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BBA - Molecular Cell Research (v.1823, #11)

Editorial Board (pp. i).

Ubiquitin Drug Discovery and Diagnostics by Christian Loch; Michael Eddins (pp. 2013-2013).

Ubiquitin-based anticancer therapy: Carpet bombing with proteasome inhibitors vs surgical strikes with E1, E2, E3, or DUB inhibitors by Michael R. Mattern; Jian Wu; Benjamin Nicholson (pp. 2014-2021).

The proteasome inhibitor bortezomib remains the only ubiquitin pathway effector to become a drug (VELCADE®) and has become a successful treatment for hematological malignancies. While producing a global cellular effect, proteasome inhibitors have not triggered the catastrophe articulated initially in terms such as “buildup of cellular garbage”. Proteasome inhibitors, in fact, do have a therapeutic window, although in the case of the prototype bortezomib it is small owing to peripheral neuropathy, myelosuppression and, as recently reported, cardiotoxicity [1]. Currently, several second-generation molecules are undergoing clinical evaluation to increase this window. An alternative strategy is to target ubiquitin pathway enzymes acting at non-proteasomal sites—E1, E2, and E3, associated with ubiquitin conjugation, and deubiquitylating enzymes (“DUBs”)—that act locally on selected targets rather than on the whole cell. Inhibitors (or activators, in some cases) of these enzymes should be developable as selective antitumor agents with toxicity profiles superior to that of bortezomib. Various therapeutic hypotheses follow from known cellular mechanisms of these target enzymes; most hypotheses relate to cancer, reminiscent of the FDA-approved protein kinase inhibitors now marketed. Since ubiquitin tagging controls the cellular content, activity, or compartmentation of proteins associated with disease, inhibitors or activators of ubiquitin conjugation or deconjugation are predicted to have an impact on disease. For practical and empirical reasons, inhibitors of ubiquitin pathway enzymes have been the favored therapeutic avenue. In approximately the time that has elapsed since the approval of bortezomib in 2003, there has been some progress in developing potential anticancer drugs that target various ubiquitin pathway enzymes. An E1 inhibitor and inhibitors of E3 are now in clinical trial, with some objective responses reported. Appropriate assays and/or rational design may uncover improved inhibitors of these enzymes, as well as E2 and DUBs, for further development. Presently, it should become clear whether one or both of the two general strategies for ubiquitin-based drug discovery will lead to truly superior new medicines for cancer and other diseases. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.► Proteasome inhibitors (VELCADE®) are efficacious anticancer drugs. ► Since approval of VELCADE®, other ubiquitin targets have been used in drug discovery efforts. ► To date, more progress has been made with proteasome inhibitors than with these enzymes. ► Possible reasons for this difference are considered.

Keywords: Proteasome inhibitor; Ubiquitin; E3 ligase; Deubiquitylase; Anticancer drugs

Ubiquitination and phosphorylation in the regulation of NOD2 signaling and NOD2-mediated disease by Justine T. Tigno-Aranjuez; Derek W. Abbott (pp. 2022-2028).

The immune system is exquisitely balanced. It has the ability to effectively respond to and control infections while at the same time preventing inappropriate responses to self and environmental antigens. When this response goes awry, either through a failure to activate the immune response, or failure to terminate it, inflammatory pathology results. Posttranslational modifications (PTMs) such as ubiquitination and phosphorylation help ensure that the delicate balance underlying immune signal transduction is maintained. Ubiquitination and phosphorylation affect localization, activity, stability, and interactions of various components of the immune signal transduction machinery. Moreover, ubiquitination and phosphorylation are tightly linked, with one PTM affecting the other. Therefore, in order to find potential therapies for many immune-related pathologies, it is necessary to understand not only how the immune response is activated by ubiquitination and phosphorylation, but also how it is regulated by these PTMs at different stages of the response. An excellent system to study such activation and regulation is the NOD2 pathway. Dysregulation of NOD2 signaling is involved in the pathogenesis of a variety of inflammatory disorders including Crohn's disease, early onset sarcoidosis, and Blau syndrome. More recently NOD2 has been implicated in the development of autoimmune disease, allergy and asthma. This review will focus on what is currently known about how ubiquitination and phosphorylation regulate NOD2 signaling with particular emphasis on novel in vitro substrates which may serve as potential in vivo therapeutic targets for hyperactive NOD2 states. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.► The NOD2 pathway is regulated by phosphorylation and ubiquitination. ► A number of disease settings may benefit from inhibition of NOD2 signaling. ► We describe potential therapeutic targets in the NOD2 pathway. ► We describe currently available inhibitors of NOD2 signaling.

Keywords: NOD2; RIP2; IAP; Ubiquitination; Phosphorylation

Detection of ubiquitin–proteasome enzymatic activities in cells: Application of activity-based probes to inhibitor development by Holger B. Kramer; Benjamin Nicholson; Benedikt M. Kessler; Mikael Altun (pp. 2029-2037).

Background: Synthetic probes that mimic natural substrates can enable the detection of enzymatic activities in a cellular environment. One area where such activity-based probes have been applied is the ubiquitin–proteasome pathway, which is emerging as an important therapeutic target. A family of reagents has been developed that specifically label deubiquitylating enzymes (DUBs) and facilitate characterization of their inhibitors. Scope of review: Here we focus on the application of probes for intracellular DUBs, a group of specific proteases involved in the ubiquitin proteasome system. In particular, the functional characterization of the active subunits of this family of proteases that specifically recognize ubiquitin and ubiquitin-like proteins will be discussed. In addition we present the potential and design of activity-based probes targeting kinases and phosphatases to study phosphorylation. Major conclusions: Synthetic molecular probes have increased our understanding of the functional role of DUBs in living cells. In addition to the detection of enzymatic activities of known members, activity-based probes have contributed to a number of functional assignments of previously uncharacterized enzymes. This method enables cellular validation of the specificity of small molecule DUB inhibitors. General significance: Molecular probes combined with mass spectrometry-based proteomics and cellular assays represent a powerful approach for discovery and functional validation, a concept that can be expanded to other enzyme classes. This addresses a need for more informative cell-based assays that are required to accelerate the drug development process. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.► Activity-based chemical probe screen for specificity of small molecule deubiquitylating enzyme inhibitors in cells. ► Cell-based profiling of inhibitors specific for deubiquitylating enzymes by mass spectrometry. ► Overview of proteasome, deubiquitylating enzyme, kinase, and phosphatase probes.

Keywords: Abbreviations; ABP; Activity-based probe; ADP; Adenosine diphosphate; ATP; Adenosine triphosphate; DUB; Deubiquitylating enzyme; E1; Ubiquitin activating enzyme; E2; Ubiquitin conjugating enzyme; E3; Ubiquitin-protein ligase; HAUbBr2; HA-tagged ubiquitin ethyl bromide; HAUbVME; HA-tagged ubiquitin vinyl methyl ester; HAUbVS; HA-tagged ubiquitin vinyl sulfone; IP; Immunoprecipitation; MS; Mass Spectrometry; PTM; Post-Translational Modification; PTP; Protein Tyrosine Phosphatase; SDS-PAGE; Sodium dodecylsulfate polyacrylamide gel electrophoresis; SPPS; Solid Phase Peptide Synthesis; Ub; Ubiquitin; Ubl; Ubiquitin-like protein; UCH-L1; Ubiquitin carboxyl terminal hydrolase isozyme L1; UCH-L3; Ubiquitin carboxyl terminal hydrolase isozyme L3; UCH-L5; Ubiquitin carboxyl terminal hydrolase isozyme L5; USP7; Ubiquitin specific processing protease 7Ubiquitin; Small molecular inhibitor; Deubiquitinating enzyme; Ubiquitin specific protease; Proteomics; Active site-directed molecular probe

Development of a homogeneous AlphaLISA ubiquitination assay using ubiquitin binding matrices as universal components for the detection of ubiquitinated proteins by Stephen Schneider; Hao Chen; Jin Tang; Renee Emkey; Paul S. Andrews (pp. 2038-2045).

The Ubiquitin Proteasome Pathway (UPP) has become a target rich pathway for therapeutic intervention as its role in pathogenic disease is better understood. In particular many E3 ligases within this pathway have been implicated in cancer, inflammation and metabolic diseases. It has been of great interest to develop biochemical assays to identify inhibitors of catalytic E3 ubiquitination activity as a means of abrogating the disease mechanism. Here we describe a homogeneous biochemical assay that utilizes native ubiquitin andTandem-repeatedUbiquitin-BindingEntities (TUBEs) as a detection technology for ubiquitination activity. We developed a TUBEs based proximity AlphaLisa® assay for Mdm2, which is an E3 ligase that negatively regulates p53 tumor suppressor protein. We further demonstrate that this assay strategy or design can also be applied to the development of assays to detect autoubiquitination of other E3 ligases that are also of interest for therapeutic intervention. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.► A homogenous autoubiquitination AlphaLisa assay was developed. ► Tandem Ubiquitin Binding Entities (TUBES) allows for the use of wild type ubiquitin. ► We demonstrate the universality of this approach with 3 E3s from 2 ligase classes.

Keywords: Abbreviations; UPP; ubiquitin proteasome pathway; E1; ubiquitin activating enzyme; E2; ubiquitin conjugating enzyme; E3; ubiquitin ligase; ECL; electrochemiluminescence; HECT; Homologous to E6AP Carboxy Terminus; RING; Really Interesting New Gene; Smurf1; SM; ad; U; biquitin; R; egulatory; F; actor 1; TGF-β; transforming growth factor beta; DTT; Dithiothreitol; BSA; bovine serum albumin; GST; glutathione-s-transferase; UBA; ubiquitin association domain; IL-1; Interleukine1; TLR; Toll Like Receptor; TNFR; tumor necrosis factor receptor; NFκB; Nuclear factor κB; TR-FRET; Time-Resolved Fluorescence Resonance Energy Transfer; TUBE; T; andem-repeated; U; biquitin-; B; inding; E; ntitie; s; HTS; High Throughput Screening; MS; Mass Spectrometry; DMSO; DimethylsulfoxideUbiquitin; TUBEs; AlphaLISA; Mdm2

Structural and biochemical studies of the open state of Lys48-linked diubiquitin by Ming-Yih Lai; Daoning Zhang; Nicole LaRonde-LeBlanc; David Fushman (pp. 2046-2056).

Ubiquitin (Ub) is a small protein highly conserved among eukaryotes and involved in practically all aspects of eukaryotic cell biology. Polymeric chains assembled from covalently-linked Ub monomers function as molecular signals in the regulation of a host of cellular processes. Our previous studies have shown that the predominant state of Lys48-linked di- and tetra-Ub chains at near-physiological conditions is a closed conformation, in which the Ub–Ub interface is formed by the hydrophobic surface residues of the adjacent Ub units. Because these very residues are involved in (poly)Ub interactions with the majority of Ub-binding proteins, their sequestration at the Ub–Ub interface renders the closed conformation of polyUb binding incompetent. Thus the existence of open conformation(s) and the interdomain motions opening and closing the Ub–Ub interface is critical for the recognition of Lys48-linked polyUb by its receptors. Knowledge of the conformational properties of a polyUb signal is essential for our understanding of its specific recognition by various Ub-receptors. Despite their functional importance, open states of Lys48-linked chains are poorly characterized. Here we report a crystal structure of the open state of Lys48-linked di-Ub. Moreover, using NMR, we examined interactions of the open state of this chain (at pH4.5) with a Lys48-linkage-selective receptor, the UBA2 domain of a shuttle protein hHR23a. Our results show that di-Ub binds UBA2 in the same mode and with comparable affinity as the closed state. Our data suggest a mechanism for polyUb signal recognition, whereby Ub-binding proteins select specific conformations out of the available ensemble of polyUb chain conformations. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.Display Omitted► We report the crystal structure of an open form of Lys48-linked di-ubiquitin. ► At acidic pH ubiquitin binds hHR23a UBA2 domain as at physiological conditions. ► The open state of Lys48 di-ubiquitin binds hHR23a UBA2 similarly to the closed state. ► Our results highlight intrinsic flexibility of polyubiquitin chains in solution. ► We propose a conformational selection mechanism of polyubiquitin signal recognition.

Keywords: Ubiquitin; Lys48-linked diubiquitin; Polyubiquitin; Ubiquitin-associated domain; Lysine-48 linkage selectivity

The small molecule inhibitor PR-619 of deubiquitinating enzymes affects the microtubule network and causes protein aggregate formation in neural cells: Implications for neurodegenerative diseases by Veronika Seiberlich; Olaf Goldbaum; Victoria Zhukareva; Christiane Richter-Landsberg (pp. 2057-2068).

A pathological hallmark of many neurodegenerative diseases is the aggregation of proteins. Protein aggregate formation may be linked to a failure of the ubiquitin proteasome system (UPS) and/or the autophagy pathway. The UPS involves the ubiquitination of proteins followed by proteasomal degradation. Deubiquitination of target proteins is performed by proteases called deubiquitinating proteins (DUBs). Inhibition of DUBs may lead to the dysregulation of homeostasis and have pathological consequences. To assess the effects of DUB-inhibition, we have used the oligodendroglial cell line, OLN-t40, stably expressing the longest human tau isoform. Cells were incubated with PR-619, a broad-range, reversible inhibitor of ubiquitin isopeptidases. Incubation with PR-619 led to morphological changes, the upregulation of heat shock proteins (HSP), including HSP70 and αB-crystallin, and to protein aggregates near the MTOC, containing ubiquitin, HSPs, and the ubiquitin binding protein p62, which may provide a link between the UPS and autophagy. Thus, inhibition of DUB activity caused stress responses and the formation of protein aggregates resembling pathological inclusions observed in aggregopathies. Furthermore, PR-619 led to the stabilization of the microtubule network, possibly through the modulation of tau phosphorylation, and small tau deposits assembled near the MTOC. Hence, organization and integrity of the cytoskeleton were affected, which is particularly important for the maintenance of the cellular architecture and intracellular transport processes, and essential for the functionality and survival of neural cells. Our data demonstrate that DUB inhibitors provide a useful tool to elucidate the manifold mechanisms of DUB functions in cells and their dysregulation in neurodegenerative diseases. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.► Inhibition of deubiquitinating enzymes impairs oligodendroglial cell integrity. ► Protein aggregates are formed as observed in neurodegenerative diseases. ► DUB inhibition leads to a stress response and accumulation of heat shock proteins. ► Ubiquitin and the ubiquitin binding protein p62 accumulate in aggregates. ► PR-619 leads to the stabilization of microtubules and tau dephosphorylation.

Keywords: Deubiquitinating enzymes; Oligodendroglia; p62; Heat shock proteins; Microtubules; Tau protein

A microarray of ubiquitylated proteins for profiling deubiquitylase activity reveals the critical roles of both chain and substrate by Christian M. Loch; James E. Strickler (pp. 2069-2078).

Substrate ubiquitylation is a reversible process critical to cellular homeostasis that is often dysregulated in many human pathologies including cancer and neurodegeneration. Elucidating the mechanistic details of this pathway could unlock a large store of information useful to the design of diagnostic and therapeutic interventions. Proteomic approaches to the questions at hand have generally utilized mass spectrometry (MS), which has been successful in identifying both ubiquitylation substrates and profiling pan-cellular chain linkages, but is generally unable to connect the two. Interacting partners of the deubiquitylating enzymes (DUBs) have also been reported by MS, although substrates of catalytically competent DUBs generally cannot be. Where they have been used towards the study of ubiquitylation, protein microarrays have usually functioned as platforms for the identification of substrates for specific E3 ubiquitin ligases. Here, we report on the first use of protein microarrays to identify substrates of DUBs, and in so doing demonstrate the first example of microarray proteomics involving multiple (i.e., distinct, sequential and opposing) enzymatic activities. This technique demonstrates the selectivity of DUBs for both substrate and type (mono- versus poly-) of ubiquitylation. This work shows that the vast majority of DUBs are monoubiquitylated in vitro, and are incapable of removing this modification from themselves. This work also underscores the critical role of utilizing both ubiquitin chains and substrates when attempting to characterize DUBs. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.►DUB activity depends on both substrate and the type of ubiquitin modification. ►DUBs are monoubiquitylated in vitro. ►Most DUBs do not appear capable of auto-deubiquitylation. ►E3s are hyper-activated for auto-ubiquitylation in the presence of DUBs. ►Microarray proteomics for substrate identification of enzymes that reverse PTMs

Keywords: Abbreviations; DUB; deubiquitylase; MS; mass spectrometry; PTM; post-translational modification; ATP; adenosine triphosphate; K48; the forty-eighth amino acid of ubiquitin, a lysine; K63; the sixty-third amino acid of ubiquitin, a lysine; TUBEs; tandem ubiquitin binding entities; c; catalytic (for example, USP2c is the catalytic core domain only of USP2); SDS PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresisUbiquitin; Deubiquitylase; Protein microarray; TUBEs; DUB Chip; Monoubiquitylated

Bioluminescence assay platform for selective and sensitive detection of Ub/Ubl proteases by Steven J. Orcutt; Jian Wu; Michael J. Eddins; Craig A. Leach; James E. Strickler (pp. 2079-2086).

As the importance of ubiquitylation in certain disease states becomes increasingly apparent, the enzymes responsible for removal of ubiquitin (Ub) from target proteins, deubiquitylases (DUBs), are becoming attractive targets for drug discovery. For rapid identification of compounds that alter DUB function, in vitro assays must be able to provide statistically robust data over a wide dynamic range of both substrate and enzyme concentrations during high throughput screening (HTS). The most established reagents for HTS are Ubs with a quenched fluorophore conjugated to the C-terminus; however, a luciferase-based strategy for detecting DUB activity (DUB-Glo™, Promega) provides a wider dynamic range than traditional fluorogenic reagents. Unfortunately, this assay requires high enzyme concentrations and lacks specificity for DUBs over other isopeptidases (e.g. desumoylases), as it is based on an aminoluciferin (AML) derivative of a peptide derived from the C-terminus of Ub (Z-RLRGG-). Conjugation of aminoluciferin to a full-length Ub (Ub-AML) yields a substrate that has a wide dynamic range, yet displays detection limits for DUBs 100- to 1000-fold lower than observed with DUB-Glo™. Ub-AML was even a sensitive substrate for DUBs (e.g. JosD1 and USP14) that do not show appreciable activity with DUB-Glo™. Aminoluciferin derivatives of hSUMO2 and NEDD8 were also shown to be sensitive substrates for desumoylases and deneddylases, respectively. Ub/Ubl-AML substrates are amenable to HTS (Z’=0.67) yielding robust signal, and providing an alternative drug discovery platform for Ub/Ubl isopeptidases. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.► We have generated a novel class of Ub/Ubl-aminoluciferin substrates for DUBs. ► These reagents display greater sensitivity and selectivity than existing DUB substrates. ► Ub/Ubl-aminoluciferins are highly amenable to high throughput screening (HTS). ► Use of these substrates allows for HTS of DUBs that have low activity for other such reagents.

Keywords: Abbreviations; Ub; ubiquitin; DUB; deubiquitylase; HTS; high throughput screening; AML; aminoluciferin; USP; ubiquitin specific protease; UCH; ubiquitin carboxyterminal hydrolase; SUMO; small ubiquitin-like modifier; NEDD8; nerounal precursor cell expressed developmently down-regulated 8; AMC; aminomethylcoumarin; Rho110; rhodamine 110Ubiquitin; SUMO; Luciferin; Deubiquitylation; Protease assay; Desumoylation

Proteasome protease mediated regulation of cytokine induction and inflammation by Nilofer Qureshi; David C. Morrison; Julia Reis (pp. 2087-2093).

We have previously demonstrated that proteasome serves as a central regulator of inflammation and macrophage function. Until recently, proteasomes have generally been considered to play a relatively passive role in the regulation of cellular activity, i.e., any ubiquitinated protein was considered to be in discriminatively targeted for degradation by the proteasome. We have demonstrated, however, by using specific proteasome protease inhibitors and knockout mice lacking specific components of immunoproteasomes, that proteasomes (containing X, Y, and Z protease subunits) and immunoproteasomes (containing LMP7, LMP2, and LMP10 protease subunits) have well-defined functions in cytokine induction and inflammation based on their individual protease activities. We have also shown that LPS-TLR mediated signaling in the murine RAW 264.7 macrophage cell line results in the replacement of macrophage immunoproteasomal subunits. Such modifications serve as pivotal regulators of LPS-induced inflammation. Our findings support the relatively novel concept that defects in structure/function of proteasome protease subunits caused by genetic disorders, aging, diet, or drugs may well have the potential to contribute to modulation of proteasome activity. Of particular relevance, we have identified quercetin and resveratrol, significant constituents present in berries and in red wine respectively, as two novel proteasome inhibitors that have been previously implicated as disease-modifying natural products. We posit that natural proteasome inhibitors/activators can potentially be used as therapeutic response modifiers to prevent/treat diseases through pathways involving the ubiquitin–proteasome pathway (UP-pathway), which likely functions as a master regulator involved in control of overall inflammatory responses. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.► The proteasome serves as a pivotal regulator of agonist-induced inflammation and cytokine induction. ► Agonist treatment of RAW 264.7 cells induces changes in relative proteolytic activity of the proteasomes. ► Proteasomes and immunoproteasomes have well-defined functions affecting multiple signaling pathways. ► Several proteins are ubiquitinated and modulated during cellular treatment with LPS. ► Quercetin and resveratrol are novel natural proteasome inhibitors.

Keywords: Proteasome; Cytokines; Resveratrol; Quercetin; Proteasome inhibitors; LMP knockout mice

Analysis of ubiquitin E3 ligase activity using selective polyubiquitin binding proteins by Jeffrey G. Marblestone; James P. LaRocque; Michael R. Mattern; Craig A. Leach (pp. 2094-2097).

The ubiquitin proteasome pathway controls the cellular degradation of ~80–90% of the proteome in a highly regulated manner. In this pathway, E3 ligases are responsible for the conjugation of ubiquitin to protein substrates which can lead to their destruction by the 26S proteasome. Aberrant E3 ligases have been implicated in several diseases and are widely recognized as attractive targets for drug discovery. As researchers continue to characterize E3 ligases, additional associations with various disease states are being exposed. The availability of assays that allow rapid analysis of E3 ligase activity is paramount to both biochemical studies and drug discovery efforts aimed at E3 ligases. To address this need, we have developed a homogenous assay for monitoring ubiquitin chain formation using Tandem Ubiquitin Binding Entities (TUBEs). TUBEs bind selectively to polyubiquitin chains versus mono-ubiquitin thus enabling the detection of polyubiquitin chains in the presence of mono-ubiquitin. This assay reports on the proximity between the protein substrate and TUBEs as a result of polyubiquitin chain formation by an E3 ligase. This homogenous assay is a step forward in streamlining an approach for characterizing and quantitating E3 ligase activity in a rapid and cost effective manner. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.► Demonstration of the assays' capability to detect polyubiquitin chains relative to free ubiquitin using TUBEs by TR-FRET ► Profiling ubiquitin E3 ligases, demonstrating dose dependency in a homogenous manner ► Exhibiting kinetic analysis of E3 ligase activity in real time

Keywords: Ubiquitin; E3 ligase; Assay; MuRF1; TRIM25; Drug discovery

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BBA - Molecular Cell Research (v.1823, #11)