title: Global Profiling of Huntingtin-associated protein E (HYPE)-Mediated AMPylation through a Chemical Proteomic Approach.
abstract: AMPylation 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.
authors.affiliation: From the ‡Department of Chemistry, Imperial College London, Exhibition Road, London SW7 2AZ, UK; ¶Current address: The Institute of Cancer Research, Division of Cancer Biology, 237 Fulham Road, London SW3 6JB, UK.
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Broncel
authors.affiliation: From the ‡Department of Chemistry, Imperial College London, Exhibition Road, London SW7 2AZ, UK;
authors.forename: Remigiusz A
authors.initials: RA
authors.lastname: Serwa
authors.affiliation: §Division of Biosciences, Institute of Structural and Molecular Biology, University College London, Gower Street, London WC1E 6BT, UK;
authors.forename: Tom D
authors.initials: TD
authors.lastname: Bunney
authors.affiliation: §Division of Biosciences, Institute of Structural and Molecular Biology, University College London, Gower Street, London WC1E 6BT, UK;
authors.forename: Matilda
authors.initials: M
authors.lastname: Katan
authors.affiliation: From the ‡Department of Chemistry, Imperial College London, Exhibition Road, London SW7 2AZ, UK; e.tate@imperial.ac.uk.
authors.forename: Edward W
authors.initials: EW
authors.lastname: Tate
mesh_terms: D000595:Amino Acid Sequence / Q000187:drug effects; D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics / Q000382:microbiology; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism; D000091342:Endoplasmic Reticulum Chaperone BiP; D054884:Host-Pathogen Interactions; D006801:Humans; D007239:Infections / Q000235:genetics / Q000382:microbiology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D009713:Nucleotidyltransferases; D011499:Protein Processing, Post-Translational / Q000235:genetics; D040901:Proteomics; D053719:Tandem Mass Spectrometry; D014166:Transferases / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D008565:Membrane Proteins; D000242:Cyclic AMP; D014166:Transferases; C541422:FICD protein, human; D009713:Nucleotidyltransferases; D020559:Monomeric GTP-Binding Proteins