title: Mass spectrometry analysis of the oxidation states of the pro-oncogenic protein anterior gradient-2 reveals covalent dimerization via an intermolecular disulphide bond.
abstract: Anterior Gradient-2 (AGR2) is a component of a pro-oncogenic signalling pathway that can promote p53 inhibition, metastatic cell migration, limb regeneration, and cancer drug-resistance. AGR2 is in the protein-disulphide isomerase superfamily containing a single cysteine (Cys-81) that forms covalent adducts with its client proteins. We have found that mutation of Cysteine-81 attenuates its biochemical activity in its sequence-specific peptide docking function, reduces binding to Reptin, and reduces its stability in cells. As such, we evaluated how chemical oxidation of its cysteine affects its biochemical properties. Recombinant AGR2 spontaneously forms covalent dimers in the absence of reductant whilst DTT promotes dimer to monomer conversion. Mutation of Cysteine-81 to alanine prevents peroxide catalysed dimerization of AGR2 in vitro, suggesting a reactive cysteine is central to covalent dimer formation. Both biochemical assays and ESI mass spectrometry were used to demonstrate that low levels of a chemical oxidant promote an intermolecular disulphide bond through formation of a labile sulfenic acid intermediate. However, higher levels of oxidant promote sulfinic or sulfonic acid formation thus preventing covalent dimerization of AGR2. These data together identify the single cysteine of AGR2 as an oxidant responsive moiety that regulates its propensity for oxidation and its monomeric-dimeric state. This has implications for redox regulation of the pro-oncogenic functions of AGR2 protein in cancer cells.
authors.affiliation: School of Chemistry, Edinburgh, Scotland EH4 2XR, United Kingdom. Electronic address: david.clarke@ed.ac.uk.
authors.forename: David J
authors.initials: DJ
authors.lastname: Clarke
authors.affiliation: University of Edinburgh, Institute of Genetics and Molecular Medicine, Division of Cancer Biology, Edinburgh, Scotland EH4 2XR, United Kingdom.
authors.forename: Euan
authors.initials: E
authors.lastname: Murray
authors.affiliation: Regional Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, 656 53 Brno, Czech Republic.
authors.forename: Jakub
authors.initials: J
authors.lastname: Faktor
authors.affiliation: University of Edinburgh, Institute of Genetics and Molecular Medicine, Division of Cancer Biology, Edinburgh, Scotland EH4 2XR, United Kingdom.
authors.forename: Aiman
authors.initials: A
authors.lastname: Mohtar
authors.affiliation: Regional Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, 656 53 Brno, Czech Republic.
authors.forename: Borek
authors.initials: B
authors.lastname: Vojtesek
authors.affiliation: School of Chemistry, Edinburgh, Scotland EH4 2XR, United Kingdom.
authors.forename: C Logan
authors.initials: CL
authors.lastname: MacKay
authors.affiliation: School of Chemistry, Edinburgh, Scotland EH4 2XR, United Kingdom.
authors.forename: Pat Langridge
authors.initials: PL
authors.lastname: Smith
authors.affiliation: University of Edinburgh, Institute of Genetics and Molecular Medicine, Division of Cancer Biology, Edinburgh, Scotland EH4 2XR, United Kingdom; Regional Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, 656 53 Brno, Czech Republic. Electronic address: ted.hupp@ed.ac.uk.
authors.forename: Ted R
authors.initials: TR
authors.lastname: Hupp
mesh_terms: D000074183:ATPases Associated with Diverse Cellular Activities; D000595:Amino Acid Sequence / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D006801:Humans; D061986:MCF-7 Cells; D013058:Mass Spectrometry; D009088:Mucoproteins; D009154:Mutation; D009369:Neoplasms / Q000737:chemistry / Q000235:genetics* / Q000473:pathology; D015513:Oncogene Proteins; D010084:Oxidation-Reduction; D055503:Protein Multimerization / Q000235:genetics*; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D013434:Sulfenic Acids / Q000378:metabolism
chemical_list: C502296:AGR2 protein, human; D002352:Carrier Proteins; D004220:Disulfides; D009088:Mucoproteins; D015513:Oncogene Proteins; D011506:Proteins; D013434:Sulfenic Acids; D000074183:ATPases Associated with Diverse Cellular Activities; D004265:DNA Helicases; C122079:RUVBL2 protein, human; D003545:Cysteine
keywords: Anterior Gradient-2; Aptamers; Cancer; Protein mass spectrometry; Therapeutics; p53