title: Fluorescent peptide sensors for tyrosylprotein sulfotransferase activity.
abstract: Tyrosine sulfurylation is a post-translational modification important for protein-protein interactions in the extracellular space that are instrumental in cell adhesion, cell signaling, immune responses, and pathogen recognition of host cells. Tyrosine sulfurylation is catalyzed by the tyrosylprotein sulfotransferases (TPSTs), and in humans there are two isoforms: hTPST1 and hTPST2. The study of hTPST function and the development of small molecule probes to examine the role of hTPSTs in cell biology have been delayed by the absence of a continuous direct assay for hTPST activity. We have developed a fluorescent peptide-based assay to directly monitor tyrosine sulfurylation in real time. TPST-mediated tyrosine sulfurylation of the peptides disrupts fluorophore quenching and results in increased fluorescence emission. The assay can be used to study TPST enzymatic activity, and we show that recombinant hTPSTs are active in the absence of divalent metal ions and that optimal activity is at pH 6.0. We further show that the assay can also be used to identify inhibitors of tyrosine sulfurylation. A clear understanding of hTPST function in normal cell biology and in disease states will require the identification of small molecule inhibitors or probes to modulate enzymatic activity, and our results will facilitate that process.
authors.affiliation: Center for Drug Design, Academic Health Center, University of Minnesota, Minneapolis, MN 55455, USA.
authors.forename: Wenbo
authors.initials: W
authors.lastname: Zhou
authors.affiliation: Center for Drug Design, Academic Health Center, University of Minnesota, Minneapolis, MN 55455, USA.
authors.forename: Benjamin P
authors.initials: BP
authors.lastname: Duckworth
authors.affiliation: Center for Drug Design, Academic Health Center, University of Minnesota, Minneapolis, MN 55455, USA. Electronic address: gerag012@umn.edu.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Geraghty
mesh_terms: D000595:Amino Acid Sequence; D015374:Biosensing Techniques / Q000379:methods*; D003067:Coenzymes / Q000494:pharmacology; D057075:Enzyme Assays / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D015238:Sulfotransferases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*
chemical_list: D003067:Coenzymes; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D010455:Peptides; D011505:Protein-Tyrosine Kinases; D015238:Sulfotransferases; C039079:protein-tyrosine sulfotransferase
keywords: Fluorescence quenching; Fluorescent peptide; Kinase; Sulfotransferase; TPST; Tyrosine