicits regression of squamous cell lung carcinoma.

Lung squamous cell carcinoma (LSCC) is a considerable global health burden, with an incidence of over 600,000 cases per year. Treatment options are limited, and patient's 5-year survival rate is less than 5%. The ubiquitin-specific protease 28 (USP28) has been implicated in tumourigenesis through its stabilization of the oncoproteins c-MYC, c-JUN, and Δp63. Here, we show that genetic inactivation of Usp28-induced regression of established murine LSCC lung tumours. We developed a small molecule that inhibits USP28 activity in the low nanomole range. While displaying cross-reactivity against the closest homologue USP25, this inhibitor showed a high degree of selectivity over other deubiquitinases. USP28 inhibitor treatment resulted in a dramatic decrease in c-MYC, c-JUN, and Δp63 proteins levels and consequently induced substantial regression of autochthonous murine LSCC tumours and human LSCC xenografts, thereby phenocopying the effect observed by genetic deletion. Thus, USP28 may represent a promising therapeutic target for the treatment of squamous cell lung carcinoma.

USP30 sets a trigger threshold for PINK1-PARKIN amplification of mitochondrial ubiquitylation.

The mitochondrial deubiquitylase USP30 negatively regulates the selective autophagy of damaged mitochondria. We present the characterisation of an N-cyano pyrrolidine compound, FT3967385, with high selectivity for USP30. We demonstrate that ubiquitylation of TOM20, a component of the outer mitochondrial membrane import machinery, represents a robust biomarker for both USP30 loss and inhibition. A proteomics analysis, on a SHSY5Y neuroblastoma cell line model, directly compares the effects of genetic loss of USP30 with chemical inhibition. We have thereby identified a subset of ubiquitylation events consequent to mitochondrial depolarisation that are USP30 sensitive. Within responsive elements of the ubiquitylome, several components of the outer mitochondrial membrane transport (TOM) complex are prominent. Thus, our data support a model whereby USP30 can regulate the availability of ubiquitin at the specific site of mitochondrial PINK1 accumulation following membrane depolarisation. USP30 deubiquitylation of TOM complex components dampens the trigger for the Parkin-dependent amplification of mitochondrial ubiquitylation leading to mitophagy. Accordingly, PINK1 generation of phospho-Ser65 ubiquitin proceeds more rapidly in cells either lacking USP30 or subject to USP30 inhibition.

Insights into ClpXP proteolysis: heterooligomerization and partial deactivation enhance chaperone affinity and substrate turnover in Listeria monocytogenes.

Caseinolytic proteases (ClpP) are important for recognition and controlled degradation of damaged proteins. While the majority of bacterial organisms utilize only a single ClpP, Listeria monocytogenes expresses two isoforms (LmClpP1 and LmClpP2). LmClpPs assemble into either a LmClpP2 homocomplex or a LmClpP1/2 heterooligomeric complex. The heterocomplex in association with the chaperone ClpX, exhibits a boost in proteolytic activity for unknown reasons. Here, we use a combined chemical and biochemical strategy to unravel two activation principles of LmClpPs. First, determination of apparent affinity constants revealed a 7-fold elevated binding affinity between the LmClpP1/2 heterocomplex and ClpX, compared to homooligomeric LmClpP2. This tighter interaction favors the formation of the proteolytically active complex between LmClpX and LmClpP1/2 and thereby accelerating the overall turnover. Second, screening a diverse library of fluorescent labeled peptides and proteins with various ClpP mutants allowed the individual analysis of substrate preferences for both isoforms within the heterocomplex. In addition to Leu and Met, LmClpP2 preferred a long aliphatic chain (2-Aoc) in the P1 position for cleavage. Strikingly, design and synthesis of a corresponding 2-Aoc chloromethyl ketone inhibitor resulted in stimulation of proteolysis by 160% when LmClpP2 was partially alkylated on 20% of the active sites. Determination of apparent affinity constants also revealed an elevated complex stability between partially modified LmClpP2 and the cognate chaperone LmClpX. Thus, the stimulation of proteolysis through enhanced binding to the chaperone seems to be a characteristic feature of LmClpPs.