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Products and Drug Discovery Services July 2013 Ubiquigent Ltd provides high quality Services / HTS Assay Development reagents, kits, and drug discovery services to the research community Ubiquigent offers expert custom services to researchers interested in ubiquitin/ to help investigators unravel the ubiquitin-like protein (Ubl) cascade drug discovery. We have access to a suite of complexity of, and pursue new thera- automation robotics to help us deliver the highest quality services to you spe- peutic target opportunities across the cifically designed to help accelerate your drug discovery efforts in this field. ubiquitin cascade. The Company is located adjacent to the Medical Research Council Protein Phos- phorylation and Ubiquitylation Unit (PPU), based at the University of Dundee, Scotland, profiler™ NEW HTS Compatible Assay Kits DUB UK. This provides Ubiquigent with access to Compound Screening Service state-of-the-art services and capabilities HTS compatible assay kits have been devel- within both the PPU and other departments oped to therapeutically relevant ubiquitin sys- Ubiquigent can help you with your DUB (deu- of the College of Life Sciences. The PPU is tem targets including: Inducer of LDL (Low biquitylase) drug discovery programmes by Density Lipoprotein) Receptor (IDOL)-cataly- one of Ubiquigent’s founding partners. determining the selectivity and potency of your sed LDLR (LDL Receptor) substrate ubiquity- inhibitors. We offer the DUBprofiler service in ei- lation and CHIP autoubiquitylation assays. ther single concentration profiling or IC50 analy- sis mode. HTS Compatible WHAT YOU DO: INSIDE Custom Assay Development • Send us your compound(s). Services As pathways and functions are unraveled, • Request single concentration profiling and/ new and attractive ubiquitin/Ubl cascade drug or IC50 analysis (electronic submission). Kits discovery targets are coming to light with rel- evance across many key therapeutic areas. WHAT WE DO: With access to a wide range of reagents and NEW >> HTS Assay Kits benefiting from our experience working with • Determine the selectivity and specificity of these pathways, Ubiquigent’s Custom Assay your deubiquitylase (DUB) inhibitor lead E1 Activating Enzymes Development team supports pharmaceutical compound(s). and biotechnology companies in translating • Electronically report fully quality con- E2 Conjugating Enzymes these discoveries into lead programmes. trolled data to you (single concentration % activities or IC50s). E3 Ligases We will work closely and collaboratively with you throughout our partnership. Our approach SERVICE HIGHLIGHTS: is highly flexible; from providing high quality Deconjugating Enzymes activity validated reagents to full-scale proj- • 35 DUBs on the panel and expanding. ect outsourcing developing required screen- • Any scale and combination of screening Ubiquitin/Ubl Binding Proteins ing and deconvolution assays and supplying available; compound(s) vs DUB(s). the reagents to enable you to conduct your • 2 week service cycle. Ubiquitin/Ubl Modifiers, subsequent HTS campaign in-house. Derivatives and Substrates Ubiquigent have successfully developed and DUBprofiler™-HT Cell Lysates delivered HTS-compatible assays and kits enabling leading pharmaceutical compa- Ubiquigent offers DUBprofiler-HT for the screen- nies to conduct screening campaigns against ing of larger numbers of compound (>50) ver- Kinases ubiquitin system targets (DUBs, E3s and oth- sus the DUBprofiler panel. In this service we can ers) of high therapeutic value. Furthermore offer high precision and accuracy ultra-low Antibodies Ubiquigent has developed and supplied many volume compound dispensing (down to 2.5 deconvolution and specificity profiling as- nl) to enable addition of the assay reagents says to help characterize the hits from these directly to the compound in 100% DMSO. This screens. A number of our assays have been Please visit us at is particularly suited to screens where com- developed from first principles starting with pound solubility is a concern. www.ubiquigent.com little or no data from the literature to guide the for the latest information on construction of the assay. Ubiquigent can re- new products and services. search, design, manage and deliver the entire programme to help you to achieve success in your ubiquitin system HTS campaigns. Ubiquigent Ltd - Linking Ubiquitin Research to Drug Discovery www.ubiquigent.com The Ubiquitin Cascade Product Categories are web-interactive Kinases E1 E2 E3 DCE Ub/Ubl Anti-Ub-conjugate Anti-Ub-conjugate DCE UBP DCE: deconjugating enzyme Ub: ubiquitin Ubl: ubiquitin-like protein Protein Ubiquitylation: Ubiquitylation, like phosphorylation, de- uitin chains and deubiquitylases may act on Although still a post-translational modi- scribes a reversible post-translational pro- either mono-ubiquitylated or poly-ubiqui- fication, albeit involving a functional protein tein modification. Ubiquitylation or ‘ubiquit- tylated substrates to remove the ubiquitin rather than a functional group, ubiquityla- ination’ may control the protein substrate’s monomers or chains respectively. tion is a much more complex process than destiny – in respect of its turnover – or its The enzymes of the ubiquitylation pathway phosphorylation mainly due to the ability of signalling functionality. It is a process that play a pivotal role in a number of cellular ubiquitin to form polyubiquitin chains of a refers to the covalent attachment - through processes including, but not exclusively, the variety of different linkage types and com- it’s C-terminus - of a small, 76 amino acid targeted proteasomal degradation of sub- plexity, but also because there are further protein called ubiquitin to the epsilon-amino strate proteins. Three classes of enzymes are related ubiquitin-like (Ubl; including SUMO, group of a lysine residue residing within a involved in the process of substrate ubiqui- NEDD8, ISG15, and FAT10) proteins that substrate protein – which may also be anoth- tylation; activating enzymes (E1s), conjugat- may each follow a similar specific enzymatic er ubiquitin molecule. This results in either ing enzymes (E2s) and protein ligases (E3s). cascade but resulting in different outcomes mono- or poly-ubiquitylation of the substrate; Ubiquitylation of substrate proteins depends for the Ubl modified target substrate. At a the latter being where chains of ubiquitin are on the sequential action of these three en- further level of system complexity modifica- attached to the substrate protein. Ubiquitin zymes. In an ATP-dependent first step, an E1 tions of either the E3 ligase or the substrate may also form chains through attachment enzyme forms a thioester linkage with ubiq- may alter their ability to modify substrates to the amino terminus of a further ubiquitin uitin which is then transferred to the sulphy- or themselves be modified; for example by to generate linear poly-ubiquitin chains. The dryl group of the active-site cysteine on an NEDDylation (NEDD8 being a further Ubl) in structure of the chain determines whether E2 enzyme forming a ubiquitin-thioester in- the case of E3 ligases or phosphorylation in a ubiquitylated protein modulates a specific termediate. An E3 then acts as an adaptor to the case of both E3 ligases and substrates. signalling cascade or may become degraded bind both substrate protein and E2 ‘loaded’ in a proteasomal or lysosomal dependent with ubiquitin. The E3 facilitates isopeptide manner. Mono-ubiquitylated proteins may bond formation between ubiquitin and the be further ubiquitylated to form polyubiq- substrate protein. 2 Ubiquigent Ltd - Linking Ubiquitin Research to Drug Discovery www.ubiquigent.com E2scan™ Kit version 2 NEW - now containing 34 E2s The E2scan Kit is designed to facilitate the identification of E2 conjugating Why use the E2scan kit? enzymes which support the ubiquitylation of substrate proteins catalysed by an E3 ligase and/or the auto-ubiquitylation of the E3 ligase itself. • E2 conjugating enzymes can be critical in determining the ubiquitin chain linkage type(s) generated in a ubiquitylation 1 2 3 4 5 6 7 8 9 10 11 12 reaction. A 2A 2B 2C 2D1 2D2 2D3 2D4 2E1 2E2 2E3 2F 2G1 Kit Enzymes • The linkage type can determine the fate of the ubiquitylated B 2G2 2H 2I 2J1 2J2 2K 2L3 2L6 2M 2N 2N/2V1 2N/2V2 substrate; for example to be degraded or to function in a sig- Kit Controls C 2Q 2Q2 2R1 2R2 2S 2T 2V1 2V2 2W 2Z nalling capacity (Ye and Rape (2009)). BFR Buffer scan D • Discovery of novel aspects to E2 functionality: The E2 kit 2D4 Positive Control E 2A 2B 2C 2D1 2D2 2D3 2D4 2E1 2E2 2E3 2F 2G1 was used to identify E2s that partner RNF4 in polySUMO ubiquitylation and it was discovered that Ube2W (Ubc16) is F 2G2 2H 2I 2J1 2J2 2K 2L3 2L6 2M 2N 2N/2V1 2N/2V2 an E2 with - a previously unknown - specific protein amino G 2Q 2Q2 2R1 2R2 2S 2T 2V1 2V2 2W 2Z terminal monoubiquitylation activity (Tatham et al. 2013). H BFR BFR 2D4 2D4 E2scan experiments facilitate: • the rapid identification of the E2s that couple with your E3 The kit contains an E2scan plate - a panel of 34 E2 conjugating enzymes arrayed (auto and/or substrate ubiquitylation modes) in duplicate across a 96-well plate - plus all the components (E1 enzyme, con- trol E3 ligase, ubiquitin, ATP, assay buffer) required to perform a complete • new insights regarding the E3 ligase and/or the substrate substrate ubiquitylation or auto-ubiquitylation assay using your E3 ligase and/ • the development of assays for the E3 ligase or E3 ligase/substrate combination of choice. Through the addition of a sub- strate mix (E1, E3, substrate, and ubiquitin) and then ATP to the wells of an References E2scan plate, screen the E2 conjugating enzyme panel in just one hour. Reaction Ye and Rape (2009) Nat Rev Mol Cell Biol 10, 755-764. products may be analysed as required, for example by SDS-PAGE or Western Tatham MH, Plechanovová A, Jaffray EG, Salmen H, Hay RT (2013) Biochem J 453, 137-145. blotting. E2scan Kit version 2 Cat. # 67-0005-001 1 Kit £345 DUBscan™ Kit The DUBscan kit has been flexibly designed for many potential applications.
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  • The UBE2L3 Ubiquitin Conjugating Enzyme: Interplay with Inflammasome Signalling and Bacterial Ubiquitin Ligases

    The UBE2L3 Ubiquitin Conjugating Enzyme: Interplay with Inflammasome Signalling and Bacterial Ubiquitin Ligases

    The UBE2L3 ubiquitin conjugating enzyme: interplay with inflammasome signalling and bacterial ubiquitin ligases Matthew James George Eldridge 2018 Imperial College London Department of Medicine Submitted to Imperial College London for the degree of Doctor of Philosophy 1 Abstract Inflammasome-controlled immune responses such as IL-1β release and pyroptosis play key roles in antimicrobial immunity and are heavily implicated in multiple hereditary autoimmune diseases. Despite extensive knowledge of the mechanisms regulating inflammasome activation, many downstream responses remain poorly understood or uncharacterised. The cysteine protease caspase-1 is the executor of inflammasome responses, therefore identifying and characterising its substrates is vital for better understanding of inflammasome-mediated effector mechanisms. Using unbiased proteomics, the Shenoy grouped identified the ubiquitin conjugating enzyme UBE2L3 as a target of caspase-1. In this work, I have confirmed UBE2L3 as an indirect target of caspase-1 and characterised its role in inflammasomes-mediated immune responses. I show that UBE2L3 functions in the negative regulation of cellular pro-IL-1 via the ubiquitin- proteasome system. Following inflammatory stimuli, UBE2L3 assists in the ubiquitylation and degradation of newly produced pro-IL-1. However, in response to caspase-1 activation, UBE2L3 is itself targeted for degradation by the proteasome in a caspase-1-dependent manner, thereby liberating an additional pool of IL-1 which may be processed and released. UBE2L3 therefore acts a molecular rheostat, conferring caspase-1 an additional level of control over this potent cytokine, ensuring that it is efficiently secreted only in appropriate circumstances. These findings on UBE2L3 have implications for IL-1- driven pathology in hereditary fever syndromes, and autoinflammatory conditions associated with UBE2L3 polymorphisms.