7274 Assessing the interactome of hexose-6-phosphate dehydrogenase in breast cancer cells and identifying anterior gradient protein 2 as interacting partner by using a BioID method

Abstract Disclosure: G. Sakalauskaite: None. M. Weingartner: None. T. Bock: None. J. Birk: None. M. Tsachaki: None. A. Odermatt: None. Hexose-6-phosphate dehydrogenase (H6PD) catalyzes the first two steps of the pentose-phosphate pathway in the endoplasmic reticulum (ER). It is so far the only enzyme shown to generate NADPH in the ER. H6PD is involved in physiological processes such as glucose and steroid metabolism, redox regulation, and response to ER-stress. So far, only one H6PD interacting partner, 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1), has been reported. This enzyme uses NADPH for its well-reported role in glucocorticoid activation. However, H6PD has 11β-HSD1-independent functions, such as the recently described modulation of breast cancer cell properties. Here, we adapted the BioID approach to identify novel H6PD interacting partners in the ER. First, we generated a triple negative breast cancer cell line MDA-MB 231 stably expressing a H6PD-biotin ligase fusion protein. Then, we validated the method to confirm the known interaction between H6PD and 11β-HSD1. Next, we analyzed enriched biotinylated proteins by mass spectrometry and further assessed potential candidates by co-immunoprecipitation and functional assays. The resulting interactome revealed a potential hit cluster of protein disulfide isomerase (PDI) family members, different ER-resident chaperones and luminal calcium binding proteins. Due to its association with breast cancer, we further examined the PDI Anterior gradient protein 2 (AGR2) as H6PD interacting partner. Physical interaction between AGR2 and H6PD was confirmed by co-immunoprecipitation assay in MCF7 cells. Downregulation of AGR2 by siRNA resulted in elevated H6PD protein levels but decreased activity. Furthermore, increased H6PD activity was observed when this protein was co-expressed with AGR2 in HEK-293 cells. In conclusion, the results validated the use of the BioID approach to identify protein interactions within the ER and they revealed that AGR2 physically interacts with H6PD, thereby controlling its expression and enzyme activity. Presentation: 6/3/2024