A BioID-based approach uncovers the interactome of hexose-6-phosphate dehydrogenase in breast cancer cells and identifies anterior gradient protein 2 as an interacting partner.

Abstract

Background: Hexose-6-phosphate dehydrogenase (H6PD) catalyzes the first two steps of the pentose-phosphate-pathway (PPP) within the endoplasmic reticulum, generating NADPH. H6PD modulates essential physiological processes, including energy and redox metabolism. Its sole reported interacting partner is 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1), utilizing NADPH to reactivate glucocorticoids, linking energy status with hormonal response. Previous studies showed that loss of H6PD affects breast cancer cell properties, independent of 11β-HSD1. It remains unknown whether this is due to impaired concentrations of NADPH or PPP products downstream of H6PD. To gain insight into novel roles and pathways influenced by this enzyme, we aimed to assess the H6PD interactome.

Results: We adapted the proximity-dependent Biotin Identification (BioID) method to identify novel H6PD interacting partners. First, we validated the method and confirmed the known interaction between H6PD and 11β-HSD1. Next, we constructed a triple-negative breast cancer MDA-MB-231 cell clone stably expressing a H6PD-biotin ligase fusion protein. Enriched biotinylated proteins were analyzed by mass-spectrometry and potential candidates assessed further by co-immunoprecipitation and functional assays. The resulting interactome revealed proteins of the calreticulin/calnexin cycle, unfolded-protein response (UPR) and chaperone activation pathways. Due to its known association with breast cancer, we examined the PDI Anterior gradient protein 2 (AGR2) as H6PD interacting partner. Gene set enrichment analysis revealed multiple overlapping pathways enriched in breast cancer tissues with relatively high H6PD and AGR2 expression. These included glycolysis, fatty acid metabolism, hypoxia, angiogenesis and epithelial to mesenchymal transition. Co-immunoprecipitation (Co-IP) from MCF7 cells confirmed a physical interaction between H6PD and AGR2. ARG2 knockdown in these cells increased H6PD protein levels but decreased activity. Coexpression with AGR2 in HEK-293 cells did not affect expression but enhanced H6PD activity.

Conclusion: BioID was successfully applied in the endoplasmic reticulum to identify AGR2 as H6PD interactor. This was confirmed using Co-IP from MCF7 cells endogenously expressing both proteins. The results indicate that AGR2 controls H6PD protein expression and enhances its activity. Whether higher H6PD activity due to increased AGR2 expression promotes a more aggressive cancer cell phenotype, for example by altering energy metabolism, Ca²⁺-related processes or UPR and chaperone activation pathways, warrants further investigations.