The Sequence-specific Peptide-binding Activity of the Protein Sulfide Isomerase AGR2 Directs Its Stable Binding to the Oncogenic Receptor EpCAM
| Autor: | Melanie L. Lawrence, Borek Vojtesek, Ted R. Hupp, J. Robert O’Neill, Euan Murray, M. Aiman Mohtar, Lenka Hernychová |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Peptide Peptide binding Plasma protein binding Biochemistry Analytical Chemistry 03 medical and health sciences chemistry.chemical_compound Mucoproteins Humans Binding site Protein disulfide-isomerase Peptide library Molecular Biology chemistry.chemical_classification Oncogene Proteins Chemistry Research Proteins Epithelial cell adhesion molecule Proto-Oncogene Proteins c-mdm2 Epithelial Cell Adhesion Molecule Transmembrane protein Recombinant Proteins 030104 developmental biology MCF-7 Cells Peptides Protein Binding |
| Zdroj: | Molecular & Cellular Proteomics : MCP Mohtar, M A, Hernychova, L, O'Neill, J, Lawrence, L, Murray, E, Vojtesek, B & Hupp, T R 2018, ' The sequence-specific peptide-binding activity of the protein sulfide isomerase AGR2 directs its stable binding to the oncogenic receptor EpCAM ', Molecular and Cellular Proteomics . https://doi.org/10.1074/mcp.RA118.000573 |
| ISSN: | 1535-9484 |
| DOI: | 10.1074/mcp.RA118.000573 |
| Popis: | AGR2 is an oncogenic endoplasmic reticulum (ER)-resident protein disulfide isomerase. AGR2 protein has a relatively unique property for a chaperone in that it can bind sequence-specifically to a specific peptide motif (TTIYY). A synthetic TTIYY-containing peptide column was used to affinity-purify AGR2 from crude lysates highlighting peptide selectivity in complex mixtures. Hydrogen-deuterium exchange mass spectrometry localized the dominant region in AGR2 that interacts with the TTIYY peptide to within a structural loop from amino acids 131-135 (VDPSL). A peptide binding site consensus of Tx[IL][YF][YF] was developed for AGR2 by measuring its activity against a mutant peptide library. Screening the human proteome for proteins harboring this motif revealed an enrichment in transmembrane proteins and we focused on validating EpCAM as a potential AGR2-interacting protein. AGR2 and EpCAM proteins formed a dose-dependent protein-protein interaction in vitro. Proximity ligation assaysdemonstrated that endogenous AGR2 and EpCAM protein associate in cells. Introducing a single alanine mutation in EpCAM at Tyr251 attenuated its binding to AGR2 in vitro and in cells. Hydrogen-deuterium exchange mass spectrometry was used to identify a stable binding site for AGR2 on EpCAM, adjacent to the TLIYY motif and surrounding EpCAM’s detergent binding site. These data define a dominant site on AGR2 that mediates its specific peptidebinding function. EpCAM forms a model client protein for AGR2 to study how an ER-residentchaperone can dock specifically to a peptide motif and regulate the trafficking a protein destined for the secretory pathway. |
| Databáze: | OpenAIRE |
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