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  • Journal article
    Pollard D, Berger CN, So E, Yu L, Hadavizadeh K, Jennings P, Tate E, Choudhary J, Frankel Get al., 2018,

    Broad spectrum regulation of non receptor tyrosine kinases by the bacterial ADP ribosyltransferase EspJ

    , mBio, Vol: 9, ISSN: 2150-7511

    Tyrosine phosphorylation is key for signal transduction fromexogenousstimuli, including the defence against pathogens. Conversely, pathogens cansubvert protein phosphorylation to control hostimmune responsesand facilitateinvasionanddissemination. The bacterial 23effectorsEspJand SeoC areinjected into host cellsthough a type III secretion system by enteropathogenic and enterohaemorrhagic Escherichia coli(EPEC and EHEC), Citrobacter rodentiumand Salmonellaentericawhere they inhibit Src kinase bycoupledamidation andADP-ribosylation. C. rodentium, which is used tomodel EPEC and EHEC infections in human, is a mouse pathogen triggeringcolonic crypt hyperplasia (CCH) and colitis. Enumeration of bacterial shedding and CCH confirmed that EspJ affects neither tolerance nor resistance to infection. However, comparing the proteomes of intestinal epithelial cells isolated from mice infected with wildtype C.rodentiumor C. rodentiumencoding catalyticallyinactive EspJrevealed that EspJ-induced ADP-ribosylationregulatesmultiple non-receptor tyrosine kinasesin vivo. Investigating the substrate repertoire of EspJ revealed that in HeLa and A549 Src and Csk were significantly targeted; in polarised Caco2 cells EspJ targeted Src and Csk and the Src family kinase (SFK) Yes1, while in differentiated Thp1 EspJ modifiedCsk, the SFKs Hck and Lyn, the Tec family kinases Tec and Btk, and the adapter tyrosine kinase Syk. Furthermore, Abl (HeLa and Caco2) and Lyn (Caco2) were enriched specifically in the EspJ-containing samples. Biochemical assays revealed that EspJ, the only bacterial ADP-ribosyltransferase which targets mammalian kinases,controls immune responses andthe Src/Csk signalling axis.

  • Journal article
    Lubin AS, Zubiaurre AR, Matthews H, Baumann H, Fisher FR, Morales-Sanfrutos J, Hadavizadeh KS, Nardella F, Tate EW, Baum J, Scherf A, Fuchter MJet al., 2018,

    Development of a photo-crosslinkable diaminoquinazoline inhibitor for target identification in plasmodium falciparum

    , ACS Infectious Diseases, Vol: 4, Pages: 523-530, ISSN: 2373-8227

    Diaminoquinazolines represent a privileged scaffold for antimalarial discovery, including use as putative Plasmodium histone lysine methyltransferase inhibitors. Despite this, robust evidence for their molecular targets is lacking. Here we report the design and development of a small-molecule photo-crosslinkable probe to investigate the targets of our diaminoquinazoline series. We demonstrate the effectiveness of our designed probe for photoaffinity labelling of Plasmodium lysates and identify similarities between the target profiles of the probe and the representative diaminoquinazoline BIX-01294. Initial pull-down proteomics experiments identified 104 proteins from different classes, many of which are essential, highlighting the suitability of the developed probe as a valuable tool for target identification in Plasmodium falciparum.

  • Journal article
    Lanyon-Hogg T, Faronato M, Serwa RA, Tate EWet al., 2017,

    Dynamic protein acylation: new substrates, mechanisms and drug targets

    , Trends in Biochemical Sciences, Vol: 42, Pages: 566-581, ISSN: 0968-0004

    Post-translational attachment of lipids to proteins is found in all organisms, and is important for many biological processes. Acylation with myristic and palmitic acids are among the most common lipid modifications, and understanding reversible protein palmitoylation dynamics has become a particularly important goal. Linking acyltransferase enzymes to disease states can be challenging due to a paucity of robust models, compounded by functional redundancy between many palmitoyl transferases; however, in cases such as Wnt or Hedgehog signalling, small molecule inhibitors have been identified, with some progressing to clinical trials. In this review, we present recent developments in our understanding of protein acylation in human health and disease through use of chemical tools, global profiling of acylated proteomes, and functional studies of specific protein targets.

  • Journal article
    Clulow JA, Storck EM, Lanyon-Hogg T, Kalesh KA, Jones LH, Tate EWet al., 2017,

    Competition-based, quantitative chemical proteomics in breast cancer cells identifies new target profiles for sulforaphane

    , Chemical Communications, Vol: 53, Pages: 5182-5185, ISSN: 1364-548X

    Sulforaphane is a small molecule isothiocyanate which exhibits anticancer potential, yet its biological targets remain poorly understood. Here we employ a competition-based chemical proteomics strategy to profile sulforaphane's targets and identify over 500 targets along with their relative affinities. These targets provide a new set of mediators for sulforaphane's bioactivity, and aid understanding of its complex mode of action.

  • Journal article
    Ritzefeld M, Wright MH, Tate EW, 2017,

    New developments in probing and targeting protein acylation in malaria, leishmaniasis and African sleeping sickness

    , Parasitology, Vol: 145, Pages: 157-174, ISSN: 1469-8161

    Infections by protozoan parasites, such as Plasmodium falciparum or Leishmania donovani, have a significant health, social and economic impact and threaten billions of people living in tropical and sub-tropical regions of developing countries worldwide. The increasing range of parasite strains resistant to frontline therapeutics makes the identification of novel drug targets and the development of corresponding inhibitors vital. Post-translational modifications (PTMs) are important modulators of biology and inhibition of protein lipidation has emerged as a promising therapeutic strategy for treatment of parasitic diseases. In this review we summarize the latest insights into protein lipidation in protozoan parasites. We discuss how recent chemical proteomic approaches have delivered the first global overviews of protein lipidation in these organisms, contributing to our understanding of the role of this PTM in critical metabolic and cellular functions. Additionally, we highlight the development of new small molecule inhibitors to target parasite acyl transferases.

  • Journal article
    Lanyon-Hogg T, Patel NV, Ritzefeld M, Boxall KJ, Burke R, Blagg J, Magee AI, Tate EWet al., 2017,

    Microfluidic mobility shift assay for real-time analysis of peptide n-palmitoylation

    , SLAS Discovery, Vol: 22, Pages: 418-424, ISSN: 2472-5552

    The Hedgehog pathway is a key developmental signaling pathway but is also implicated in many types of cancer. The extracellular signaling protein Sonic hedgehog (Shh) requires dual lipidation for functional signaling, whereby N-terminal palmitoylation is performed by the enzyme Hedgehog acyltransferase (Hhat). Hhat is an attractive target for small-molecule inhibition to arrest Hedgehog signaling, and methods for assaying Hhat activity are central to understanding its function. However, all existing assays to quantify lipidation of peptides suffer limitations, such as safety hazards, high costs, extensive manual handling, restriction to stopped-assay measurements, or indirect assessment of lipidation. To address these limitations, we developed a microfluidic mobility shift assay (MSA) to analyze Shh palmitoylation. MSA allowed separation of fluorescently labeled Shh amine-substrate and palmitoylated Shh amide-product peptides based on differences in charge and hydrodynamic radius, coupled with online fluorescence intensity measurements for quantification. The MSA format was employed to study Hhat-catalyzed reactions, investigate Hhat kinetics, and determine small-molecule inhibitor IC50 values. Both real-time and stopped assays were performed, with the latter achieved via addition of excess unlabeled Shh peptide. The MSA format therefore allows direct and real-time fluorescence-based measurement of acylation and represents a powerful alternative technique in the study of N-lipidation.

  • Journal article
    Demetriadou A, Morales-Sanfrutos J, Nearchou M, Baba O, Kyriacou K, Tate EW, Drousiotou A, Petrou PPet al., 2017,

    Mouse Stbd1 is N-myristoylated and affects ER-mitochondria association and mitochondrial morphology

    , Journal of Cell Science, Vol: 130, Pages: 903-915, ISSN: 1477-9137

    Starch binding domain-containing protein 1 (Stbd1) is a carbohydrate-binding protein that has been proposed to be a selective autophagy receptor for glycogen. Here, we show that mouse Stbd1 is a transmembrane endoplasmic reticulum (ER)-resident protein with the capacity to induce the formation of organized ER structures in HeLa cells. In addition to bulk ER, Stbd1 was found to localize to mitochondria-associated membranes (MAMs), which represent regions of close apposition between the ER and mitochondria. We demonstrate that N-myristoylation and binding of Stbd1 to glycogen act as major determinants of its subcellular targeting. Moreover, overexpression of non-myristoylated Stbd1 enhanced the association between ER and mitochondria, and further induced prominent mitochondrial fragmentation and clustering. Conversely, shRNA-mediated Stbd1 silencing resulted in an increase in the spacing between ER and mitochondria, and an altered morphology of the mitochondrial network, suggesting elevated fusion and interconnectivity of mitochondria. Our data unravel the molecular mechanism underlying Stbd1 subcellular targeting, support and expand its proposed function as a selective autophagy receptor for glycogen and uncover a new role for the protein in the physical association between ER and mitochondria.

  • Journal article
    French PMW, Görlitz F, Kelly D, Warren S, Alibhai D, West L, Kumar S, Alexandrov Y, Munro I, McGinty J, Talbot C, Serwa R, Thinon E, Da Paola V, Murray EJ, Stuhmeier F, Neil M, Tate E, Dunsby Cet al., 2017,

    Open source high content analysis utilizing automated fluorescence lifetime imaging microscopy

    , Jove-Journal of Visualized Experiments, Vol: 119, ISSN: 1940-087X

    We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using Förster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe herethe functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in μ Manager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

  • Journal article
    Duluc L, Ahmetaj-Shala B, Mitchell J, Abdul Salam VB, Mahomed AS, Aldabbous L, Oliver E, Iannone L, Dubois OD, Storck EM, Tate EW, Zhao L, Wilkins MR, Wojciak-Stothard Bet al., 2017,

    Tipifarnib prevents development of hypoxia-induced pulmonary hypertension

    , Cardiovascular Research, Vol: 113, Pages: 276-287, ISSN: 1755-3245

    Aims.RhoB plays a key role in the pathogenesis of hypoxia-induced pulmonary hypertension. Farnesylated RhoB promotes growth responses in cancer cells and we investigated whether inhibition of protein farnesylation will have a protective effect. Methods and Results.The analysis of lung tissues from rodent models and pulmonary hypertensive patientsshowed increased levels of protein farnesylation. Oral farnesyltransferase inhibitor tipifarnib prevented development of hypoxia-induced pulmonary hypertension in mice. Tipifarnib reduced hypoxia-induced vascular cell proliferation, increased endothelium-dependent vasodilatation and reduced vasoconstriction of intrapulmonary arteries without affecting cell viability. Protective effects of tipifarnib were associated with inhibition of Ras and RhoB, actin depolymerisation and increased eNOS expression in vitroand in vivo. Farnesylated-only RhoB (F-RhoB) increased proliferative responses in cultured pulmonary vascular cells, mimicking the effects of hypoxia, while both geranylgeranylated-only RhoB (GG-RhoB) and tipifarnib had an inhibitory effect. Label-free proteomics linked F-RhoB with cell survival, activation of cell cycle and mitochondrial biogenesis. Hypoxia increased and tipifarnib reduced the levels of F-RhoB-regulated proteins in the lung, reinforcing the importance of RhoB as a signalling mediator.Unlike simvastatin, tipifarnib did not increase the expression levels of Rho proteins.Conclusions.Our study demonstrates the importance of protein farnesylation in pulmonary vascular remodeling and provides a rationale for selective targeting of this pathway in pulmonary hypertension.

  • Journal article
    Perdios L, Lowe AR, Saladino G, Bunney TD, Thiyagarajan N, Alexandrov Y, Dunsby C, French PM, Chin JW, Gervasio FL, Tate EW, Katan Met al., 2017,

    Conformational transition of FGFR kinase activation revealed by site-specific unnatural amino acid reporter and single molecule FRET

    , Scientific Reports, Vol: 7, ISSN: 2045-2322

    Protein kinases share significant structural similarity; however, structural features alone are insufficient to explain their diverse functions. Thus, bridging the gap between static structure and function requires a more detailed understanding of their dynamic properties. For example, kinase activation may occur via a switch-like mechanism or by shifting a dynamic equilibrium between inactive and active states. Here, we utilize a combination of FRET and molecular dynamics (MD) simulations to probe the activation mechanism of the kinase domain of Fibroblast Growth Factor Receptor (FGFR). Using genetically-encoded, site-specific incorporation of unnatural amino acids in regions essential for activation, followed by specific labeling with fluorescent moieties, we generated a novel class of FRET-based reporter to monitor conformational differences corresponding to states sampled by non phosphorylated/inactive and phosphorylated/active forms of the kinase. Single molecule FRET analysis in vitro, combined with MD simulations, shows that for FGFR kinase, there are populations of inactive and active states separated by a high free energy barrier resulting in switch-like activation. Compared to recent studies, these findings support diversity in features of kinases that impact on their activation mechanisms. The properties of these FRET-based constructs will also allow further studies of kinase dynamics as well as applications in vivo.

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