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Deubiquitinating enzymes are key regulators in the ubiquitin system and an emerging class of drug targets. These proteases disassemble polyubiquitin chains and many deubiquitinases show selectivity for specific polyubiquitin linkages. However, most biochemical insights originate from studies of single diubiquitin linkages in isolation, whereas in cells all linkages coexist. To better mimick this diubiquitin substrate competition, we develop a multiplexed mass spectrometry-based deubiquitinase assay that can probe all ubiquitin linkage types simultaneously to quantify deubiquitinase activity in the presence of all potential diubiquitin substrates. For this, all eight native diubiquitins are generated and each linkage type is designed with a distinct molecular weight by incorporating neutron-encoded amino acids. Overall, 22 deubiquitinases are profiled, providing a three-dimensional overview of deubiquitinase linkage selectivity over time and enzyme concentration.

Although ubiquitin is found only in eukaryotes, several pathogenic bacteria and viruses possess proteins that hinder the host ubiquitin system.Legionella, a gram-negative intracellular bacterium, possesses an ovarian tumor (OTU) family of deubiquitinases (Lot DUBs). Herein, we describe the molecular characteristics of Lot DUBs. We elucidated the structure of LotA OTU1 domain and revealed that entire Lot DUBs possess a characteristic extended helical lobe (EHL) that is not found in other OTU-DUBs. The structural topology of EHL is the same throughout the Lot family, and it provides an S1′ ubiquitin-binding site. Moreover, the catalytic triads of Lot DUBs resemble those of the A20-type OTU-DUBs. Furthermore, we revealed a unique mechanism by which LotA OTU domains cooperate together to distinguish the length of the chain and preferentially cleaves longer K48-linked polyubiquitin chains. The LotA OTU1 domain itself cleaves K6-linked ubiquitin chains, while it is also essential for assisting the cleavage of longer K48-linked polyubiquitin chains by the OTU2 domain. Thus, this study provides novel insights into the structure and mechanism of action of Lot DUBs.

Protein ubiquitination is a key post-translational modification in regulating many fundamental cellular processes and dysregulation of these processes can give rise to a vast array of diseases. Unravelling the molecular mechanisms of ubiquitination hence is an important area in current ubiquitin research with as aim to understand this enigmatic process. The complexity of ubiquitin (Ub) signaling arises from the large variety of Ub conjugates, where Ub is attached to other Ub proteins, Ub-like proteins, and protein substrates. The chemical preparation of such Ub conjugates in high homogeneity and in adequate amounts contributes greatly to the deciphering of Ub signaling. The strength of these chemically synthesized conjugates lies in the chemo-selectivity in which they can be created that are sometimes difficult to obtain using biochemical methodology. In this review, we will discuss the progress in the chemical protein synthesis of state-of-the-art Ub and Ub-like chemical probes, their unique concepts and related discoveries in the ubiquitin field.

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.

Solid-phase peptide synthesis (SPPS) enables the synthesis of chemically modified peptides and proteins. Chemically synthesized ubiquitin(-like) proteins containing a fluorescent tag or reactive warhead have proven to be important tools in elucidating biological processes. Here, we describe the first fully synthetic method for the linear synthesis of two LC3 ubiquitin-like proteins using disaggregating building blocks and heated synthesis. Both LC3A and LC3B were synthesized and equipped with a fluorescent rhodamine tag, followed by folding of the proteins and liquid chromatography-mass spectrometry and SDS-PAGE analysis to prove that the quality of the synthetic material is comparable to expressed material.

Development of (semi-)synthetic methods to prepare ubiquitin (Ub)-based reagents has proven to be helpful in the classification of deubiquitinating proteases (DUBs). To study DUB selectivity for one or more of the eight possible poly-Ub chains, fluorogenic assay reagents have been reported relying on the appearance of a fluorescent signal upon DUB-mediated cleavage of the reagent. In this protocol, we describe the use of such an assay to profile the selectivity of TRABID, a member of the OTU family of DUBs.

Tuberculosis is a global health problem caused by infection with the Mycobacterium tuberculosis (Mtb) bacteria. Although antibiotic treatment has dramatically reduced the impact of tuberculosis on the population, the existence and spreading of drug resistant strains urgently demands the development of new drugs that target Mtb in a different manner than currently used antibiotics. The prokaryotic ubiquitin‐like protein (Pup) proteasome system is an attractive target for new drug development as it is unique to Mtb and related bacterial genera. Using a Pup‐based fluorogenic substrate, we screened for inhibitors of Dop, the Mtb depupylating protease, and identified I‐OMe‐Tyrphostin AG538 (1) and Tyrphostin AG538 (2). The hits were validated and determined to be fast‐reversible, non‐ATP competitive inhibitors. We synthesized >25 analogs of 1 and 2 and show that several of the synthesized compounds also inhibit the depupylation actions of Dop on native substrate, FabD‐Pup. Importantly, the pupylation activity of PafA, the sole Pup ligase in Mtb, was also inhibited by some of these compounds., The Mycobacterium tuberculosis (Mtb) prokaryotic ubiquitin‐like (pup) proteasome system is an attractive target for new drug development. In this study a screen was performed identifying Tyrphostins as low micromolar inhibitors of the Mtb protease Dop. To gain insight in the important functional aspects of these inhibitors, 25 new analogues were prepared and validated, in vitro. Several new compounds were able to inhibit both the depupylating activity of Dop as well as the pupylating activity of de pup ligase PafA.

Deubiquitinating enzymes function to cleave ubiquitin moieties from modified proteins, serving to maintain the pool of free ubiquitin in the cell while simultaneously impacting the fate and function of a target protein. Like all eukaryotes, Plasmodium parasites rely on the dynamic addition and removal of ubiquitin for their own growth and survival. While humans possess around 100 DUBs, Plasmodium contains ∼20 putative ubiquitin hydrolases, many of which bear little to no resemblance to those of other organisms. In this study, we characterize PfUBP-1, a large ubiquitin hydrolase unique to Plasmodium spp that has been linked to endocytosis and drug resistance. We demonstrate its ubiquitin activity, linkage specificity and assess the repercussions of point mutations associated with drug resistance on catalytic activity and parasite fitness.

The ubiquitin-proteasome system is an essential regulator of many cellular processes including controlling protein homeostasis. The degradation of proteins by the multi-subunit proteasome complex is tightly regulated through a series of checkpoints, amongst which are a set of deubiquitinating proteases (DUBs). The proteasome-associated DUBs, UCH-L5 (Ubiquitin carboxyl-terminal hydrolase isozyme L5) and USP14 (Ubiquitin-specific protease 14), and the integral-DUB in the proteasome, Rpn11, is known to regulate proteasomal degradation by deubiquitination of distinct substrates. Although selective inhibitors for USP14 and Rpn11 have been recently developed, there are no known inhibitors that selectively bind to UCH-L5. The X-ray structure of the Ubiquitin (Ub) bound to UCH-L5 shows a β-sheet hairpin in Ub that contains a crucial hydrophobic patch involved in the interaction with UCH-L5. Herein, we designed and developed both a Ub sequence-based linear- and cyclic- β-sheet hairpin peptide that was found to preferably inhibit UCH-L5. We show that these peptides have low micromolar IC50values and the cyclic peptide competes with the activity-based UbVME (Ubiquitin-Vinyl-Methyl-Ester) probe for UCH-L5, binding in a concentration-dependent manner. We further establish the selectivity profile of the cyclic peptide for UCH-L5 compared to other members of the UCH-DUB family and other cysteine DUBs in cell lysate. Furthermore, the cyclic peptide infiltrated cells resulting in the accumulation of polyUb chains, and was found to be non-toxic at the concentrations used here. Taken together, our data suggest that the cyclic peptide permeates the cell membrane, inhibits UCH-L5 by possibly blocking its deubiquitinating function, and contributes to the accumulation of polyubiquitinated substrates. The implications of inhibiting UCH-L5 in the context of the 26S proteasome render it an attractive candidate for further development as a potential selective inhibitor for therapeutic purposes.

The AAA ATPases PEX1•PEX6 extract PEX5, the peroxisomal protein shuttling receptor, from the peroxisomal membrane so that a new protein transport cycle can start. Extraction requires ubiquitination of PEX5 at residue 11 and involves a threading mechanism, but how exactly this occurs is unclear. We used a cell-free in vitro system and a variety of engineered PEX5 and ubiquitin molecules to challenge the extraction machinery. We show that PEX5 modified with a single ubiquitin is a substrate for extraction and extend previous findings proposing that neither the N- nor the C-terminus of PEX5 are required for extraction. Chimeric PEX5 molecules possessing a branched polypeptide structure at their C-terminal domains can still be extracted from the peroxisomal membrane thus suggesting that the extraction machinery can thread more than one polypeptide chain simultaneously. Importantly, we found that the PEX5-linked monoubiquitin is unfolded at a pre-extraction stage and, accordingly, an intra-molecularly cross-linked ubiquitin blocked extraction when conjugated to residue 11 of PEX5. Collectively, our data suggest that the PEX5-linked monoubiquitin is the extraction initiator and that the complete ubiquitin-PEX5 conjugate is threaded by PEX1•PEX6.

Apart from prevention using vaccinations, the management options for COVID-19 remain limited. In retrospective cohort studies, use of famotidine, a specific oral H2 receptor antagonist (antihistamine), has been associated with reduced risk of intubation and death in patients hospitalized with COVID-19. In a case series, nonhospitalized patients with COVID-19 experienced rapid symptom resolution after taking famotidine, but the molecular basis of these observations remains elusive. Here we show using biochemical, cellular, and functional assays that famotidine has no effect on viral replication or viral protease activity. However, famotidine can affect histamine-induced signaling processes in infected Caco2 cells. Specifically, famotidine treatment inhibits histamine-induced expression of Toll-like receptor 3 (TLR3) in SARS-CoV-2 infected cells and can reduce TLR3-dependent signaling processes that culminate in activation of IRF3 and the NF-kappa B pathway, subsequently controlling antiviral and inflammatory responses. SARS-CoV-2-infected cells treated with famotidine demonstrate reduced expression levels of the inflammatory mediators CCL-2 and IL6, drivers of the cytokine release syndrome that precipitates poor outcome for patients with COVID-19. Given that pharmacokinetic studies indicate that famotidine can reach concentrations in blood that suffice to antagonize histamine H2 receptors expressed in mast cells, neutrophils, and eosinophils, these observations explain how famotidine may contribute to the reduced histamine-induced inflammation and cytokine release, thereby improving the outcome for patients with COVID-19.

The enzyme-mediated construction of poly-ubiquitin (Ub) chains on target proteins leads to a variety of cellular responses and is involved in processes ranging from protein degradation to cell cycle control and immune responses. This complex post-translational modification system is under intense investigation, but generation of specific Ub chains and tools made thereof is not always trivial. We discovered that native methionine-1-linked polymeric ubiquitin chains can be constructed in a single chemical reaction. We validate correct folding and regioselective attachment of such chains using linkage specific proteases and further demonstrate that these poly-Ub chains can be converted into thioesters by the activating E1-enzyme. Subsequent ligation reactions using these in situ prepared thioesters leads to poly-ubiquitinated peptides.

Functional analysis of lysine 27-linked ubiquitin chains ((K27)Ub) is difficult due to the inability to make them through enzymatic methods and due to a lack of model tools and substrates. Here we generate a series of ubiquitin (Ub) tools to study how the deubiquitinase UCHL3 responds to (K27)Ub chains in comparison to lysine 63-linked chains and mono-Ub. From a crystal structure of a complex between UCHL3 and synthetic (K27)Ub(2), we unexpectedly discover that free (K27)Ub(2) and (K27)Ub(2)-conjugated substrates are natural inhibitors of UCHL3. Using our Ub tools to profile UCHL3's activity, we generate a quantitative kinetic model of the inhibitory mechanism and we find that (K27)Ub(2) can inhibit UCHL3 covalently, by binding to its catalytic cysteine, and allosterically, by locking its catalytic loop tightly in place. Based on this inhibition mechanism, we propose that UCHL3 and (K27)Ub chains likely sense and regulate each other in cells.

Legionnaires’ disease is caused by infection with the intracellularly replicating Gram‐negative bacterium Legionella pneumophila. This pathogen uses an unconventional way of ubiquitinating host proteins by generating a phosphoribosyl linkage between substrate proteins and ubiquitin by making use of an ADPribosylated ubiquitin (UbADPr) intermediate. The family of SidE effector enzymes that catalyze this reaction is counteracted by Legionella hydrolases, which are called Dups. This unusual ubiquitination process is important for Legionella proliferation and understanding these processes on a molecular level might prove invaluable in finding new treatments. Herein, a modular approach is used for the synthesis of triazole‐linked UbADPr, and analogues thereof, and their affinity towards the hydrolase DupA is determined and hydrolysis rates are compared to natively linked UbADPr. The inhibitory effects of modified Ub on the canonical eukaryotic E1‐enzyme Uba1 are investigated and rationalized in the context of a high‐resolution crystal structure reported herein. Finally, it is shown that synthetic UbADPr analogues can be used to effectively pull‐down overexpressed DupA from cell lysate., Modular modifications: Analogues of ADPribosylated ubiquitin, including a nonhydrolyzable methylenebisphosphonate version, are constructed and subsequently applied to investigate (de‐)ubiquitinating enzymes involved in Legionella infection. These probes are used in the study of the bacterial effector DupA and the human ubiquitin activating E1 enzyme.

The SARS‐CoV‐2 coronavirus encodes an essential papain‐like protease domain as part of its non‐structural protein (nsp)‐3, namely SARS2 PLpro, that cleaves the viral polyprotein, but also removes ubiquitin‐like ISG15 protein modifications as well as, with lower activity, Lys48‐linked polyubiquitin. Structures of PLpro bound to ubiquitin and ISG15 reveal that the S1 ubiquitin‐binding site is responsible for high ISG15 activity, while the S2 binding site provides Lys48 chain specificity and cleavage efficiency. To identify PLpro inhibitors in a repurposing approach, screening of 3,727 unique approved drugs and clinical compounds against SARS2 PLpro identified no compounds that inhibited PLpro consistently or that could be validated in counterscreens. More promisingly, non‐covalent small molecule SARS PLpro inhibitors also target SARS2 PLpro, prevent self‐processing of nsp3 in cells and display high potency and excellent antiviral activity in a SARS‐CoV‐2 infection model., Crystal structure and biochemical analysis explains the specificity of SARS‐CoV‐2 PLpro for ISG15 and longer Lys48‐linked ubiquitin chains leading to the identification of inhibitors that show promising antiviral activity in a SARS‐CoV‐2 infection model.

Biochemical, structural and functional studies on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) papain-like protease PLpro reveal that it regulates host antiviral responses by preferentially cleaving the ubiquitin-like interferon-stimulated gene 15 protein (ISG15) and identify this protease as a potential therapeutic target for coronavirus disease 2019 (COVID-19). The papain-like protease PLpro is an essential coronavirus enzyme that is required for processing viral polyproteins to generate a functional replicase complex and enable viral spread(1,2). PLpro is also implicated in cleaving proteinaceous post-translational modifications on host proteins as an evasion mechanism against host antiviral immune responses(3-5). Here we perform biochemical, structural and functional characterization of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PLpro (SCoV2-PLpro) and outline differences with SARS-CoV PLpro (SCoV-PLpro) in regulation of host interferon and NF-kappa B pathways. SCoV2-PLpro and SCoV-PLpro share 83% sequence identity but exhibit different host substrate preferences; SCoV2-PLpro preferentially cleaves the ubiquitin-like interferon-stimulated gene 15 protein (ISG15), whereas SCoV-PLpro predominantly targets ubiquitin chains. The crystal structure of SCoV2-PLpro in complex with ISG15 reveals distinctive interactions with the amino-terminal ubiquitin-like domain of ISG15, highlighting the high affinity and specificity of these interactions. Furthermore, upon infection, SCoV2-PLpro contributes to the cleavage of ISG15 from interferon responsive factor 3 (IRF3) and attenuates type I interferon responses. Notably, inhibition of SCoV2-PLpro with GRL-0617 impairs the virus-induced cytopathogenic effect, maintains the antiviral interferon pathway and reduces viral replication in infected cells. These results highlight a potential dual therapeutic strategy in which targeting of SCoV2-PLpro can suppress SARS-CoV-2 infection and promote antiviral immunity.

Stable NAD+ analogues carrying single atom substitutions in either the furanose ring or the nicotinamide part have proven their value as inhibitors for NAD+‐consuming enzymes. To investigate the potential of such compounds to inhibit the adenosine diphosphate ribosyl (ADPr) transferase activity of the Legionella SdeC enzyme, we prepared three NAD+ analogues, namely carbanicotinamide adenosine dinucleotide (c‐NAD+), thionicotinamide adenosine dinucleotide (S‐NAD+) and benzamide adenosine dinucleotide (BAD). We optimized the chemical synthesis of thionicotinamide riboside and for the first time used an enzymatic approach to convert all three ribosides into the corresponding NAD+ mimics. We thus expanded the known scope of substrates for the NRK1/NMNAT1 enzyme combination by turning all three modified ribosides into NAD+ analogues in a scalable manner. We then compared the three NAD+ mimics side‐by‐side in a single assay for enzyme inhibition on Legionella effector enzyme SdeC. The class of SidE enzymes to which SdeC belongs was recently identified to be important in bacterial virulence, and we found SdeC to be inhibited by S‐NAD+ and BAD with IC50 values of 28 and 39 μM, respectively., Modestly modified: Three stable NAD+ analogues namely, c‐NAD+, S‐NAD+ and BAD were prepared by using an optimized chemoenzymatic procedure and were compared side‐by‐side for enzyme inhibition of Legionella effector enzyme SdeC, which is important in bacterial virulence. Minimal structural variation in the furanose ring or nicotinamide part of NAD+ leads to efficient inhibitors.

Main protease and papain-like protease (PLpro) are essential coronaviral enzymes required for polypeptide processing during viral maturation. PLpro additionally cleaves proteinous post-translational modifications from host proteins to evade anti-viral immune responses. Here, we provide biochemical, structural and functional characterizations of PLpro from SARS-CoV-2 (PLproCoV2) and reveal differences to that of SARS (PLproSARS) in controlling interferon (IFN) and NF-kB pathways. PLproCoV2 and PLproSARS share 83% sequence identity, yet they differ in their host substrate preferences: PLproCoV2 predominantly cleaves the ubiquitin-like protein ISG15 off from host proteins, while PLproSARS preferentially targets ubiquitin chains. The crystal structure of PLproCoV2 in complex with ISG15 explains the affinity and higher specificity through distinctive binding to ISG15’s unique amino-terminal ubiquitin-like domain, and enabled the identification of GRL-0617 as a non-covalent candidate inhibitor for PLproCoV2. In human cells, PLproCoV2 cleaves ISG15 from interferon responsive factor 3 (IRF3), blocks its nuclear translocation, and reduces type I interferon responses, whereas PLproSARS preferentially mediates deubiquitination of critical components of the NF-kB pathway. Pharmacological inhibition of PLproCoV2 blocks the virus-induced cytopathogenic effect upon infection with SARS-CoV-2, fosters the anti- viral interferon pathway and reduces viral release from infected cells. We propose that therapeutic targeting of PLproCoV2 can suppress SARS-CoV-2 infection and promote anti-viral immunity.

Virtually all aspects of cell biology are regulated by a ubiquitin code where distinct ubiquitin chain architectures guide the binding events and itineraries of modified substrates. Various combinations of E2 and E3 enzymes accomplish chain formation by forging isopeptide bonds between the C terminus of their transiently linked donor ubiquitin and a specific nucleophilic amino acid on the acceptor ubiquitin, yet it is unknown whether the fundamental feature of most acceptors—the lysine side chain—affects catalysis. Here, use of synthetic ubiquitins with non-natural acceptor site replacements reveals that the aliphatic side chain specifying reactive amine geometry is a determinant of the ubiquitin code, through unanticipated and complex reliance of many distinct ubiquitin-carrying enzymes on a canonical acceptor lysine. [Figure not available: see fulltext.] © 2020, The Author(s), under exclusive licence to Springer Nature America, Inc.

Post-translational modification of substrate proteins plays crucial roles in the regulation of their activity, cellular localization, and ability to be recognized by other proteins. One of those modifications involves the installment of adenosine-diphosphate-ribose (ADPr) onto nucleophilic side-chain groups of amino acid residues. This highly dynamic process is regulated by ADP-ribosyl transferases (ARTs) that install the ADPr-molecules on selected proteins and poly(ADP-ribosyl) glycohydrolases (PARGs) and (ADP-ribosyl) hydrolases (ARHs) that trim down and remove ADPr-chains. In this mini-review, the most recent advances in the chemical synthesis of ADPr-conjugates, poly-ADP-ribose, ADPr-peptides, and -proteins, and other tools to investigate ADPr-biology are discussed.

Legionella pneumophila causes a severe pneumonia known as Legionnaires’ disease. During the infection, Legionella injects more than 300 effector proteins into host cells. Among them are enzymes involved in altering the host-ubiquitination system. Here, we identified two LegionellaOTU (ovarian tumor)-like deubiquitinases (LOT-DUBs; LotB [Lpg1621/Ceg23] and LotC [Lpg2529]). The crystal structure of the LotC catalytic core (LotC14-310) was determined at 2.4 Å. Unlike the classical OTU-family, the LOT-family shows an extended helical lobe between the Cys-loop and the variable loop, which defines them as a unique class of OTU-DUBs. LotB has an additional ubiquitin-binding site (S1’), which enables the specific cleavage of Lys63-linked polyubiquitin chains. By contrast, LotC only contains the S1 site and cleaves different species of ubiquitin chains. MS analysis of LotB and LotC identified different categories of host-interacting proteins and substrates. Together, our results provide new structural insights into bacterial OTU-DUBs and indicate distinct roles in host–pathogen interactions.

BackgroundCross-presentation of exogenous antigens in HLA-class I molecules by professional antigen presenting cells (APCs) is crucial for CD8+ T cell function. Recent murine studies show that several non-professional APCs, including cancer-associated fibroblasts (CAFs) also possess this capacity. Whether human CAFs are able to cross-present exogenous antigen, which molecular pathways are involved in this process and how this ultimately affects tumor-specific CD8+ T cell function is unknown.MethodsIn this study, we investigated the ability of human colorectal cancer (CRC)-derived CAFs to cross-present neoantigen-derived synthetic long peptides (SLPs), corresponding to tumor-derived mutant peptides, and how this affects tumor-specific T-cell function. Processing of the SLP was studied by targeting components of the cross-presentation machinery through CRISPR/Cas9 and siRNA-mediated genetic ablation to identify the key molecules involved in fibroblast-mediated cross-presentation. Multispectral flow cytometry and killing assays were performed to study the effect of fibroblast cross-presentation on T cell function.ResultsHere, we show that human CRC-derived CAFs display an enhanced capacity to cross-present neoantigen-derived SLPs when compared with normal colonic fibroblasts. Cross-presentation of antigens by fibroblasts involved the lysosomal protease cathepsin S. Cathepsin S expression by CAFs was detected in situ in human CRC tissue, was upregulated in ex vivo cultured CRC-derived CAFs and showed increased expression in normal fibroblasts after exposure to CRC-conditioned medium. Cognate interaction between CD8+ T cells and cross-presenting CAFs suppressed T cell function, reflected by decreased cytotoxicity, reduced activation (CD137) and increased exhaustion (TIM3, LAG3 and CD39) marker expression.ConclusionThese data indicate that CAFs may directly suppress tumor-specific T cell function in an antigen-dependent fashion in human CRC.

Current methods to prepare adenosine diphosphate ribosylated (ADPr) peptides are not generally applicable due to the labile nature of this post-translational modification and its incompatibility with strong acidic conditions used in standard solid-phase peptide synthesis. A general strategy is presented to prepare ADPr peptide analogues based on a copper-catalyzed click reaction between an azide-modified peptide and an alkyne-modified ADPr counterpart. The scope of this approach was expanded to proteins by preparing two ubiquitin ADPr analogues carrying the biological relevant α-glycosidic linkage. Biochemical validation using Legionella effector enzyme SdeA shows that clicked ubiquitin ADPr is well-tolerated and highlights the potential of this strategy to prepare ADPr proteins.

Plasmodium parasites are the causative agents of malaria, a disease with wide public health repercussions. Increasing drug resistance and the absence of a vaccine make finding new chemotherapeutic strategies imperative. Components of the ubiquitin and ubiquitin-like pathways have garnered increased attention as novel targets given their necessity to parasite survival. Understanding how these pathways are regulated in Plasmodium and identifying differences to the host is paramount to selectively interfering with parasites. Here, we focus on Nedd8 modification in Plasmodium falciparum, given its central role to cell division and DNA repair, processes critical to Plasmodium parasites given their unusual cell cycle and requirement for refined repair mechanisms. By applying a functional chemical approach, we show that deNeddylation is controlled by a different set of enzymes in the parasite versus the human host. We elucidate the molecular determinants of the unusual dual ubiquitin/Nedd8 recognition by the essential PfUCH37 enzyme and, through parasite transgenics and drug assays, determine that only its ubiquitin activity is critical to parasite survival. Our experiments reveal interesting evolutionary differences in how neddylation is controlled in higher versus lower eukaryotes, and highlight the Nedd8 pathway as worthy of further exploration for therapeutic targeting in antimalarial drug design., Author summary Ubiquitin and ubiquitin-like post-translational modifications are evolutionarily conserved and involved in fundamental cellular processes essential to all eukaryotes. As such, enzymatic components of these pathways present attractive targets for therapeutic intervention for both chronic and communicable diseases. Nedd8 modification of cullin ubiquitin E3 ligases is critical to the viability of eukaryotic organisms and mediates cell cycle progression and DNA damage repair. Given the complex lifecycle and unusual replication mechanisms of the malaria parasite, one would expect neddylation to be of central importance to its survival, yet little is known about this pathway in Plasmodium. Here we present our findings on how Nedd8 removal is controlled in Plasmodium falciparum and how this pathway differs to that of its human host.

One of the enigmas in the ubiquitin (Ub) field is the requirement for a poly-Ub chain as a proteasomal targeting signal. The canonical chain appears to be longer than the distance between the two Ub-binding proteasomal receptors. Furthermore, genetic manipulation has shown that one receptor subunit is sufficient, which suggests that a single Ub can serve as a degradation signal. To shed light on this mystery, we chemically synthesized tetra-Ub, di-Ub (K(48)-based), and mono-Ub adducts of HA-α-globin, where the distal or proximal Ub moieties were tagged differentially with either Myc or Flag. When incubated in a crude cell extract, the distal Ub moiety in the tetra-Ub adduct was mostly removed by deubiquitinating enzymes (DUBs) and reconjugated to other substrates in the extract. In contrast, the proximal moiety was most likely degraded with the substrate. The efficacy of degradation was proportionate to the chain length; while tetra-Ub globin was an efficient substrate, with mono-Ub globin, we observed rapid removal of the Ub moiety with almost no degradation of the free globin. Taken together, these findings suggest that the proximal moieties are necessary for securing the association of the substrate with the proteasome along the proteolytic process, whereas the distal moieties are important in protecting the proximal moieties from premature deubiquitination. Interestingly, when the same experiment was carried out using purified 26S proteasome, mono- and tetra-Ub globin were similarly degraded, highlighting the roles of the entire repertoire of cellular DUBs in regulating the degradation of proteasomal substrates.

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.

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.

Deubiquitinating enzymes (DUBs) recognize and cleave linkage-specific polyubiquitin (polyUb) chains, but mechanisms underlying specificity remain elusive in many cases. The severe acute respiratory syndrome (SARS) coronavirus papain-like protease (PLpro) is a DUB that cleaves ISG15, a two-domain Ub-like protein, and Lys48-linked polyUb chains, releasing diUb(Lys48) products. To elucidate this specificity, we report the 2.85 Å crystal structure of SARS PLpro bound to a diUb(Lys48) activity-based probe. SARS PLpro binds diUb(Lys48) in an extended conformation via two contact sites, S1 and S2, which are proximal and distal to the active site, respectively. We show that specificity for polyUb(Lys48) chains is predicated on contacts in the S2 site and enhanced by an S1-S1' preference for a Lys48 linkage across the active site. In contrast, ISG15 specificity is dominated by contacts in the S1 site. Determinants revealed for polyUb(Lys48) specificity should prove useful in understanding PLpro deubiquitinating activities in coronavirus infections.

Summary Ubiquitin chains are important post-translational modifications that control a large number of cellular processes. Chains can be formed via different linkages, which determines the type of signal they convey. Deubiquitylating enzymes (DUBs) regulate ubiquitylation status by trimming or removing chains from attached proteins. DUBs can contain several ubiquitin-binding pockets, which confer specificity toward differently linked chains. Most tools for monitoring DUB specificity target binding pockets on opposing sides of the active site; however, some DUBs contain additional pockets. Therefore, reagents targeting additional pockets are essential to fully understand linkage specificity. We report the development of active site-directed probes and fluorogenic substrates, based on non-hydrolyzable diubiquitin, that are equipped with a C-terminal warhead or a fluorogenic activity reporter moiety. We demonstrate that various DUBs in lysates display differential reactivity toward differently linked diubiquitin probes, as exemplified by the proteasome-associated DUB USP14. In addition, OTUD2 and OTUD3 show remarkable linkage-specific reactivity with our diubiquitin-based reagents., Graphical Abstract, Highlights • Protease-resistant diUb probes bind to DUB S1-S2 sites and react at the proximal end • First kinetic assay showing proximal end cleavage by DUBs using diUb-based substrates • OTUD3 binds K11-linked diUb and OTUD2 binds K11- and K33-linked diUb in S1-S2 pockets • Kinetics suggest different mechanisms for polyUb cleavage by OTUD2 and OTUD3, Deubiquitylating enzymes (DUBs) use multiple ubiquitin-binding pockets to disassemble polyubiquitin chains. Most tools probing DUB specificity target pockets adjacent to the active site. The protease-resistant diUb probes described here monitor linkage specificity of an additional S2 pocket and provide kinetics that explain polyubiquitin chain specificity.

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.

An optimized large scale and highly reproducible route to orthogonally protected γ-thiolysine is reported. Its utility in the synthesis of bifunctional ubiquitin monomers is demonstrated. These ubiquitin synthons are employed in polymerization reactions giving access to synthetic poly-ubiquitin chains of defined linkage.

A ribosyltriazole ring-fused cyclooctyne was prepared and converted into the corresponding phosphoramidite, which was applied in the automated synthesis of DNA and RNA oligomers. Ensuing strain-promoted alkyne–azide cycloaddition of the obtained oligonucleotides to fluorescent azides yielded the corresponding fluorescent oligonucleotide conjugates.

Nucleic acid-templated reactions are frequently explored tools in nucleic acid diagnosis. To enable a separation-free DNA detection, the reactive probe molecules require conjugation with reporter groups that provide measurable changes of an observable parameter upon reaction. A widely used, generic read-out method is based on fluorescence resonance energy transfer (FRET) between two appended dyes. Yet, spectral cross-talk usually limits the achievable enhancements of the FRET signal in DNA-directed chemistries. We describe a DNA-triggered transfer reaction which provides for strong increases of a fluorescent signal caused by FRET. The method may involve DNA- and PNA-based probes and is based upon a proximity-triggered transfer reaction which leads to the covalent fixation of a fluorescence dye on the surface of a quantum dot (QD). The transfer reaction brings the dye closer to the QD than hybridization alone. The resulting FRET signal is a specific monitor of the reaction and allows efficient discrimination of single base mismatched templates. Of note, the 35-fold increase of the FRET signal is measured at 310 nm apparent Stokes shift and turnover in template provides a means for signal amplification.

The glycosylation properties of ribofuranosyl N-phenyltrifluoroacetimidates toward carboxamide side chains of asparagine and glutamine were investigated. Conditions were found that promote nearly exclusive formation of the α-anomerically configured N-glycosides. The strategy allows for the synthesis of Fmoc-amino acids suitably modified for the preparation of ADP-ribosylated peptides. Furthermore, ribosylation of serine with these donors proved to be completely α-selective, and for the first time, α-ribosylated glutamic and aspartic acid, the naturally occurring sites for poly-ADP-ribosylation, were synthesized.

Small proteins called viral protein genome-linked (VPg), attached to the 5'-end of the viral RNA genome are found as common structure in the large family of picornaviruses. The replication of these viruses is primed by this VPg protein linked to a single uridylyl residue. We report a general procedure to obtain such nucleoproteins employing a pre-uridylylated tyrosine building block in an on-line solid phase-based approach.

A method to ribosylate adenosine on the 2' hydroxyl function in an α-selective fashion and in good yield is presented. Protective groups chosen for the acceptor and donor used in this glycosylation not only direct α-selectivity but also allow the construction of a fully orthogonally protected building block for the future assembly of oligo-ADP-ribosylated peptides and proteins.

Phosphitylation of the side chain hydroxyl function of Fmoc protected tyrosine with 5'-phosphoramidites of suitably protected cytidine, adenosine, and guanosine, followed by oxidation gave three novel nucleotidylated amino acid building blocks. After protective group manipulation, these building blocks were used in a solid phase peptide synthesis to afford the nucleotidylated poliovirus proteins VPgpC, VPgpA, and VPgpG.

Enteroviruses (EV) uridylylate a peptide, VPg, as the first step in their replication. VPgpUpU, found free in infected cells, serves as the primer for RNA elongation. The abilities of four polymerases (3D pol ), from EV-species A–C, to uridylylate VPgs that varied by up to 60% of their residues were compared. Each 3D pol was able to uridylylate all five VPgs using polyA RNA as template, while showing specificity for its own genome encoded peptide. All 3D pol uridylylated a consensus VPg representing the physical chemical properties of 31 different VPgs. Thus the residues required for uridylylation and the enzymatic mechanism must be similar in diverse EV. As VPg-binding sites differ in co-crystal structures, the reaction is probably done by a second 3D pol molecule. The conservation of polymerase residues whose mutation reduces uridylylation but not RNA elongation is compared.

The synthesis of an array of 2-azidoalkoxy substituted 7-hydro-8-oxoadenines is described. The relation of the structure of these compounds and their ability to induce maturation of dendritic cells is evaluated.

Small proteins called viral protein genome-linked (VPg), attached to the 5'-end of the viral RNA genome are found as common structure in the large family of picornaviruses. The replication of these viruses is primed by this VPg protein linked to a single uridylyl residue. We report a general procedure to obtain such nucleoproteins employing a pre-uridylylated tyrosine building block in an on-line solid phase-based approach.

A previously described mammalian cell activity, called VPg unlinkase, specifically cleaves a unique protein–RNA covalent linkage generated during the viral genomic RNA replication steps of a picornavirus infection. For over three decades, the identity of this cellular activity and its normal role in the uninfected cell had remained elusive. Here we report the purification and identification of VPg unlinkase as the DNA repair enzyme, 5′-tyrosyl–DNA phosphodiesterase-2 (TDP2). Our data show that VPg unlinkase activity in different mammalian cell lines correlates with their differential expression of TDP2. Furthermore, we show that recombinant TDP2 can cleave the protein–RNA linkage generated by different picornaviruses without impairing the integrity of viral RNA. Our results reveal a unique RNA repair-like function for TDP2 and suggest an unusual role in host–pathogen interactions for this cellular enzyme. On the basis of the identification of TDP2 as a potential antiviral target, our findings may lead to the development of universal therapeutics to treat the millions of individuals afflicted annually with diseases caused by picornaviruses, including myocarditis, aseptic meningitis, encephalitis, hepatitis, and the common cold.