Imperial College London

Prof Ed Tate

Faculty of Natural SciencesDepartment of Chemistry

Professor of Chemical Biology
 
 
 
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Contact

 

+44 (0)20 7594 3752e.tate Website CV

 
 
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Assistant

 

Ms Agnes Lee +44 (0)20 7594 9852

 
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Location

 

301BMolecular Sciences Research HubWhite City Campus

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Summary

 

Publications

Publication Type
Year
to

143 results found

Lim C, Ha KP, Clarke R, Gavin L-A, Cook D, Hutton J, Sutherell C, Edwards A, Evans L, Tate E, Lanyon-Hogg Tet al., Identification of a potent small-molecule inhibitor of bacterial DNA repair that potentiates quinolone antibiotic activity in methicillin-resistant Staphylococcus aureus, Bioorganic and Medicinal Chemistry, ISSN: 0968-0896

JOURNAL ARTICLE

Birtley JR, Alomary M, Zanini E, Antony J, Maben Z, Weaver G, von Arx C, Mura M, Marinho AT, Lu H, Morecroft E, Karali E, Chayen N, Tate E, Jurewicz M, Stern L, Recchi C, Gabra Het al., 2019, Inactivating mutations and X-ray crystal structure of the tumor suppressor OPCML reveal cancer-associated functions, Nature Communications, ISSN: 2041-1723

JOURNAL ARTICLE

Schlott AC, Mayclin S, Reers AR, Coburn-Flynn O, Bell AS, Green J, Knuepfer E, Charter D, Bonnert R, Campo B, Burrows J, Lyons-Abbott S, Staker BL, Chung C-W, Myler PJ, Fidock DA, Tate EW, Holder AAet al., 2019, Structure-Guided Identification of Resistance Breaking Antimalarial N‑Myristoyltransferase Inhibitors., Cell Chem Biol

The attachment of myristate to the N-terminal glycine of certain proteins is largely a co-translational modification catalyzed by N-myristoyltransferase (NMT), and involved in protein membrane-localization. Pathogen NMT is a validated therapeutic target in numerous infectious diseases including malaria. In Plasmodium falciparum, NMT substrates are important in essential processes including parasite gliding motility and host cell invasion. Here, we generated parasites resistant to a particular NMT inhibitor series and show that resistance in an in vitro parasite growth assay is mediated by a single amino acid substitution in the NMT substrate-binding pocket. The basis of resistance was validated and analyzed with a structure-guided approach using crystallography, in combination with enzyme activity, stability, and surface plasmon resonance assays, allowing identification of another inhibitor series unaffected by this substitution. We suggest that resistance studies incorporated early in the drug development process help selection of drug combinations to impede rapid evolution of parasite resistance.

JOURNAL ARTICLE

Storck Saha E, Morales Sanfrutos J, Serwa R, Panyain N, Lanyon-Hogg T, Tolmachova T, Ventimiglia L, Martin-Serrano J, Seabra M, Wojciak-Stothard B, Tate Eet al., 2019, Dual chemical probes enable quantitative system-wide analysis of protein prenylation and prenylation dynamics, Nature Chemistry, ISSN: 1755-4330

Post-translational farnesylation or geranylgeranylation at a C-terminal cysteine residue regulates the localization and function of over 100 proteins, including the Ras isoforms, and is a therapeutic target in diseases including cancer and infection. Here, we report global and selective profiling of prenylated proteins in living cells enabled by the development of isoprenoid analogues YnF and YnGG in combination with quantitative chemical proteomics. Eighty prenylated proteins were identified in a single human cell line, 64 for the first time at endogenous abundance without metabolic perturbation. We further demonstrate that YnF and YnGG enable direct identification of post-translationally processed prenylated peptides, proteome-wide quantitative analysis of prenylation dynamics and alternative prenylation in response to four different prenyltransferase inhibitors, and quantification of defective Rab prenylation in a model of the retinal degenerative disease choroideremia.

JOURNAL ARTICLE

Kallemeijn W, Lueg G, Faronato M, Hadavizadeh K, Goya Grocin A, Song O-R, Howell M, Dinnis C, Tate Eet al., Validation and invalidation of chemical probes for the human N-myristoyltransferases, Cell Chemical Biology, ISSN: 2451-9456

On-target, cell-active chemical probes are of fundamental importance in both chemical and cell biology, whereas the application of poorly-characterised probes often leads to invalid conclusions.Human N-myristoyltransferase (NMT) has attracted increasing interest as a target in cancer and infectious diseases; here we report an in-depth comparison of five compounds widely applied as human NMT inhibitors, using a combination of quantitative whole-proteome N-myristoylation profiling, biochemical enzyme assays, cytotoxicity, in-cell protein synthesis and cell cycle assays. We find that N-myristoylation is unaffected by 2-hydroxymyristic acid (100 μM), D-NMAPPD (30 μM) or Tris-DBA palladium (10 μM), with the latter compounds causing cytotoxicity through mechanisms unrelated to NMT. In contrast, drug-like inhibitors IMP-366 (DDD85646) and IMP-1088 delivered complete and specific inhibition of N-myristoylation in a range of cell lines at 1 μM and 100 nM, respectively. This study enables the selection of appropriate on-target probes for future studies and suggests the need for reassessment of previous studies which used off-target compounds.

JOURNAL ARTICLE

Jamshidiha M, Pérez-Dorado I, Murray JW, Tate EW, Cota E, Read RJet al., 2019, Coping with strong translational noncrystallographic symmetry and extreme anisotropy in molecular replacement with Phaser: human Rab27a, Acta Crystallographica Section D Structural Biology, Vol: 75, Pages: 342-353, ISSN: 2059-7983

Data pathologies caused by effects such as diffraction anisotropy and translational noncrystallographic symmetry (tNCS) can dramatically complicate the solution of the crystal structures of macromolecules. Such problems were encountered in determining the structure of a mutant form of Rab27a, a member of the Rab GTPases. Mutant Rab27a constructs that crystallize in the free form were designed for use in the discovery of drugs to reduce primary tumour invasiveness and metastasis. One construct, hRab27a<jats:sup>Mut</jats:sup>, crystallized within 24 h and diffracted to 2.82 Å resolution, with a unit cell possessing room for a large number of protein copies. Initial efforts to solve the structure using molecular replacement by <jats:italic>Phaser</jats:italic> were not successful. Analysis of the data set revealed that the crystals suffered from both extreme anisotropy and strong tNCS. As a result, large numbers of reflections had estimated standard deviations that were much larger than their measured intensities and their expected intensities, revealing problems with the use of such data at the time in <jats:italic>Phaser</jats:italic>. By eliminating extremely weak reflections with the largest combined effects of anisotropy and tNCS, these problems could be avoided, allowing a molecular-replacement solution to be found. The lessons that were learned in solving this structure have guided improvements in the numerical analysis used in <jats:italic>Phaser</jats:italic>, particularly in identifying diffraction measurements that convey very little information content. The calculation of information content could also be applied as an alternative to ellipsoidal truncation. The post-mortem analysis also revealed an oversight in accounting for measurement errors in the fast rotation function. While the crystal of mutant Rab27a is not amenable to drug screening, the structure can guide new modifications to obtain more sui

JOURNAL ARTICLE

Tapodi A, Clemens DM, Uwineza A, Goldberg MW, Thinon E, Heal WP, Tate EW, Nemeth-Cahalan K, Vorontsova I, Jarrin M, Hall JE, Quinlan RAet al., 2019, BFSP1 C-terminal domains released by post-translational processing events can alter significantly the calcium regulation of AQP0 water permeability., Exp Eye Res

BFSP1 (beaded filament structural protein 1, filensin) is a cytoskeletal protein expressed in the eye lens. It binds AQP0 in vitro and its C-terminal sequences have been suggested to regulate the water channel activity of AQP0. A myristoylated fragment from the C-terminus of BFSP1 was found in AQP0 enriched fractions. Here we identify BFSP1 as a substrate for caspase-mediated cleavage at several C-terminal sites including D433. Cleavage at D433 exposes a cryptic myristoylation sequence (434-451). We confirm that this sequence is an excellent substrate for both NMT1 and 2 (N-myristoyl transferase). Thus caspase cleavage may promote formation of myristoylated fragments derived from the BFSP1 C-terminus (G434-S665). Myristoylation at G434 was, however, not required for plasma membrane association, but biochemical fractionation and immunogold labeling confirmed that C-terminal BFSP1 fragments containing the myristoylation sequence colocalised with AQP0 in the same plasma membrane compartments of lens fibre cells. To determine the functional significance of the association of BFSP1 G434-S665 sequences with AQP0, we measured AQP0 water permeability in Xenopus oocytes co-transfected with transcripts expressing both AQP0 and various naturally occurring C-terminus fragments of BFSP1. We found that different fragments dramatically alter the response of AQP0 to different concentrations of Ca2+. The full-length C-terminal fragment (G434-S665) eliminates calcium regulation altogether. Shorter fragments enhance regulation by elevated calcium or reverse the wild type response. In particular, elimination of the myristoylation site by the mutation G434A reverses the order of water permeability sensitivity to different Ca2+ concentrations.

JOURNAL ARTICLE

Hong WD, Benayoud F, Nixon GL, Ford L, Johnston KL, Clare RH, Cassidy A, Cook DAN, Siu A, Shiotani M, Webborn PJH, Kavanagh S, Aljayyoussi G, Murphy E, Steven A, Archer J, Struever D, Frohberger SJ, Ehrens A, Huebner MP, Hoerauf A, Roberts AP, Hubbard ATM, Tate EW, Serwa RA, Leung SC, Qie L, Berry NG, Gusovsky F, Hemingway J, Turner JD, Taylor MJ, Ward SA, O'Neill PMet al., 2019, AWZ1066S, a highly specific anti-Wolbachia drug candidate for a short-course treatment of filariasis, Proceedings of the National Academy of Sciences of the United States of America, Vol: 116, Pages: 1414-1419, ISSN: 0027-8424

Onchocerciasis and lymphatic filariasis are two neglected tropical diseases that together affect ∼157 million people and inflict severe disability. Both diseases are caused by parasitic filarial nematodes with elimination efforts constrained by the lack of a safe drug that can kill the adult filaria (macrofilaricide). Previous proof-of-concept human trials have demonstrated that depleting >90% of the essential nematode endosymbiont bacterium, Wolbachia, using antibiotics, can lead to permanent sterilization of adult female parasites and a safe macrofilaricidal outcome. AWZ1066S is a highly specific anti-Wolbachia candidate selected through a lead optimization program focused on balancing efficacy, safety and drug metabolism/pharmacokinetic (DMPK) features of a thienopyrimidine/quinazoline scaffold derived from phenotypic screening. AWZ1066S shows superior efficacy to existing anti-Wolbachia therapies in validated preclinical models of infection and has DMPK characteristics that are compatible with a short therapeutic regimen of 7 days or less. This candidate molecule is well-positioned for onward development and has the potential to make a significant impact on communities affected by filariasis.

JOURNAL ARTICLE

Kaiser N, Mejuch T, Fedoryshchak R, Janning P, Tate EW, Waldmann Het al., 2019, Photoactivatable Myristic Acid Probes for UNC119-Cargo Interactions, CHEMBIOCHEM, Vol: 20, Pages: 134-139, ISSN: 1439-4227

JOURNAL ARTICLE

Goya Grocin A, Serwa R, Morales Sanfrutos J, Ritzefeld M, Tate Eet al., 2019, Whole proteome profiling of N-myristoyltransferase activity and inhibition using Sortase A, Molecular and Cellular Proteomics, Vol: 18, Pages: 115-126, ISSN: 1535-9476

N-myristoylation is the covalent addition of a 14-carbon saturated fatty acid (myristate) to the N-terminal glycine of specific protein substrates by N-myristoyltransferase (NMT) and plays an important role in protein regulation by controlling localization, stability, and interactions. We developed a novel method for whole-proteome profiling of free N-terminal glycines through labeling with S. Aureus sortase A (SrtA) and used it for assessment of target engagement by an NMT inhibitor. Analysis of the SrtA-labeling pattern with an engineered biotinylated depsipeptide SrtA substrate (Biotin-ALPET-Haa, Haa = 2-hydroxyacetamide) enabled whole proteome identification and quantification of de novo generated N-terminal Gly proteins in response to NMT inhibition by nanoLC-MS/MS proteomics, and was confirmed for specific substrates across multiple cell lines by gel-based analyses and ELISA. To achieve optimal signal over background noise we introduce a novel and generally applicable improvement to the biotin/avidin affinity enrichment step by chemically dimethylating commercial NeutrAvidin resin and combining this with two-step LysC on-bead/trypsin off-bead digestion, effectively eliminating avidin-derived tryptic peptides and enhancing identification of enriched peptides. We also report SrtA substrate specificity in whole-cell lysates for the first time, confirming SrtA promiscuity beyond its recognized preference for N-terminal glycine, and its usefulness as a tool for unbiased labeling of N-terminal glycine-containing proteins. Our new methodology is complementary to metabolic tagging strategies, providing the first approach for whole proteome gain-of signal readout for NMT inhibition in complex samples which are not amenable to metabolic tagging.

JOURNAL ARTICLE

Furniss RCD, Low WW, Mavridou DAI, Dagley LF, Webb AI, Tate E, Clements Aet al., 2018, Plasma membrane profiling during enterohemorrhagic E. coli infection reveals that the metalloprotease StcE cleaves CD55 from host epithelial surfaces, Journal of Biological Chemistry, Vol: 293, Pages: 17188-17199, ISSN: 0021-9258

Enterohemorrhagic Escherichia coli (EHEC) is one of several E. coli pathotypes that infect the intestinal tract and cause disease. Formation of the characteristic attaching and effacing (A/E) lesion on the surface of infected cells causes significant remodelling of the host cell surface, however limited information is available about changes at the protein level. Here we employed "plasma membrane profiling", a quantitative cell-surface proteomics technique, to identify host proteins whose cell-surface levels are altered during infection. Using this method, we quantified more than 1100 proteins, 280 of which showed altered cell-surface levels after exposure to EHEC. 22 host proteins were significantly reduced on the surface of infected epithelial cells. These included both known and unknown targets of EHEC infection. The complement decay-accelerating factor CD55 exhibited the greatest reduction in cell surface levels during infection. We showed by flow cytometry and Western blot analysis that CD55 is cleaved from the cell surface by the EHEC-specific protease StcE, and found that StcE-mediated CD55 cleavage results in increased neutrophil adhesion to the apical surface of intestinal epithelial cells. This suggests that StcE alters host epithelial surfaces to depress neutrophil transepithelial migration during infection. This work is the first report of the global manipulation of the epithelial cell surface by a bacterial pathogen and illustrates the power of quantitative cell-surface proteomics in uncovering critical aspects of bacterial infection biology.

JOURNAL ARTICLE

Wang Z, Grosskurth SE, Cheung T, Petteruti P, Zhang J, Wang X, Wang W, Gharahdaghi F, Wu J, Su N, Howard RT, Mayo M, Widzowski D, Scott DA, Johannes JW, Lamb ML, Lawson D, Dry JR, Lyne PD, Tate EW, Zinda M, Mikule K, Fawell SE, Reimer C, Chen Het al., Pharmacological inhibition of PARP6 triggers multipolar spindle formation and demonstrates therapeutic effects in breast cancer, Cancer Research, Vol: 78, Pages: 6691-6702, ISSN: 1538-7445

PARP proteins represent a class of post-translational modification enzymes with diverse cellular functions. Targeting PARPs has proven to be efficacious clinically, but exploration of the therapeutic potential of PARP inhibition has been limited to targeting poly(ADP-ribose) generating PARP, including PARP1/2/3 and tankyrases. The cancer-related functions of mono(ADP-ribose) generating PARP, including PARP6, remain largely uncharacterized. Here, we report a novel therapeutic strategy targeting PARP6 using the first reported PARP6 inhibitors. By screening a collection of PARP compounds for their ability to induce mitotic defects, we uncovered a robust correlation between PARP6 inhibition and induction of multipolar spindle (MPS) formation, which was phenocopied by PARP6 knockdown. Treatment with AZ0108, a PARP6 inhibitor with a favorable pharmacokinetic profile, potently induced the MPS phenotype, leading to apoptosis in a subset of breast cancer cells in vitro and antitumor effects in vivo. In addition, Chk1 was identified as a specific substrate of PARP6 and was further confirmed by enzymatic assays and by mass spectrometry. Furthermore, when modification of Chk1 was inhibited with AZ0108 in breast cancer cells, we observed marked upregulation of p-S345 Chk1 accompanied by defects in mitotic signaling. Together, these results establish proof-of-concept antitumor efficacy through PARP6 inhibition and highlight a novel function of PARP6 in maintaining centrosome integrity via direct ADP-ribosylation of Chk1 and modulation of its activity.

JOURNAL ARTICLE

De Vita E, Schuler P, Lovell S, Lohbeck J, Kullmann S, Rabinovich E, Sananes A, Hessling B, Hamon V, Papo N, Hess J, Tate EW, Gunkel N, Miller AKet al., 2018, Depsipeptides Featuring a Neutral P1 Are Potent Inhibitors of Kallikrein-Related Peptidase 6 with On-Target Cellular Activity, JOURNAL OF MEDICINAL CHEMISTRY, Vol: 61, Pages: 8859-8874, ISSN: 0022-2623

JOURNAL ARTICLE

Benns HJ, Tate EW, Child MA, 2018, Activity-Based Protein Profiling for the Study of Parasite Biology., Curr Top Microbiol Immunol, Vol: 420, Pages: 155-174, ISSN: 0070-217X

Parasites exist within most ecological niches, often transitioning through biologically and chemically complex host environments over the course of their parasitic life cycles. While the development of technologies for genetic engineering has revolutionised the field of functional genomics, parasites have historically been less amenable to such modification. In light of this, parasitologists have often been at the forefront of adopting new small-molecule technologies, repurposing drugs into biological tools and probes. Over the last decade, activity-based protein profiling (ABPP) has evolved into a powerful and versatile chemical proteomic platform for characterising the function of enzymes. Central to ABPP is the use of activity-based probes (ABPs), which covalently modify the active sites of enzyme classes ranging from serine hydrolases to glycosidases. The application of ABPP to cellular systems has contributed vastly to our knowledge on the fundamental biology of a diverse range of organisms and has facilitated the identification of potential drug targets in many pathogens. In this chapter, we provide a comprehensive review on the different forms of ABPP that have been successfully applied to parasite systems, and highlight key biological insights that have been enabled through their application.

JOURNAL ARTICLE

Beard R, Stucki A, Schmitt M, Py G, Grundschober C, Gee A, Tate EWet al., 2018, Building bridges for highly selective, potent and stable oxytocin and vasopressin analogs, Bioorganic and Medicinal Chemistry, Vol: 26, Pages: 3039-3045, ISSN: 0968-0896

Oxytocin (OT) is an exciting potential therapeutic agent, but it is highly sensitive to modification and suffers extensive degradation at elevated temperature and in vivo. Here we report studies towards OT analogs with favorable selectivity, affinity and potency towards the oxytocin receptor (OTR), in addition to improving stability of the peptide by bridging the disulfide region with substituted dibromo-xylene analogs. We found a sensitive structure-activity relationship in which meta-cyclized analogs (dOTmeta) gave highest affinity (50 nM Ki), selectivity (34-fold), and agonist potency (34 nM EC50, 87-fold selectivity) towards OTR. Surprisingly, ortho-cyclized analogs demonstrated OTR and vasopressin V1a receptor subtype affinity (220 nM and 69 nM, respectively) and pharmacological activity (294 nM and 35 nM, respectively). V1a binding and selectivity for ortho-cyclized peptides could be improved 6-fold by substituting a neutral residue at position 8 with a basic amino acid, providing potent antagonists (14 nM IC50) that displayed no activation of the OTR. Furthermore, xylene-bridged analogs demonstrated increased stability compared to OT at elevated temperature, demonstrating promising therapeutic potential for these analogs which warrants further study.

JOURNAL ARTICLE

Riviere F, Dian C, Perez-Dorado I, Ritzefeld M, Shen J, Cota E, Meinnel T, Tate EW, Giglione Cet al., 2018, Mechanistic insight into HsNMT1-mediated acylation, Publisher: WILEY, Pages: 421-422, ISSN: 2211-5463

CONFERENCE PAPER

Tate EW, 2018, Protein N terminal modifications: from chemical biology to drug discovery, Publisher: WILEY, Pages: 72-73, ISSN: 2211-5463

CONFERENCE PAPER

Mousnier A, Bell AS, Swieboda DP, Morales-Sanfrutos J, Pérez-Dorado I, Brannigan JA, Newman J, Ritzefeld M, Hutton JA, Guedán A, Asfor AA, Robinson SW, Hopkins-Navratilova I, Wilkinson AJ, Johnston SL, Leatherbarrow RJ, Tuthill TJ, Solari R, Tate EWet al., 2018, Fragment-derived inhibitors of human N-myristoyltransferase block capsid assembly and replication of the common cold virus, Nature Chemistry, Vol: 10, Pages: 599-606, ISSN: 1755-4330

Rhinoviruses are the pathogens most often responsible for the common cold, and are a frequent cause of exacerbations in asthma, chronic obstructive pulmonary disease and cystic fibrosis. Here we report discovery of IMP-1088, a picomolar dual inhibitor of the human N-myristoyltransferases NMT1 and NMT2, and use it to demonstrate that pharmacological inhibition of host cell N-myristoylation rapidly and completely prevents rhinoviral replication without inducing cytotoxicity. Identification of cooperative binding between weak-binding fragments led to rapid inhibitor optimization through fragment reconstruction, structure-guided fragment linking, and conformational control over linker geometry. We show that inhibition of co-translational myristoylation of a specific virus-encoded protein (VP0) by IMP-1088 potently blocks a key step in viral capsid assembly, delivering low nanomolar antiviral activity against multiple rhinovirus strains, poliovirus and foot-and-mouth disease virus, and protection of cells against virus-induced killing, highlighting the potential of host myristoylation as a drug target in picornaviral infections.

JOURNAL ARTICLE

Craven G, Affron D, Allen C, Matthies S, Greener J, Morgan R, Tate E, Armstrong A, Mann Det al., 2018, High-throughput kinetic analysis for target-directed covalent ligand discovery, Angewandte Chemie, Vol: 57, Pages: 5257-5261, ISSN: 1521-3757

Cysteine-­reactive small molecules are used as chemical probes of biological systems and as medicines. Identifying high-­quality covalent ligands requires comprehensive kinetic analysis to distinguish selective binders from pan-­reactive compounds. Here we describe quantitative irreversible tethering(qIT), a general method for screening cysteine-­reactive small moleculesbased upon the maximization of kinetic selectivity. We apply this method prospectively to discover covalent fragments that target the clinically important cell cycle regulator Cdk2. Crystal structures of the inhibitor complexes validate the approach and guide further optimization. The power of this technique is highlighted by the identification of a Cdk2-­selective allosteric (type IV) kinase inhibitor whose novel mode-­of-­action could be exploited therapeutically.

JOURNAL ARTICLE

Alzahofi N, Robinson CL, Welz T, Page EL, Briggs DA, Stainthorp AK, Reekes J, Elbe DA, Straub F, Tate EW, Goff PS, Sviderskaya EV, Cantero M, Montoliu L, Bailly M, Kerkhoff E, Hume ANet al., 2018, Rab27a co-ordinates actin-dependent transport by controlling organelle-associated motors and track assembly proteins., Publisher: Cold Spring Harbor Laboratory

<jats:p>Cell biologists generally consider that microtubules and actin play complementary roles in long- and short-distance transport in animal cells. On the contrary, using melanosomes of melanocytes as a model, we recently discovered that motor myosin-Va, works with dynamic actin tracks, to drive long-range organelle dispersion in microtubule depleted cells. This suggests that in animals, as in yeast and plants, myosin/actin can drive long-range transport. Here we show that SPIRE1/2 and formin-1 (FMN1) proteins generate actin tracks required for myosin-Va-dependent transport in melanocytes. Moreover we show that, in addition to melanophilin/myosin-Va, Rab27a can recruit SPIRE1/2 to melanosomes, thereby integrating motor and track assembly activity at the organelle membrane. Based on this we suggest a model in which organelles and force generators (motors and track assemblers) are linked forming a cell-wide network that allows their collective activity to rapidly disperse the population of organelles long-distance throughout the cytoplasm.</jats:p>

WORKING PAPER

Schlott AC, Holder AA, Tate EW, 2018, N-myristoylation as a drug target in malaria: exploring the role of N-myristoyltransferase substrates in the inhibitor mode of action, ACS Infectious Diseases, Vol: 4, Pages: 449-457, ISSN: 2373-8227

Malaria continues to be a significant cause of death and morbidity worldwide, and there is a need for new antimalarial drugs with novel targets. We have focused as a potential target for drug development on N-myristoyl transferase (NMT), an enzyme that acylates a wide range of substrate proteins. The NMT substrates in Plasmodium falciparum include some proteins that are common to processes in eukaryotes such as secretory transport and others that are unique to apicomplexan parasites. Myristoylation facilitates a protein interaction with membranes that may be strengthened by further lipidation, and the inhibition of NMT results in incorrect protein localization and the consequent disruption of function. The diverse roles of NMT substrates mean that NMT inhibition has a pleiotropic and severe impact on parasite development, growth, and multiplication. To study the mode of action underlying NMT inhibition, it is important to consider the function of proteins upstream and downstream of NMT. In this work, we therefore present our current perspective on the different functions of known NMT substrates as well as compare the inhibition of cotranslational myristoylation to the inhibition of known targets upstream of NMT.

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

Beard R, Singh N, Grundschober C, Gee AD, Tate EWet al., 2018, High-yielding 18F radiosynthesis of a novel oxytocin receptor tracer, a probe for nose-to-brain oxytocin uptake in vivo, Chemical Communications, ISSN: 1359-7345

A novel Al18F labelled peptide tracer for PET imaging of oxytocin receptor has been accessed through a high radiochemical yield approach. This tracer showed comparable affinity and higher selectivity and stability compared to oxytocin, and was used to demonstrate direct nose-to-brain uptake following intranasal administration, a common yet controversial delivery route for oxytocin-based therapeutics.

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

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