Characterization of the AGR2 Interactome Uncovers New Players of Protein Disulfide Isomerase Network in Cancer Cells

Pavla Bouchalova,Lucia Sommerova,David Potesil,Andrea Martisova,Petr Lapcik,Veronika Koci,Alex Scherl,Petr Vonka,Joan Planas-Iglesias,Eric Chevet,Pavel Bouchal,Roman Hrstka

doi:10.1016/j.mcpro.2021.100188

Abstract

Anterior gradient 2 (AGR2) is an endoplasmic reticulum (ER)-resident protein disulfide isomerase (PDI) known to be overexpressed in many human epithelial cancers and is involved in cell migration, cellular transformation, angiogenesis, and metastasis. This protein inhibits the activity of the tumor suppressor p53, and its expression levels can be used to predict cancer patient outcome. However, the precise network of AGR2-interacting partners and clients remains to be fully characterized. Herein, we used label-free quantification and also stable isotope labeling with amino acids in cell culture–based LC–MS/MS analyses to identify proteins interacting with AGR2. Functional annotation confirmed that AGR2 and its interaction partners are associated with processes in the ER that maintain intracellular metabolic homeostasis and participate in the unfolded protein response, including those associated with changes in cellular metabolism, energy, and redox states in response to ER stress. As a proof of concept, the interaction between AGR2 and PDIA3, another ER-resident PDI, was studied in more detail. Pathway analysis revealed that AGR2 and PDIA3 play roles in protein folding in ER, including post-translational modification and in cellular response to stress. We confirmed the AGR2–PDIA3 complex formation in cancer cells, which was enhanced in response to ER stress. Accordingly, molecular docking characterized potential quaternary structure of this complex; however, it remains to be elucidated whether AGR2 rather contributes to PDIA3 maturation in ER, the complex directly acts in cellular signaling, or mediates AGR2 secretion. Our study provides a comprehensive insight into the protein–protein interaction network of AGR2 by identifying functionally relevant proteins and related cellular and biochemical pathways associated with the role of AGR2 in cancer cells.

Highlights

Protein-protein interactions (PPIs) are essential for the correct structure and function of a vast majority of protein complexes
Anterior gradient 2 (AGR2) complexes with interacting proteins were targeted using AGR2-specific peptide E7, while F4 peptide served as a negative control (Fig. 1A, B)
HADDOCK clustered 315-333 structures in 22-24 clusters representing in oo between 78 and 83.75% of the analyzed docking solutions across the triplicates in the case of AGR2 pr protein in complex with E7 peptide, while F4 peptide only rendered 234-247 structures in 24-30 e- clusters representing 58-61% of the analyzed docking solutions across the triplicates (Fig. S2, S3). r These results show a clear trend for a more cohesive docking of E7 peptide compared to that of F4, l P which might be interpreted as a hint for better binding as well

Summary

Filter Aided Sample Preparation

Ambiguous Interaction Restraint Fraction of Common Contact Binding Affinity Dock(ing) Root-Mean-Square Deviation Cytosolic Fraction ImmunoPrecipitation Horse Radish Peroxidase Negative Control Proximity Ligation Assay. Endoplasmic Reticulum Associated Degradation Epithelial Cell Adhesion Molecule Epidermal Growth Factor Receptor mammalian Target Of Rapamycin Complex 1 control Heat Shock Protein PYruvate Carboxylase Endoplasmic Reticulum-to-CYtosol-Signaling f CALReticulin oo HeterogeNous RiboNucleoProtein U Journal Pre-pr 75kDa Glucose Regulated Protein stress-70 protein (mitochondrial)

Introduction

Results

Majority protein IDs

ID name abbreviation Protein name