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The sequence-specific peptide-binding activity of the protein sulfide isomerase AGR2 directs its’ stable binding to the oncogenic receptor EpCAM

ABSTRACT: 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 peptide motif (TTIYY). A synthetic TTIYY-containing peptide column can be 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 alanine mutagenized synthetic peptide library. Screening the human proteome for proteins harboring this consensus motif revealed an enrichment in transmembrane proteins and we focus on validating EpCAM as one such oncogenic protein. Recombinant AGR2 and EpCAM proteins formed a dose-dependent protein-protein interaction in vitro. Proximity ligation assays demonstrated 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 TILYY motif and surrounding EpCAM’s detergent binding site. Together, these data define a dominant peptide-binding site on AGR2 that mediates its specific peptide-binding function. A model client protein, EpCAM, is proposed for AGR2 to study how an ER-resident chaperone can dock specifically and regulate assembly of a protein destined for the secretory pathway.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Escherichia Coli

TISSUE(S): Epithelial Cell

DISEASE(S): Breast Cancer

SUBMITTER: Lenka Hernychova

LAB HEAD: Ted Hupp

PROVIDER: PXD005782 | Pride | 2018-01-22

REPOSITORIES: Pride

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The Sequence-specific Peptide-binding Activity of the Protein Sulfide Isomerase AGR2 Directs Its Stable Binding to the Oncogenic Receptor EpCAM.

Mohtar M Aiman MA Hernychova Lenka L O'Neill J Robert JR Lawrence Melanie L ML Murray Euan E Vojtesek Borek B Hupp Ted R TR

Molecular & cellular proteomics : MCP 20180116 4

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 t ...[more]

PMID: 29339412

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	AGR2_S134A_10min.raw	Raw
	AGR2_S134A_1h.raw	Raw
	AGR2_S134A_1min.raw	Raw
	AGR2_S134A_30min.raw	Raw
	AGR2_S134A_30s.raw	Raw