Byrne, Dominic 
ORCID: 0000-0001-5197-345X, Li, Yong, Ngamlert, Pawin, Ramakrishnan, Krithika ORCID: 0000-0002-4844-0975, Eyers, Claire ORCID: 0000-0002-3223-5926, Wells, Carrow, Drewry, David, Zuercher, William, Berry, Neil ORCID: 0000-0003-1928-0738, Fernig, David ORCID: 0000-0003-4875-4293 et al (show 1 more authors)
(2018)
New tools for evaluating protein tyrosine sulphation: Tyrosyl Protein Sulphotransferases (TPSTs) are novel targets for RAF protein kinase inhibitors
[Preprint]
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Byrne BJ 2018 Protein sulfation 2435.full.pdf - Open Access published version Download (11MB) | Preview |
Abstract
<h4>ABSTRACT</h4> Protein tyrosine sulphation is a post-translational modification (PTM) best known for regulating extracellular protein-protein interactions. Tyrosine sulphation is catalysed by two Golgi-resident enzymes termed Tyrosyl Protein Sulpho Transferases (TPSTs) 1 and 2, which transfer sulphate from the co-factor PAPS (3’-phosphoadenosine 5’-phosphosulphate) to a context-dependent tyrosine in a protein substrate. A lack of quantitative tyrosine sulphation assays has hampered the development of chemical biology approaches for the identification of small molecule inhibitors of tyrosine sulphation. In this paper, we describe the development of a non-radioactive mobility-based enzymatic assay for TPST1 and TPST2, through which the tyrosine sulphation of synthetic fluorescent peptides can be rapidly quantified. We exploit ligand binding and inhibitor screens to uncover a susceptibility of TPST1 and 2 to different classes of small molecules, including the anti-angiogenic compound suramin and the kinase inhibitor rottlerin. By screening the Published Kinase Inhibitor Set (PKIS), we identified oxindole-based inhibitors of the Ser/Thr kinase RAF as low micromolar inhibitors of TPST1/2. Interestingly, unrelated RAF inhibitors, exemplified by the dual BRAF/VEGFR2 inhibitor RAF265, were also TPST inhibitors in vitro . We propose that target-validated protein kinase inhibitors could be repurposed, or redesigned, as more-specific TPST inhibitors to help evaluate the sulphotyrosyl proteome. Finally, we speculate that mechanistic inhibition of cellular tyrosine sulphation might be relevant to some of the phenotypes observed in cells exposed to anionic TPST ligands and RAF protein kinase inhibitors. <h4>SUMMARY STATEMENT</h4> We develop new assays to quantify tyrosine sulphation by the human tyrosine sulphotransferases TPST1 and 2. TPST1 and 2 catalytic activities are inhibited by protein kinase inhibitors, suggesting new starting points to synthesise (or repurpose) small molecule compounds to evaluate biological TPST using chemical biology.
| Item Type: | Preprint |
|---|---|
| Uncontrolled Keywords: | 4613 Theory Of Computation, 3101 Biochemistry and Cell Biology, 31 Biological Sciences, 3404 Medicinal and Biomolecular Chemistry, 34 Chemical Sciences, 46 Information and Computing Sciences, Cancer, Biotechnology, 5.1 Pharmaceuticals |
| Divisions: | Faculty of Health & Life Sciences Faculty of Science & Engineering > School of Physical Sciences Faculty of Health & Life Sciences > Inst. Systems, Molec & Integrative Biology > Inst. Systems, Molec & Integrative Biology |
| Depositing User: | Symplectic Admin |
| Date Deposited: | 25 Mar 2024 08:46 |
| Last Modified: | 06 Nov 2025 17:58 |
| DOI: | 10.1101/296707 |
| Related Websites: | |
| URI: | https://livrepository.liverpool.ac.uk/id/eprint/3179836 |
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