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Journal articleThinon E, Morales Sanfrutos J, Mann D, et al., 2016,
N-Myristoyltransferase Inhibition Induces ER-Stress, Cell Cycle Arrest, and Apoptosis in Cancer Cells
, ACS Chemical Biology, Vol: 11, Pages: 2165-2176, ISSN: 1554-8937N-Myristoyltransferase (NMT) covalently attaches a C14-fatty acid to the N-terminal glycine of proteins and has been proposed as a therapeutic target in cancer. We have recently shown that selective NMT inhibition leads to dose-responsive loss of N-myristoylation on more than 100 protein targets in cells, and cytotoxicity in cancer cells. N-myristoylation lies upstream of multiple pro-proliferative and oncogenic pathways, but to date the complex substrate specificity of NMT has limited determination of which diseases are most likely to respond to a selective NMT inhibitor. We describe here the phenotype of NMT inhibition in HeLa cells, and show that cells die through apoptosis following or concurrent with accumulation in G1 phase. We used quantitative proteomics to map protein expression changes for more than 2700 proteins in response to treatment with an NMT inhibitor in HeLa cells, and observed down-regulation of proteins involved in cell cycle regulation, and up-regulation of proteins involved in the endoplasmic reticulum stress and unfolded protein response, with similar results in breast (MCF-7, MDA-MB-231) and colon (HCT116) cancer cell lines. This study describes the cellular response to NMT inhibition at the proteome level, and provides a starting point for selective targeting of specific diseases with NMT inhibitors, potentially in combination with other targeted agents.
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Journal articleWright MH, Paape D, Price HP, et al., 2016,
Global profiling and inhibition of protein lipidation in vector andhost stages of the sleeping sickness parasite Trypanosoma brucei
, ACS Infectious Diseases, Vol: 2, Pages: 427-441, ISSN: 2373-8227The enzyme N-myristoyltransferase (NMT) catalyses the essential fatty acylation ofsubstrate proteins with myristic acid in eukaryotes and is a validated drug target in theparasite Trypanosoma brucei, the causative agent of African trypanosomiasis (sleepingsickness). N-Myristoylation typically mediates membrane localisation of proteins and isessential to the function of many. However, only a handful of proteins are experimentallyvalidated as N-myristoylated in T. brucei. Here, we perform metabolic labelling with analkyne-tagged myristic acid analogue, enabling the capture of lipidated proteins in insect and host life stages of T. brucei. We further compare this with a longer chain palmitate analogueto explore the chain length-specific incorporation of fatty acids into proteins. Finally, wecombine the alkynyl-myristate analogue with NMT inhibitors and quantitative chemicalproteomics to globally define N-myristoylated proteins in the clinically relevant bloodstreamform parasites. This analysis reveals five ARF family small GTPases, calpain-like proteins,phosphatases and many uncharacterized proteins as substrates of NMT in the parasite,providing a global view of the scope of this important protein modification and furtherevidence for the crucial and pleiotropic role of NMT in the cell.
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Journal articleAlbrow VE, Grimley RL, Clulow J, et al., 2016,
Design and development of histone deacetylase (HDAC) chemical probes for cell-based profiling
, Molecular Biosystems, Vol: 12, Pages: 1781-1789, ISSN: 1742-206XHistone deacetylases (HDACs) contribute to regulation of gene expression by mediating higher-order chromatin structures. They assemble into large multiprotein complexes that regulate activity and specificity. We report the development of small molecule probes with class IIa and pan-HDAC activity that contain photoreactive crosslinking groups and either a biotin reporter, or a terminal alkyne handle for subsequent bioorthogonal ligation. The probes retained inhibitory activity against recombinant HDAC proteins and caused an accumulation of acetylated histone and tubulin following cell treatment. The versatility of the probes has been demonstrated by their ability to photoaffinity modify HDAC targets in vitro. An affinity enrichment probe was used in conjunction with mass spectrometry proteomics to isolate HDACs and their interacting proteins in a native proteome. The performance of the probes in recombinant versus cell-based systems highlights issues for the development of chemoproteomic technologies targeting class IIa HDACs in particular.
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Journal articleSo EC, Schroeder GN, Carson D, et al., 2016,
The Rab-binding profiles of bacterial virulence factors during infection
, Journal of Biological Chemistry, Vol: 291, Pages: 5832-5843, ISSN: 1083-351XLegionella pneumophila, the causativeagent of Legionnaire’s disease, uses its typeIV secretion system to translocate over 300effector proteins into host cells. Theseeffectors subvert host cell signalingpathways to ensure bacterial proliferation.Despite their importance for pathogenesis,the roles of most of the effectors are yet tobe characterized. Key to understanding thefunction of effectors is the identification ofhost proteins they bind during infection. Wepreviously developed a novel tandemaffinitypurification (TAP) approach usinghexahistidine and BirA-specificbiotinylation tags for isolating translocatedeffector complexes from infected cellswhose composition were subsequentlydeciphered by mass spectrometry. Here wefurther advanced the workflow for the TAPapproach and determined the infectiondependentinteractomes of the effectorsSidM and LidA, which were previouslyreported to promiscuously bind multiple RabGTPases in vitro. In this study we defined astringent subset of Rab GTPases targeted bySidM and LidA during infection, comprisingof Rab1A, 1B, 6 and 10; in addition, LidAtargets Rab14 and 18. Taken together, thisstudy illustrates the power of this approachto profile the intracellular interactomes ofbacterial effectors during infection
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Journal articleLanyon-Hogg T, Masumoto N, Bodakh G, et al., 2016,
Synthesis and characterisation of 5-acyl-6,7-dihydrothieno[3,2-c]pyridine inhibitors of Hedgehog acyltransferase
, Data in Brief, Vol: 7, Pages: 257-281, ISSN: 2352-3409In this data article we describe synthetic and characterisation data for four members of the 5-acyl-6,7-dihydrothieno[3,2-c]pyridine (termed “RU-SKI”) class of inhibitors of Hedgehog acyltransferase, including associated NMR spectra for final compounds. RU-SKI compounds were selected for synthesis based on their published high potencies against the enzyme target. RU-SKI 41 (9a), RU-SKI 43 (9b), RU-SKI 101 (9c), and RU-SKI 201 (9d) were profiled for activity in the related article “Click chemistry armed enzyme linked immunosorbent assay to measure palmitoylation by Hedgehog acyltransferase” (Lanyon-Hogg et al., 2015) [1]. 1H NMR spectral data indicate different amide conformational ratios between the RU-SKI inhibitors, as has been observed in other 5-acyl-6,7-dihydrothieno[3,2-c]pyridines. The synthetic and characterisation data supplied in the current article provide validated access to the class of RU-SKI inhibitors.
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Journal articlePerdios L, Bunney TD, Warren SC, et al., 2016,
Time-resolved FRET reports FGFR1 dimerization and formation of a complex with its effector PLCγ1.
, Advances in Biological Regulation, Vol: 60, Pages: 6-13, ISSN: 2212-4934In vitro and in vivo imaging of protein tyrosine kinase activity requires minimally invasive, molecularly precise optical probes to provide spatiotemporal mechanistic information of dimerization and complex formation with downstream effectors. We present here a construct with genetically encoded, site-specifically incorporated, bioorthogonal reporter that can be selectively labelled with exogenous fluorogenic probes to monitor the structure and function of fibroblast growth factor receptor (FGFR). GyrB.FGFR1KD.TC contains a coumermycin-induced artificial dimerizer (GyrB), FGFR1 kinase domain (KD) and a tetracysteine (TC) motif that enables fluorescent labelling with biarsenical dyes FlAsH-EDT2 and ReAsH-EDT2. We generated bimolecular system for time-resolved FRET (TR-FRET) studies, which pairs FlAsH-tagged GyrB.FGFR1KD.TC and N-terminal Src homology 2 (nSH2) domain of phospholipase Cγ (PLCγ), a downstream effector of FGFR1, fused to mTurquoise fluorescent protein (mTFP). We demonstrated phosphorylation-dependent TR-FRET readout of complex formation between mTFP.nSH2 and GyrB.FGFR1KD.TC. By further application of TR-FRET, we also demonstrated formation of the GyrB.FGFR1KD.TC homodimer by coumermycin-induced dimerization. Herein, we present a spectroscopic FRET approach to facilitate and propagate studies that would provide structural and functional insights for FGFR and other tyrosine kinases.
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Journal articleBroncel M, Serwa RA, Bunney TD, et al., 2015,
Global profiling of Huntingtin-associated protein E (HYPE)-mediated AMPylation through a chemical proteomic approach
, Molecular & Cellular Proteomics, Vol: 15, Pages: 715-725, ISSN: 1535-9484AMPylation of mammalian small GTPases by bacterial virulence factors can be a key step in bacterial infection of host cells, and constitutes a potential drug target. This posttranslational modification also exists in eukaryotes, and AMP transferase activity was recently assigned to HYPE Filamentation induced by cyclic AMP domain containing protein (FICD) protein, which is conserved from Caenorhabditis elegans to humans. In contrast to bacterial AMP transferases, only a small number of HYPE substrates have been identified by immunoprecipitation and mass spectrometry approaches, and the full range of targets is yet to be determined in mammalian cells. We describe here the first example of global chemoproteomic screening and substrate validation for HYPE-mediated AMPylation in mammalian cell lysate. Through quantitative mass-spectrometry-based proteomics coupled with novel chemoproteomic tools providing MS/MS evidence of AMP modification, we identified a total of 25 AMPylated proteins, including the previously validated substrate endoplasmic reticulum (ER) chaperone BiP (HSPA5), and also novel substrates involved in pathways of gene expression, ATP biosynthesis, and maintenance of the cytoskeleton. This dataset represents the largest library of AMPylated human proteins reported to date and a foundation for substrate-specific investigations that can ultimately decipher the complex biological networks involved in eukaryotic AMPylation.
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Journal articleCharlton T, Kovacs-Simon A, Michell S, et al., 2015,
Quantitative lipoproteomics in Clostridium difficile reveals a role for lipoproteins in sporulation
, Chemistry & Biology, Vol: 22, ISSN: 1074-5521Bacterial lipoproteins are surface exposed, anchored to the membrane by Sdiacylglyceryl modification of the N-terminal cysteine thiol. They play important roles inmany essential cellular processes and in bacterial pathogenesis. For example,Clostridium difficile is a Gram-positive anaerobe that causes severe gastrointestinaldisease, however, its lipoproteome remains poorly characterized. Here we describe theapplication of metabolic tagging with alkyne-tagged lipid analogues, in combinationwith quantitative proteomics, to profile protein lipidation across diverse C. difficilestrains and on inactivation of specific components of the lipoprotein biogenesispathway. These studies provide the first comprehensive map of the C. difficilelipoproteome, demonstrate the existence of two active lipoprotein signal peptidasesand provide insights into lipoprotein function, implicating the lipoproteome intransmission of this pathogen.
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Journal articleSchroeder GN, Frankel G, Tate EW, et al., 2015,
The Legionella pneumophila effector LpdA is a palmitoylated phospholipase D virulence factor
, Infection and Immunity, Vol: 83, Pages: 3989-4002, ISSN: 1098-5522Legionella pneumophila is a bacterial pathogen that thrives in alveolar macrophages, causing a severe pneumonia. The virulence of L. pneumophila depends on its Dot/Icm type IV secretion system (T4SS), which delivers more than 300 effector proteins into the host, where they rewire cellular signaling to establish a replication-permissive niche, the Legionella-containing vacuole (LCV). Biogenesis of the LCV requires substantial redirection of vesicle trafficking and remodeling of intracellular membranes. In order to achieve this, several T4SS effectors target regulators of membrane trafficking, while others resemble lipases. Here, we characterized LpdA, a phospholipase D effector, which was previously proposed to modulate the lipid composition of the LCV. We found that ectopically expressed LpdA was targeted to the plasma membrane and Rab4- and Rab14-containing vesicles. Subcellular targeting of LpdA required a C-terminal motif, which is posttranslationally modified by S-palmitoylation. Substrate specificity assays showed that LpdA hydrolyzed phosphatidylinositol, -inositol-3- and -4-phosphate, and phosphatidylglycerol to phosphatidic acid (PA) in vitro. In HeLa cells, LpdA generated PA at vesicles and the plasma membrane. Imaging of different phosphatidylinositol phosphate (PIP) and organelle markers revealed that while LpdA did not impact on membrane association of various PIP probes, it triggered fragmentation of the Golgi apparatus. Importantly, although LpdA is translocated inefficiently into cultured cells, an L. pneumophila ΔlpdA mutant displayed reduced replication in murine lungs, suggesting that it is a virulence factor contributing to L. pneumophila infection in vivo.
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Journal articleSo EC, Mattheis C, Tate EW, et al., 2015,
Creating a customized intracellular niche: subversion of host cell signaling by Legionella type IV secretion system effectors
, Canadian Journal of Microbiology, Vol: 61, Pages: 617-635, ISSN: 1480-3275The Gram-negative facultative intracellular pathogen Legionella pneumophila infects a wide range of different protozoa in the environment and also human alveolar macrophages upon inhalation of contaminated aerosols. Inside its hosts, it creates a defined and unique compartment, termed the Legionella-containing vacuole (LCV), for survival and replication. To establish the LCV, L. pneumophila uses its Dot/Icm type IV secretion system (T4SS) to translocate more than 300 effector proteins into the host cell. Although it has become apparent in the past years that these effectors subvert a multitude of cellular processes and allow Legionella to take control of host cell vesicle trafficking, transcription, and translation, the exact function of the vast majority of effectors still remains unknown. This is partly due to high functional redundancy among the effectors, which renders conventional genetic approaches to elucidate their role ineffective. Here, we review the current knowledge about Legionella T4SS effectors, highlight open questions, and discuss new methods that promise to facilitate the characterization of T4SS effector functions in the future.
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