Analysis of ubiquitin E3 ligase activity using selective polyubiquitin binding proteins.

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.

Bioluminescence assay platform for selective and sensitive detection of Ub/Ubl proteases.

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.

Deubiquitylase, deSUMOylase, and deISGylase activity microarrays for assay of substrate preference and functional modifiers.

Microarray-based proteomics expanded the information potential of DNA arrays to the level of protein translation and interaction, but so far, not much beyond. Although enzymatic activity from immobilized proteins has been reliably studied using surface plasmon resonance, a microarray of catalytically competent enzymes would facilitate high throughput, parallel study of their function. The ability to localize activity from soluble substrates has frustrated development of such an array. Here, we report the novel use of previously developed, highly specific suicide substrates for three families of enzymes: deubiquitylases, deSUMOylases, and deISGylases. We show specificity of each family to its cognate substrate, and demonstrate utility of the array in a secondary screen of small molecule inhibitors.

Tissue-specific expression and post-translational modifications of plant- and bacterial-type phosphoenolpyruvate carboxylase isozymes of the castor oil plant, Ricinus communis L.

This study employs transcript profiling together with immunoblotting and co-immunopurification to assess the tissue-specific expression, protein:protein interactions, and post-translational modifications (PTMs) of plant- and bacterial-type phosphoenolpyruvate carboxylase (PEPC) isozymes (PTPC and BTPC, respectively) in the castor plant, Ricinus communis. Previous studies established that the Class-1 PEPC (PTPC homotetramer) of castor oil seeds (COS) is activated by phosphorylation at Ser-11 and inhibited by monoubiquitination at Lys-628 during endosperm development and germination, respectively. Elimination of photosynthate supply to developing COS by depodding caused the PTPC of the endosperm and cotyledon to be dephosphorylated, and then subsequently monoubiquitinated in vivo. PTPC monoubiquitination rather than phosphorylation is widespread throughout the castor plant and appears to be the predominant PTM of Class-1 PEPC that occurs in planta. The distinctive developmental patterns of PTPC phosphorylation versus monoubiquitination indicates that these two PTMs are mutually exclusive. By contrast, the BTPC: (i) is abundant in the inner integument, cotyledon, and endosperm of developing COS, but occurs at low levels in roots and cotyledons of germinated COS, (ii) shows a unique developmental pattern in leaves such that it is present in leaf buds and young expanding leaves, but undetectable in fully expanded leaves, and (iii) tightly interacts with co-expressed PTPC to form the novel and allosterically-desensitized Class-2 PEPC heteromeric complex. BTPC and thus Class-2 PEPC up-regulation appears to be a distinctive feature of rapidly growing and/or biosynthetically active tissues that require a large anaplerotic flux from phosphoenolpyruvate to replenish tricarboxylic acid cycle C-skeletons being withdrawn for anabolism.

Ubiquitin/SUMO modification of PCNA promotes replication fork progression in Xenopus laevis egg extracts.

The homotrimeric DNA replication protein proliferating cell nuclear antigen (PCNA) is regulated by both ubiquitylation and sumoylation. We study the appearance and the impact of these modifications on chromosomal replication in frog egg extracts. Xenopus laevis PCNA is modified on lysine 164 by sumoylation, monoubiquitylation, and diubiquitylation. Sumoylation and monoubiquitylation occur during the replication of undamaged DNA, whereas diubiquitylation occurs specifically in response to DNA damage. When lysine 164 modification is prevented, replication fork movement through undamaged DNA slows down and DNA polymerase delta fails to associate with replicating chromatin. When sumoylation alone is prevented, replication occurs normally and neither monoubiquitylation nor sumoylation are required for the replication of simple single-strand DNA templates. Our findings expand the repertoire of functions for PCNA ubiquitylation and sumoylation by elucidating a role for these modifications during the replication of undamaged DNA. Furthermore, they suggest that PCNA monoubiquitylation serves as a molecular gas pedal that controls the speed of replisome movement during S phase.

The CCR2/MCP-1 Chemokine Pathway and Lung Adenocarcinoma.

Host anti-tumor immunity can be hindered by various mechanisms present within the tumor microenvironment, including the actions of myeloid-derived suppressor cells (MDSCs). We investigated the role of the CCR2/MCP-1 pathway in MDSC-associated tumor progression in murine lung cancer models. Phenotypic profiling revealed maximal expression of CCR2 by tumor-resident MDSCs, and MCP-1 by transplanted TC1 tumor cells, respectively. Use of CCR2-knockout (CCR2-KO) mice showed dependence of tumor growth on CCR2 signaling. Tumors in CCR2-KO mice had fewer CCR2low MDSCs, CD4 T cells and Tregs than WT mice, and increased infiltration by CD8 T cells producing IFN-γ and granzyme-B. Effects were MDSC specific, since WT and CCR2-KO conventional T (Tcon) cells had comparable proliferation and production of inflammatory cytokines, and suppressive functions of WT and CCR2-KO Foxp3+ Treg cells were also similar. We used a thioglycolate-induced peritonitis model to demonstrate a role for CCR2/MCP-1 in trafficking of CCR2+ cells to an inflammatory site, and showed the ability of a CCR2 antagonist to inhibit such trafficking. Use of this CCR2 antagonist promoted anti-tumor immunity and limited tumor growth. In summary, tumor cells are the prime source of MCP-1 that promotes MDSC recruitment, and our genetic and pharmacologic data demonstrate that CCR2 targeting may be an important component of cancer immunotherapy.

Detection and characterization of SUMO protease activity using a sensitive enzyme-based reporter assay.

In this chapter we describe a novel, sensitive, homogenous high throughput reporter-based in vitro assay for SUMO protease activity developed by Progenra, Inc. A reporter construct was created by fusing His(6)-tagged small ubiquitin-like modifier (SUMO) to the amino terminus of the reporter enzyme phospholipase A(2) (PLA(2)). Following cleavage by a member of the sentrin specific proteases (SENPs), free PLA(2) is able to turn over its substrate, resulting in the release of a fluorescent product which is readily quantifiable using a fluorimeter or a fluorescence plate reader. The utility of this SUMO-CHOP-Reporter assay platform is demonstrated by its ability to determine K(m) values and to characterize inhibitors of SUMO proteases.

Glucocorticoid regulation of human eosinophil gene expression.

Molecular analysis of steroid-regulated gene expression in freshly isolated human eosinophils is difficult due to the inherent high rate of spontaneous apoptosis and elevated levels of endogenous ribonucleases. To circumvent these limitations, we determined if the human eosinophilic cell line EoL-1 could serve as an in vitro model of glucocorticoid signaling. We found by optimizing growth conditions in low serum-containing media that dexamethasone (Dex) treatment of EoL-1 cells induced an apoptotic pathway that was inhibited by interleukin-5 (IL-5). Moreover, gene expression profiling using RNA from untreated EoL-1 cells and from freshly isolated human eosinophils identified 380 commonly expressed genes, including the eosinophil markers granule major basic protein, prostaglandin-endoperoxide synthase 1 and arachidonate 15-lipoxygenase. Expression profiling was performed using EoL-1 cells that had been treated with dexamethasone for 0, 4, 12, 24 and 48h identifying 162 genes as differentially expressed. Two of the most highly upregulated genes based on expression profiling were the transcription factor Ets-2 and the MHC Class II genes (Q, R, and P). Expression of these genes in EoL-1 cells was shown to be dexamethasone-induced at the RNA and protein levels which is consistent with the known function of Ets-2 in controlling cell cycle progression and the role of MHC Class II antigens in mediating eosinophil functions.

RING-finger type E3 ubiquitin ligase inhibitors as novel candidates for the treatment of rheumatoid arthritis.

Rheumatoid arthritis (RA) significantly affects quality of life. We recently cloned synoviolin, a RING-type E3 ubiquitin ligase implicated in the endoplasmic reticulum-associated degradation (ERAD) pathway. Synoviolin is highly expressed in rheumatoid synovial cells and may be involved in the pathogenesis of RA. Inhibition of synoviolin activity is a potentially useful therapeutic approach for the treatment of RA. We conducted a high-throughput screen of small molecules to find inhibitors of synoviolin autoubiquitination activity. We identified two classes of small molecules, named LS-101 and LS-102, which inhibited synoviolin activity. LS-102 selectively inhibited synoviolin enzymatic activity, while LS-101 inhibited a broad array of RING-type E3 ligases. Moreover, these inhibitors suppressed the proliferation of rheumatoid synovial cells, and significantly reduced the severity of disease in a mouse model of RA. Our results suggest that inhibition of synoviolin is a potentially useful approach in the treatment of RA.

A small molecule inhibitor of ubiquitin-specific protease-7 induces apoptosis in multiple myeloma cells and overcomes bortezomib resistance.

Bortezomib therapy has proven successful for the treatment of relapsed/refractory, relapsed, and newly diagnosed multiple myeloma (MM); however, dose-limiting toxicities and the development of resistance limit its long-term utility. Here, we show that P5091 is an inhibitor of deubiquitylating enzyme USP7, which induces apoptosis in MM cells resistant to conventional and bortezomib therapies. Biochemical and genetic studies show that blockade of HDM2 and p21 abrogates P5091-induced cytotoxicity. In animal tumor model studies, P5091 is well tolerated, inhibits tumor growth, and prolongs survival. Combining P5091 with lenalidomide, HDAC inhibitor SAHA, or dexamethasone triggers synergistic anti-MM activity. Our preclinical study therefore supports clinical evaluation of USP7 inhibitor, alone or in combination, as a potential MM therapy.

Characterization of selective ubiquitin and ubiquitin-like protease inhibitors using a fluorescence-based multiplex assay format.

The reversible conjugation of ubiquitin and ubiquitin-like (UbL) proteins to protein substrates plays a critical role in the regulation of many cellular pathways. The removal of ubiquitin from target proteins is performed by ubiquitin proteases also known as deubiquitylases (DUBs). Owing to their substrate specificity and the central role ubiquitylation plays in cell signaling pathways, DUB are attractive targets for therapeutic development. The development of DUB inhibitors requires assays that are amenable to high-throughput screening and provide rapid assessment of inhibitor selectivity. Determination of inhibitor selectivity at an early stage of drug discovery will reduce drug failure in the clinic as well as reduce overall drug development costs. We have developed two novel assays, UbL-Enterokinase light chain and UbL-Granzyme B, for quantifying ubiquitin and UbL protease activity. In our quest to discover and characterize novel chemical entities, we have combined these assays with a previously developed assay in a multiplex format. This multiplex format allows for the detection of three distinct protease activities simultaneously, in a single well. We have demonstrated that the multiplex format is able to distinguish between selective and nonselective protease inhibitors. Specifically, we have used this assay format to characterize P022077, a selective ubiquitin-specific protease 7 inhibitor discovered at Progenra.

The ubiquitin-specific protease USP34 regulates axin stability and Wnt/ß-catenin signaling.

Wnt proteins control multiple cell behaviors during development and tissue homeostasis. However, pathological activation of Wnt signaling is the underlying cause of various human diseases. The ubiquitin-proteasome system plays important regulatory functions within the Wnt pathway by regulating the activity of several of its core components. Hence, multiple E3 ubiquitin ligases have been implicated in its regulation. Less is known, however, about the role of ubiquitin-specific proteases in Wnt signaling. Analysis of purified axin-containing protein complexes by liquid chromatography-tandem mass spectrometry revealed the presence of the ubiquitin protease USP34. Our results indicate that USP34 functions downstream of the ß-catenin destruction complex to control the stability of axin and opposes its tankyrase-dependent ubiquitination. Reflecting on the requirement for tight control of axin homeostasis during Wnt signaling, interfering with USP34 function by RNA interference leads to the degradation of axin and to the inhibition of ß-catenin-mediated transcription. Given the numerous human diseases exhibiting spurious Wnt pathway activation, the development of USP34 inhibitors may offer a novel therapeutic opportunity.

Targeting the ubiquitin E3 ligase MuRF1 to inhibit muscle atrophy.

Progressive muscle wasting, also known as myopathy or muscle atrophy is a debilitating and life-threatening disorder. Myopathy is a pathological condition of many diseases including cancer, diabetes, COPD, and AIDS and is a natural consequence of inactivity and aging (sarcopenia). Muscle atrophy occurs when there is a net loss of muscle mass resulting in a change in the balance between protein synthesis and protein degradation. The ubiquitin pathway and specific ubiquitin pathway enzymes have been directly implicated in the progression of atrophy. The ubiquitin E3 ligase Muscle-specific RING Finger E3 ligase (MuRF1) is upregulated and increases protein degradation and muscle wasting in numerous muscle atrophy models. The inhibition of MuRF1 could be a novel mechanism to prevent or reverse muscle wasting associated with various pathologies. We screened a small molecule library for inhibitors to MuRF1 activity and identified P013222, an inhibitor of MuRF1 autoubiquitylation. Further, P013222 was shown to inhibit MuRF1-dependent substrate ubiquitylation, and was active in inhibiting MuRF1 in a cellular atrophy model. Thus MuRF1 can be targeted in a specific manner and produce positive results in cellular atrophy models.

Activity-based chemical proteomics accelerates inhibitor development for deubiquitylating enzymes.

Converting lead compounds into drug candidates is a crucial step in drug development, requiring early assessment of potency, selectivity, and off-target effects. We have utilized activity-based chemical proteomics to determine the potency and selectivity of deubiquitylating enzyme (DUB) inhibitors in cell culture models. Importantly, we characterized the small molecule PR-619 as a broad-range DUB inhibitor, and P22077 as a USP7 inhibitor with potential for further development as a chemotherapeutic agent in cancer therapy. A striking accumulation of polyubiquitylated proteins was observed after both selective and general inhibition of cellular DUB activity without direct impairment of proteasomal proteolysis. The repertoire of ubiquitylated substrates was analyzed by tandem mass spectrometry, identifying distinct subsets for general or specific inhibition of DUBs. This enabled identification of previously unknown functional links between USP7 and enzymes involved in DNA repair.

Novel approach for characterizing ubiquitin E3 ligase function.

The ubiquitin-proteasome system is central to the regulation of numerous cellular events, and dysregulation may lead to disease pathogenesis. E3 ubiquitin ligases typically function in concert with E1 and E2 enzymes to recruit specific substrates, thereby coordinating their ubiquitylation and subsequent proteasomal degradation or cellular activity. E3 ligases have been implicated in a wide range of pathologies, and monitoring their activity in a rapid and cost-effective manner would be advantageous in drug discovery. The relative lack of high-throughput screening (HTS)-compliant E3 ligase assays has significantly hindered the discovery of E3 inhibitors. Herein, the authors describe a novel HTS-compliant E3 ligase assay platform that takes advantage of a ubiquitin binding domain's inherent affinity for polyubiquitin chains, permitting the analysis of ubiquitin chain formation in an E3 ligase-dependent manner. This assay has been used successfully with members of both the RING and HECT families, demonstrating the platform's broad utility for analyzing a wide range of E3 ligases. The utility of the assay platform is demonstrated by the identification of inhibitors of the E3 ligase CARP2. As the number of E3 ligases associated with various disease states increases, the ability to quantitate the activity of these enzymes in an expeditious manner becomes imperative in drug discovery.

Regulatory monoubiquitination of phosphoenolpyruvate carboxylase in germinating castor oil seeds.

Phosphoenolpyruvate carboxylase (PEPC) is a tightly regulated enzyme situated at the core of plant C-metabolism. Although its anaplerotic role and control by allosteric effectors, reversible phosphorylation, and oligomerization have been well documented in the endosperm of developing castor oil seeds (COS), relatively little is known about PEPC in germinating COS. The initial phase of COS germination was accompanied by elevated PEPC activity and accumulation of comparable amounts of pre-existing 107-kDa and inducible 110-kDa immunoreactive PEPC polypeptides (p107 and p110, respectively). A 440-kDa PEPC heterotetramer composed of an equivalent ratio of non-phosphorylated p110 and p107 subunits was purified from germinated COS. N-terminal microsequencing, mass spectrometry, and immunoblotting revealed that both subunits arose from the same gene (RcPpc3) that encodes the p107 subunit of a phosphorylated 410-kDa PEPC homotetramer in developing COS but that p110 is a monoubiquitinated form of p107. Tandem mass spectrometry sequencing of a diglycinated tryptic peptide identified Lys-628 as p110's monoubiquitination site. This residue is conserved in vascular plant PEPCs and is proximal to a PEP-binding/catalytic domain. Incubation with a human deubiquitinating enzyme (USP-2 core) converted the p110:p107 PEPC heterotetramer into a p107 homotetramer while significantly reducing the enzyme's K(m)(PEP) and sensitivity to allosteric activators (hexose-Ps, glycerol-3-P) and inhibitors (malate, aspartate). Monoubiquitination is a non-destructive and reversible post-translational modification involved in the control of diverse processes such as transcription, endocytosis, and signal transduction. The current study demonstrates that tissue-specific monoubiquitination of a metabolic enzyme can also occur and that this modification influences its kinetic and regulatory properties.

Characterization of ubiquitin and ubiquitin-like-protein isopeptidase activities.

Conjugation or deconjugation of ubiquitin (Ub) or ubiquitin-like proteins (UBLs) to or from cellular proteins is a multifaceted and universal means of regulating cellular physiology, controlling the lifetime, localization, and activity of many critical proteins. Deconjugation of Ub or UBL from proteins is performed by a class of proteases called isopeptidases. Herein is described a readily quantifiable novel isopeptidase assay platform consisting of Ub or UBL fused to the reporter enzyme phospholipase A(2) (PLA(2)). Isopeptidase activity releases PLA(2), which cleaves its substrate, generating a signal that is linear with deubiquitylase (DUB) concentration and is able to discriminate DUB, deSUMOylase, deNEDDylase, and deISGylase activities. The power and sensitivity of the UBL-PLA(2) assay are demonstrated by its ability to differentiate the contrasting deISGylase and DUB activities of two coronavirus proteases: severe acute respiratory syndrome papain-like protease (SARS-CoV PLpro) and NL63 CoV papain-like protease 2 (PLP2). Furthermore, direct comparisons with the current Ub-7-amino-4-methylcoumarin (Ub-AMC) assay demonstrated that the Ub-PLA(2) assay is an effective tool for characterizing modulators of isopeptidase activity. This observation was expanded by profiling the inhibitory activity of the nonselective isopeptidase inhibitor NSC 632839 against DUBs and deSUMOylases. Taken together, these studies illustrate the utility of the reporter-based approach to measuring isopeptidase activity.

Phosphorylation of phosphatase inhibitor-2 at centrosomes during mitosis.

Inhibitor-2 (I-2) is a regulator of protein phosphatase type-1 (PP1), known to be phosphorylated in vitro by multiple kinases. In particular Thr72 is a Thr-Pro phosphorylation site conserved from yeast to human, but there is no evidence that this phosphorylation responds to any physiological signals. Here, we used electrophoretic mobility shift and immunoblotting with a site-specific phospho-Thr72 antibody to establish Thr72 phosphorylation in HeLa cells and show a 25-fold increase in phosphorylation during mitosis. Mass spectrometry demonstrated I-2 in actively growing HeLa cells was also phosphorylated at three other sites, Ser120, Ser121, and an additional Ser located between residues 70 and 90. In vitro kinase assays using recombinant I-2 as a substrate showed that the Thr72 kinase(s) was activated during mitosis, and sensitivity to kinase inhibitors indicated that the principal I-2 Thr72 kinase was not GSK3 but instead a member of the cyclin-dependent protein kinase family. Immunocytochemistry confirmed Thr72 phosphorylation of I-2 during mitosis, with peak intensity at prophase, and revealed subcellular concentration of the phospho-Thr72 I-2 at centrosomes. Together, the data show dynamic changes in I-2 phosphorylation during mitosis and localization of phosphorylated I-2 at centrosomes, suggesting involvement in mammalian cell division.