189 results found
Tate E, Lanyon-Hogg T, Ritzefeld M, et al., 2019, Acylation-coupled lipophilic induction of polarisation (Acyl-cLIP): a universal assay for lipid transferase and hydrolase enzymes, Chemical Science, ISSN: 2041-6520
Posttranslational attachment of lipids to proteins is important for many cellular functions, and the enzymes responsible for these modifications are implicated in many diseases, from cancer to neurodegeneration. Lipid transferases and hydrolases are increasingly tractable therapeutic targets, but present unique challenges for high-throughput biochemical enzyme assays which hinder development of new inhibitors. We present Acylation-coupled Lipophilic Induction of Polarisation (Acyl-cLIP) as the first universally applicable biochemical lipidation assay, exploiting the hydrophobic nature of lipidated peptides to drive a polarised fluorescence readout. Acyl-cLIP allows sensitive, accurate, real-time measurement of S- or N-palmitoylation, N-myristoylation, S-farnesylation or S-geranylgeranylation. Furthermore, it is applicable to transfer and hydrolysis reactions, and we demonstrate its extension to a high-throughput screening format. We anticipate that Acyl-cLIP will greatly expedite future drug discovery efforts against these challenging targets.
Lanyon-Hogg T, Patel NV, Ritzefeld M, et 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.
Rodgers U, Lanyon-Hogg T, Masumoto N, et al., 2016, Characterization of hedgehog acyltransferase inhibitors identifies a small molecule probe for hedgehog signaling by cancer cells, ACS Chemical Biology, Vol: 11, Pages: 3256-3262, ISSN: 1554-8937
The Sonic Hedgehog (Shh) signaling pathway plays a critical role during embryonic development and cancer progression. N-terminal palmitoylation of Shh by Hedgehog acyltransferase (Hhat) is essential for efficient signaling, raising interest in Hhat as a novel drug target. A recently identified series of dihydrothienopyridines has been proposed to function via this mode of action; however, the lead compound in this series (RUSKI-43) was subsequently shown to possess cytotoxic activity unrelated to canonical Shh signaling. To identify a selective chemical probe for cellular studies, we profiled three RUSKI compounds in orthogonal cell-based assays. We found that RUSKI-43 exhibits off-target cytotoxicity, masking its effect on Hhat-dependent signaling, hence results obtained with this compound in cells should be treated with caution. In contrast, RUSKI-201 showed no off-target cytotoxicity, and quantitative whole-proteome palmitoylation profiling with a bioorthogonal alkyne-palmitate reporter demonstrated specific inhibition of Hhat in cells. RUSKI-201 is the first selective Hhat chemical probe in cells and should be used in future studies of Hhat catalytic function.
Lanyon-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: 2352-3409
In 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) . 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.
Lanyon-Hogg T, Masumoto N, Bodakh G, et al., 2015, Click chemistry armed enzyme-linked immunosorbent assay to measure palmitoylation by hedgehog acyltransferase, Analytical Biochemistry, Vol: 490, Pages: 66-72, ISSN: 1096-0309
Hedgehog signaling is critical for correct embryogenesis and tissue development. However, on maturation, signaling is also found to be aberrantly activated in many cancers. Palmitoylation of the secreted signaling protein sonic hedgehog (Shh) by the enzyme hedgehog acyltransferase (Hhat) is required for functional signaling. To quantify this important posttranslational modification, many in vitro Shh palmitoylation assays employ radiolabeled fatty acids, which have limitations in terms of cost and safety. Here we present a click chemistry armed enzyme-linked immunosorbent assay (click–ELISA) for assessment of Hhat activity through acylation of biotinylated Shh peptide with an alkyne-tagged palmitoyl-CoA (coenzyme A) analogue. Click chemistry functionalization of the alkyne tag with azido-FLAG peptide allows analysis through an ELISA protocol and colorimetric readout. This assay format identified the detergent n-dodecyl β-d-maltopyranoside as an improved solubilizing agent for Hhat activity. Quantification of the potency of RU-SKI small molecule Hhat inhibitors by click–ELISA indicated IC50 values in the low- or sub-micromolar range. A stopped assay format was also employed that allows measurement of Hhat kinetic parameters where saturating substrate concentrations exceed the binding capacity of the streptavidin-coated plate. Therefore, click–ELISA represents a nonradioactive method for assessing protein palmitoylation in vitro that is readily expandable to other classes of protein lipidation.
Broncel M, Serwa RA, Ciepla P, et al., 2015, Myristoylation profiling in human cells and zebrafish., Data in Brief, Vol: 4, Pages: 379-383, ISSN: 2352-3409
Human cells (HEK 293, HeLa, MCF-7) and zebrafish embryos were metabolically tagged with an alkynyl myristic acid probe, lysed with an SDS buffer and tagged proteomes ligated to multifunctional capture reagents via copper-catalyzed alkyne azide cycloaddition (CuAAC). This allowed for affinity enrichment and high-confidence identification, by delivering direct MS/MS evidence for the modification site, of 87 and 61 co-translationally myristoylated proteins in human cells and zebrafish, respectively. The data have been deposited to ProteomeXchange Consortium (Vizcaíno et al., 2014 Nat. Biotechnol., 32, 223-6) (PXD001863 and PXD001876) and are described in detail in Multifunctional reagents for quantitative proteome-wide analysis of protein modification in human cells and dynamic protein lipidation during vertebrate development׳ by Broncel et al., Angew. Chem. Int. Ed.
Broncel M, Serwa RA, Ciepla P, et al., 2015, Multifunctional Reagents for Quantitative Proteome-Wide Analysis of Protein Modification in Human Cells and Dynamic Profiling of Protein Lipidation During Vertebrate Development, Angewandte Chemie-International Edition, Vol: 54, Pages: 5948-5951, ISSN: 1521-3773
Novel multifunctional reagents were applied incombination with a lipid probe for affinity enrichment ofmyristoylated proteins and direct detection of lipid-modifiedtryptic peptides by mass spectrometry. This method enableshigh-confidence identification of the myristoylated proteomeon an unprecedented scale in cell culture, and allowed the firstquantitative analysis of dynamic changes in protein lipidationduring vertebrate embryonic development.
Masumoto N, Lanyon-Hogg T, Rodgers UR, et al., 2015, Membrane bound O-acyltransferases and their inhibitors, Biochemical Society Transactions, Vol: 43, Pages: 246-252, ISSN: 1470-8752
Since the identification of the membrane-bound O-acyltransferase (MBOATs) protein family in the early2000s, three distinct members [porcupine (PORCN), hedgehog (Hh) acyltransferase (HHAT) and ghrelin Oacyltransferase(GOAT)] have been shown to acylate specific proteins or peptides. In this review, topologydetermination, development of assays to measure enzymatic activities and discovery of small moleculeinhibitors are compared and discussed for each of these enzymes.
Ciepla P, Magee AI, Tate EW, 2015, Cholesterylation: a tail of hedgehog, BIOCHEMICAL SOCIETY TRANSACTIONS, Vol: 43, Pages: 262-267, ISSN: 0300-5127
Lanyon-Hogg T, Ritzefeld M, Masumoto N, et al., 2015, Modulation of Amide Bond Rotamers in 5-Acyl-6,7-dihydrothieno[3,2-c]pyridines, Journal of Organic Chemistry, Vol: 80, Pages: 4370-4377, ISSN: 1520-6904
2-Substituted N-acyl-piperidine is a widespread and important structuralmotif, found in approximately 500 currently available structures, and present in nearly30 pharmaceutically active compounds. Restricted rotation of the acyl substituent insuch molecules can give rise to two distinct chemical environments. Here wedemonstrate, using NMR studies and density functional theory modeling of the lowestenergy structures of 5-acyl-6,7-dihydrothieno[3,2-c]pyridine derivatives, that the amideE:Z equilibrium is affected by non-covalent interactions between the amide oxygen andadjacent aromatic protons. Structural predictions were used to design molecules that promote either the E- or Z-amideconformation, enabling preparation of compounds with a tailored conformational ratio, as proven by NMR studies. Analysis ofthe available X-ray data of a variety of published N-acyl-piperidine-containing compounds further indicates that these moleculesare also clustered in the two observed conformations. This finding emphasizes that directed conformational isomerism hassignificant implications for the design of both small molecules and larger amide-containing molecular architectures.
Konitsiotis AD, Jovanovic B, Ciepla P, et al., 2015, Topological Analysis of Hedgehog Acyltransferase, a Multipalmitoylated Transmembrane Protein, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 290, Pages: 3293-3307
Koleva MV, Rothery S, Spitaler M, et al., 2015, Sonic hedgehog multimerization: A self-organizing event driven by post-translational modifications?, MOLECULAR MEMBRANE BIOLOGY, Vol: 32, Pages: 65-74, ISSN: 0968-7688
Ciepla P, Konitsiotis AD, Serwa RA, et al., 2014, New chemical probes targeting cholesterylation of Sonic Hedgehog in human cells and zebrafish, Chemical Science, Vol: 5, Pages: 4249-4259, ISSN: 2041-6520
Sonic Hedgehog protein (Shh) is a morphogen molecule important in embryonic development and in theprogression of many cancer types in which it is aberrantly overexpressed. Fully mature Shh requiresattachment of cholesterol and palmitic acid to its C- and N-termini, respectively. The study of lipidatedShh has been challenging due to the limited array of tools available, and the roles of theseposttranslational modifications are poorly understood. Herein, we describe the development andvalidation of optimised alkynyl sterol probes that efficiently tag Shh cholesterylation and enable itsvisualisation and analysis through bioorthogonal ligation to reporters. An optimised probe was shown tobe an excellent cholesterol biomimetic in the context of Shh, enabling appropriate release of tagged Shhfrom signalling cells, formation of multimeric transport complexes and signalling. We have used thisprobe to determine the size of transport complexes of lipidated Shh in culture medium and expressionlevels of endogenous lipidated Shh in pancreatic ductal adenocarcinoma cell lines through quantitativechemical proteomics, as well as direct visualisation of the probe by fluorescence microscopy anddetection of cholesterylated Hedgehog protein in developing zebrafish embryos. These sterol probesprovide a set of novel and well-validated tools that can be used to investigate the role of lipidation onactivity of Shh, and potentially other members of the Hedgehog protein family
Konitsiotis AD, Chang S-C, Jovanovic B, et al., 2014, Attenuation of Hedgehog Acyltransferase-Catalyzed Sonic Hedgehog Palmitoylation Causes Reduced Signaling, Proliferation and Invasiveness of Human Carcinoma Cells, PLOS ONE, Vol: 9, ISSN: 1932-6203
McDonald G, Deepak S, Miguel L, et al., 2014, Normalizing glycosphingolipids restores function in CD4(+) T cells from lupus patients, JOURNAL OF CLINICAL INVESTIGATION, Vol: 124, Pages: 712-724, ISSN: 0021-9738
McDonald G, Miguel L, Hall C, et al., 2012, Targeting Glycosphingolipid Biosynthesis Normalises T Lymphocyte Function in Patients with Systemic Lupus Eyrthematosus, Annual Scientific Meeting of the American-College-of-Rheumatology (ACR) and Association-of-Rheumatology-Health-Professionals (ARHP), Publisher: WILEY-BLACKWELL, Pages: S370-S370, ISSN: 0004-3591
Konitsiotis A, Chang S, Masumoto N, et al., 2012, Hhat a Potential New Target in Treatment of Pancreatic Cancer, 22nd Biennial Congress of the European-Association-for-Cancer-Research, Publisher: ELSEVIER SCI LTD, Pages: S149-S149, ISSN: 0959-8049
Vincent J-P, Magee T, 2012, Tiki Casts a Spell on Wnt, CELL, Vol: 149, Pages: 1426-1427, ISSN: 0092-8674
Plaza-Menacho I, Morandi A, Mologni L, et al., 2011, Focal Adhesion Kinase (FAK) Binds RET Kinase via Its FERM Domain, Priming a Direct and Reciprocal RET-FAK Transactivation Mechanism, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 286, Pages: 17292-17302
Miguel L, Owen DM, Lim C, et al., 2011, Primary Human CD4(+) T Cells Have Diverse Levels of Membrane Lipid Order That Correlate with Their Function, JOURNAL OF IMMUNOLOGY, Vol: 186, Pages: 3505-3516, ISSN: 0022-1767
Salmond RJ, Filby A, Pirinen N, et al., 2011, Mislocalization of Lck impairs thymocyte differentiation and can promote development of thymomas, BLOOD, Vol: 117, Pages: 108-117, ISSN: 0006-4971
Heal WP, Jovanovic B, Bessin S, et al., 2011, Bioorthogonal chemical tagging of protein cholesterylation in living cells, CHEMICAL COMMUNICATIONS, Vol: 47, Pages: 4081-4083, ISSN: 1359-7345
McGinty J, Talbot C, Owen D, et al., 2011, Fluorescence Lifetime Imaging Microscopy, Endoscopy and Tomography, Editors: Boas, Pitris, Ramanujam, ISBN: 1420090364
Zeidman R, Buckland G, Cebecauer M, et al., 2011, DHHC2 is a protein S-acyl transferase for Lck, Molecular Membrane Biology, Vol: 28, Pages: 473-486
Lck is a non-receptor tyrosine kinase of the Src family that is essential for T cell activation. Dual N-terminal acylation of Lck with myristate (N-acylation) and palmitate (S-acylation) is essential for its membrane association and function. Reversible S-acylation of Lck is observed in vivo and may function as a control mechanism. Here we identify the DHHC family protein S-acyltransferase DHHC2 as an enzyme capable of palmitoylating of Lck in T cells. Reducing the DHHC2 level in Jurkat T cells using siRNA causes decreased Lck S-acylation and partial dislocation from membranes, and conversely overexpression of DHHC2 increases S-acylation of an Lck surrogate, LckN10-GFP. DHHC2 localizes primarily to the endoplasmic reticulum and Golgi apparatus suggesting that it is involved in S-acylation of newly-synthesized or recycling Lck involved in T cell signalling.
Chang SC, Mulloy B, Magee AI, 2011, Two distinct sites in sonic hedgehog combine for heparan sulfate interactions and cell signaling functions, Journal of Biological Chemistry
Hedgehog (Hh) proteins are morphogens that mediate many developmental processes. Hh signaling is significant for many aspects of embryonic development, whereas dysregulation of this pathway is associated with several types of cancer. Hh proteins require heparan sulfate proteoglycans (HSPGs) for their normal distribution and signaling activity. Here we have used molecular modelling to examine the heparin-binding domain of Shh. In biochemical and cell biological assays the importance of specific residues of the putative heparin-binding domain for signaling were assessed. It was determined that key residues in hShh involved in heparin and HSPG syndecan-4 binding and biological activity included the well known cationic Cardin-Weintraub motif (lysine 32-lysine38), but also a previously unidentified major role for lysine 178. The activity of Shh mutated in these residues was tested by quantitation of alkaline phosphatase activity in C3H10T1/2 cells differentiating into osteoblasts and hShh-inducible gene expression in PANC1 human pancreatic ductal adenocarcinoma (PDAC) cells. Mutated hShhs such as K37/38S, K178S and particularly K37/38/178S that could not interact with heparin efficiently had reduced signaling activity compared to wild type hShh or a control mutation (K74S). In addition, the mutant hShh proteins supported reduced proliferation and invasion of PANC1 cells compared with control hShh proteins, following endogenous hShh depletion by RNAi knockdown. The data correlated with reduced Shh multimerization where the K37/38 and/or K178 mutations were examined. These studies provide a new insight into the functional roles of hShh interactions with HSPGs, which may allow targeting this aspect of hShh biology in, for example, PDAC.
Miguel L, Owen DM, Lim C, et al., 2010, Primary human CD4(+) T cells have diverse levels of membrane lipid order that dictate their function, Annual Congress of the British-Society-for-Immunology, Publisher: WILEY-BLACKWELL, Pages: 77-77, ISSN: 0019-2805
Owen DM, Gaus K, Magee AI, et al., 2010, Dynamic organization of lymphocyte plasma membrane: lessons from advanced imaging methods, IMMUNOLOGY, Vol: 131, Pages: 1-8, ISSN: 0019-2805
Owen DM, Oddos S, Kumar S, et al., 2010, High plasma membrane lipid order imaged at the immunological synapse periphery in live T cells, MOLECULAR MEMBRANE BIOLOGY, Vol: 27, Pages: 178-189, ISSN: 0968-7688
Cebecauer M, Spitaler M, Serge A, et al., 2010, Signalling complexes and clusters: functional advantages and methodological hurdles, JOURNAL OF CELL SCIENCE, Vol: 123, Pages: 309-320, ISSN: 0021-9533
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