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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 within the endoplasmic reticulum generating NADPH through this process. H6PD is involved in essential physiological processes, including energy and redox metabolism as well as redox and hormonal regulation. H6PD sole reported interacting partner is 11b-hydroxysteroid dehydrogenase 1 (11b-HSD1), utilizing NADPH for its well-known role in glucocorticoid activation. Increasing evidence suggests that H6PD contributes to breast cancer progression. However, H6PD interactions with other proteins besides 11b-HSD1 and the mechanisms and by which this protein participates in breast cancer progression are not yet well understood. Therefore, to unravel pathways and mechanisms by which H6PD promotes breast cancer progression, it is essential to first identify the interactome of this protein. Results: We adapted the proximity-dependent Biotin Identification (BioID) method to identify novel H6PD interacting partners within the endoplasmic reticulum. First, we validated the method and confirmed the known interaction between H6PD and 11b-HSD1. Next, we constructed a triple-negative breast cancer (TNBC) 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 by co-immunoprecipitation and functional assays. The resulting interactome revealed luminal protein disulfide isomerases (PDI), chaperones and calcium binding proteins. Due to its association with breast cancer, we examined the PDI Anterior gradient protein 2 (AGR2) as H6PD interacting partner. Gene enrichment analysis indicated multiple overlapping pathways being enriched in breast cancer tissue with relatively high H6PD or AGR2 expression. Co-immunoprecipitation (Co-IP) from MCF7 cells confirmed a physical interaction between the two proteins. Downregulation of ARG2 increased H6PD protein levels but decreased activity. Furthermore, co-expression with AGR2 in HEK-293 cells enhanced H6PD activity. Conclusion: BioID identified AGR2 to interact with H6PD, which was confirmed using Co-IP from MCF7 cells expressing endogenous levels of the two proteins. The results indicate that AGR2 controls H6PD protein expression and enhances its enzymatic activity. Whether a higher H6PD activity due to increased AGR2 expression promotes a more aggressive cancer cell phenotype warrants further investigations.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Alexander Schmidt

PROVIDER: MSV000096699 | MassIVE | Thu Dec 19 01:42:00 GMT 2024

SECONDARY ACCESSION(S): PXD059039

REPOSITORIES: MassIVE

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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

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