Abstract Protein ubiquitylation has emerged as a key regulatory mechanism of innate and adaptive immune systems. In particular, Cbl-b (Casitas B-lineage lymphoma proto-oncogene b), a RING finger E3-ubiquitin ligase primarily expressed in immune cells, acts as an intracellular checkpoint and a master negative regulator of both CTLs and NK cells. As such, Cbl-b is a novel target for developing small molecule cancer immunotherapy agents. Nedd4-family E3 ligases including Itch are known to negatively regulate inflammatory immune responses by suppressing both TH2 and TH17 differentiation and cytokine production. Modulators of Itch are expected to have anti-inflammatory properties. In addition, deubiquitylases such as USP7 and USP4 are implicated in modulating immune responses. Thus, several ubiquitin pathway enzymes have emerged as therapeutic targets for a variety of immune disorders. Therapeutic modulation of these ubiquitin pathway enzymes is expected to alter the ubiquitylation of their substrate proteins and documentation of such changes is critical to establish mechanism of action in the cell and develop physiological screens for target validation and drug discovery. Current methods to study polyubiquitin on substrates are cumbersome, inefficient, and low throughput. Here we describe a powerful technology that allows selective and quantitative analysis of polyubiquitylated proteins in high throughput format in the cell. Validation is provided using small molecule modulators of E3 ligase and deubiquitylase enzymes. This technology allows detection of changes in highly dynamic poly-ubiquitylated substrates, thus elucidating the role of these enzymes in the cell and the mechanism of action of drug molecules.

Ubiquitylation occurs through isopeptide linkage between the C‐terminus of Ub and the ε‐amino group of a lysine residue on the target substrate. Ub itself has seven Lys residues (K6, K11, K27, K29, K33, K48, and K63), each of which can participate in further ubiquitylation, generating poly‐Ub chains. The ability of Ub to form polymers through various lysines as well as the NH2‐terminus appears to be central to the versatility of this system in regulating cellular processes. The most extensively characterized of these polymers are linked through either K48 or K63. K48‐linked polyUb predominantly targets proteins for proteasomal degradation, whereas K63‐linked polyUb appears to regulate protein function, subcellular localization, or protein‐protein interactions. A growing body of evidence now implicates K11‐linked polyUb in mitotic regulation and endoplasmic reticulum associated degradation (ERAD). It is apparent from the above, that different poly‐ubiquitin linkages convey different information to the cell and suggests strongly that the cell contains elements capable of decoding this information, i.e. linkage specific ubiquitin binding domains. Development of Poly‐Ubiquitin selective tools will revolutionize the field of ubiquitin. Most UBDs described to date show little ability to discriminate between different linkages; although, it is possible to construct linkage specific tandem ubiquitin binding entities (TUBEs) by manipulating the spacing and rigidity of the linkers between tandem UBDs. We and our collaborators have constructed a series of K63‐specific UBDs and demonstrated their utility for Far Western blotting and “pull‐down” type experiments for evaluating the levels and identities of proteins bearing K63‐linked polyubiquitin chains under a variety of treatments. We will describe the results of proteomic experiments in which we identified new classes of UBDs. When validated, this information will have a major impact on our understanding of the functioning of the Ubiquitin‐Proteasome Pathway and suggest new targets for intervention.Grant Funding Source: Supported by R43CA165561

Since the dawn of time, or at least the dawn of recombinant DNA technology (which for many of today's scientists is the same thing), investigators have been cloning and expressing heterologous proteins in a variety of different cells for a variety of different reasons. These range from cell biological studies looking at protein-protein interactions, post-translational modifications, and regulation, to laboratory-scale production in support of biochemical, biophysical, and structural studies, to large scale production of potential biotherapeutics. In parallel, fusion-tag technology has grown-up to facilitate microscale purification (pull-downs), protein visualization (epitope tags), enhanced expression and solubility (protein partners, e.g., GST, MBP, TRX, and SUMO), and generic purification (e.g., His-tags, streptag, and FLAG™-tag). Frequently, these latter two goals are combined in a single fusion partner. In this review, we examine the most commonly used fusion methodologies from the perspective of the ultimate use of the tagged protein. That is, what are the most commonly used fusion partners for pull-downs, for structural studies, for production of active proteins, or for large-scale purification? What are the advantages and limitations of each? This review is not meant to be exhaustive and the approach undoubtedly reflects the experiences and interests of the authors. For the sake of brevity, we have largely ignored epitope tags although they receive wide use in cell biology for immunopreciptation.

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.

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.

Although they are the primary determinants of substrate specificity, few E3-substrate pairs have been positively identified, and few E3’s profiled in a proteomic fashion. Praja1 is an E3 implicated in bone development and highly expressed in brain. Although it has been well studied relative to the majority of E3’s, little is known concerning the repertoire of proteins it ubiquitylates. We sought to identify high confidence substrates for Praja1 from an unbiased proteomic profile of thousands of human proteins using protein microarrays. We first profiled Praja1 activity against a panel of E2’s to identify its optimal partner in vitro. We then ubiquitylated multiple, identical protein arrays and detected putative substrates with reagents that vary in ubiquitin recognition according to the extent of chain formation. Gene ontology clustering identified putative substrates consistent with information previously known about Praja1 function, and provides clues into novel aspects of this enzyme’s function.

The preparation of sufficient amounts of high-quality protein samples is the major bottleneck for structural proteomics. The use of recombinant proteins has increased significantly during the past decades. The most commonly used host, Escherichia coli, presents many challenges including protein misfolding, protein degradation, and low solubility. A novel SUMO fusion technology appears to enhance protein expression and solubility ( http://www.lifesensors.com ). Efficient removal of the SUMO tag by SUMO protease in vitro facilitates the generation of target protein with a native N-terminus. In addition to its physiological relevance in eukaryotes, SUMO can be used as a powerful biotechnology tool for enhanced functional protein expression in prokaryotes and eukaryotes.

E3 ubiquitin ligases are the largest family of proteins in humans (~620), and mostly determine the substrate specificity of ubiquitin transfer to target proteins. De‐ubiquitylase enzymes (DUBs), in contrast, remove ubiquitin from proteins to recycle ubiquitin, to modify the nature of the linkage (editing), to terminate the signal, or to salvage protein from degradation. In between these two enzyme families is the ubiquitin itself, which is found singly on proteins as well as in chains of various length and linkage, i.e., a reference to which lysines are used to join adjacent moieties. Here we report using E1, E2, and E3 enzymes with free ubiquitin and ATP to transfer ubiquitin to immobilized proteins on microarrays. E3's were first screened against a nearly comprehensive panel of human E2's to determine optimal E2/E3 pairings. Here we show that various E3's ubiquitylate distinct but overlapping sets of protein substrates, which were also confirmed in vitro. We also demonstrate serial enzymatic activity on the microarray in which the protein substrates are first ubiquitylated, washed free of ubiquitylation machinery, then treated with DUB enzymes to profile their substrate specificities. These experiments were performed with wild‐type (untagged) ubiquitin as well as single‐lysine mutants to characterize the linkage specificities of the enzymes. Finally, we were able to differentiate monoubiquitylation and polyubiquitylation of substrates by comparing identically treated arrays visualized with Tandem Ubiquitin Binding Entities (TUBEs) which are specific to polyubiquitin, and antibody capable of recognizing both mono and poly‐ubiquitin. We believe the microarray format will prove critical to rapidly elucidating the substrates of E3 and DUB enzymes, as well as elucidating the chain preferences of both.

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.

The preparation of large amounts of recombinant protein is a major bottleneck for many areas of scientific research, including structural proteomics and therapeutic drug discovery. Recently, the Small Ubiquitin-like Modifier (SUMO)-based fusion tag technology has gained increasing use for the expression of recombinant proteins. In E. coli recombinant proteins expressed as SUMO fusions have demonstrated enhanced stability and solubility, leading to greatly increased yields (5 to 20-fold) over constructs lacking this tag. Furthermore, efficient removal of the SUMO tag by SUMO protease allows the generation of a native N-terminus of the target protein. The use of SUMO-fusion technology in E. coli, termed SUMOpro, as well as the use of SUMOstar, an adaptation of SUMO for use in eukaryotic hosts, will be reviewed. This review will also provide a comparison between SUMOpro technology and other fusion tags commonly used to enhance protein production and facilitate purification.

When expressing and purifying large quantities of soluble protein, expression difficulties often include poor yield and the formation of insoluble aggregates. Gene fusion technologies can overcome these obstacles and simplify purification and improve solubility. This article discusses the most popular fusion tags and the enzymes used to remove them, with special reference to recently introduced technologies.

Recombinant protein expression in insect cells varies greatly from protein to protein. A fusion tag that is not only a tool for detection and purification, but also enhances expression and/or solubility would greatly facilitate both structure/function studies and therapeutic protein production. We have shown that fusion of SUMO (small ubiquitin-related modifier) to several test proteins leads to enhanced expression levels in Escherichia coli. In eukaryotic expression systems, however, the SUMO tag could be cleaved by endogenous desumoylase. In order to adapt SUMO-fusion technology to these systems, we have developed an alternative SUMO-derived tag, designated SUMOstar, which is not processed by native SUMO proteases. In the present study, we tested the SUMOstar tag in a baculovirus/insect cell system with several proteins, i.e. mouse UBP43, human tryptase beta II, USP4, USP15, and GFP. Our results demonstrate that fusion to SUMOstar enhanced protein expression levels at least 4-fold compared to either the native or His6-tagged proteins. We isolated active SUMOstar tagged UBP43, USP4, USP15, and GFP. Tryptase was active following cleavage with a SUMOstar specific protease. The SUMOstar system will make significant impact in difficult-to-express proteins and especially to those proteins that require the native N-terminal residue for function.

A series of functionalized aryl boronic acids were synthesized and evaluated as potential inhibitors of factor XIa. Crystal structures of the protein-inhibitor complexes led to the design and synthesis of second generation compounds showing single digit micromolar inhibition against FXIa and selectivity against thrombin, trypsin, and FXa. (c) 2006 Elsevier Ltd. All rights reserved.

Human coagulation factor XIa (FXIa), a serine protease activated by site-specific cleavage of factor XI by thrombin, FXIIa, or autoactivation, is a critical enzyme in the amplification phase of the coagulation cascade. To investigate the potential of FXIa inhibitors as safe anticoagulants, a series of potent, selective peptidomimetic inhibitors of FXIa were designed and synthesized. Some of these inhibitors showed low nanomolar FXIa inhibitory activity with > 1000-fold FXa selectivity and > 100-fold thrombin selectivity. The X-ray structure of one of these inhibitors, 36, demonstrates its unique binding interactions with FXIa. Compound 32 caused a doubling of the activated partial thromboplastin time in human plasma at 2.4 mu M and was efficacious in a rat model of venous thrombosis. These data suggest that factor XIa plays a significant role in venous thrombosis and may be a suitable target for the development of antithrombotic therapy.