Mass spectrometry analysis of the oxidation states of the pro-oncogenic protein anterior gradient-2 reveals covalent dimerization via an intermolecular disulphide bond
Language English Country Netherlands Media print-electronic
Document type Journal Article, Research Support, Non-U.S. Gov't
Grant support
c483/a6354
Cancer Research UK - United Kingdom
g0800759
Medical Research Council - United Kingdom
BB/C511599/1
Biotechnology and Biological Sciences Research Council - United Kingdom
G0800759
Medical Research Council - United Kingdom
ETM/137
Chief Scientist Office - United Kingdom
bb/c511599/1
Biotechnology and Biological Sciences Research Council - United Kingdom
G0800675
Medical Research Council - United Kingdom
G0600329
Medical Research Council - United Kingdom
PubMed
26876500
DOI
10.1016/j.bbapap.2016.02.011
PII: S1570-9639(16)30024-3
Knihovny.cz E-resources
- Keywords
- Anterior Gradient-2, Aptamers, Cancer, Protein mass spectrometry, Therapeutics, p53,
- MeSH
- ATPases Associated with Diverse Cellular Activities MeSH
- Drug Resistance, Neoplasm genetics MeSH
- Cysteine genetics metabolism MeSH
- Disulfides chemistry metabolism MeSH
- DNA Helicases chemistry genetics metabolism MeSH
- Mass Spectrometry MeSH
- Sulfenic Acids metabolism MeSH
- Humans MeSH
- MCF-7 Cells MeSH
- Mucoproteins MeSH
- Protein Multimerization genetics MeSH
- Mutation MeSH
- Neoplasms chemistry genetics pathology MeSH
- Oncogene Proteins MeSH
- Oxidation-Reduction * MeSH
- Proteins chemistry genetics metabolism MeSH
- Amino Acid Sequence genetics MeSH
- Signal Transduction MeSH
- Carrier Proteins chemistry genetics metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- AGR2 protein, human MeSH Browser
- ATPases Associated with Diverse Cellular Activities MeSH
- Cysteine MeSH
- Disulfides MeSH
- DNA Helicases MeSH
- Sulfenic Acids MeSH
- Mucoproteins MeSH
- Oncogene Proteins MeSH
- Proteins MeSH
- RUVBL2 protein, human MeSH Browser
- Carrier Proteins MeSH
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.
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