| Project Detail |
The endoplasmic reticulum (ER) regulates energy metabolism, protein folding and redox reactions. Unfolded-protein response (UPR) and ER-stress due to impaired ER function have been associated with various diseases, and it is crucial to uncover the mechanisms controlling ER functions. The NAD(P)H/NAD(P)+ redox couple is essential for many biological functions but its regulation in the ER and the relevance of luminal NADPH for physiological functions like UPR control, calcium signaling, and metabolism of carbohydrates, lipids, steroids, and xenobiotics are insufficiently understood.Hexose-6-phosphate dehydrogenase (H6PD) catalyzes the first two steps of the pentose-phosphate pathway (PPP) in the ER and generates NADPH. It constitutes a link between cellular energy status and hormonal response by supplying NADPH for 11b-hydroxysteroid dehydrogenase 1 (11b-HSD1)-dependent glucocorticoid activation. Other luminal enzymes using NADPH and PPP enzymes downstream of H6PD are poorly characterized. Importantly, the effects seen in breast cancer cells after H6PD silencing and the skeletal myopathy of H6PD knockout (KO) mice are 11b-HSD1-independent, and the underlying mechanisms remain to be elucidated. Based on our results, we hypothesize that 1) H6PD plays a role in the metabolic reprogramming of cancer cells, promoting resistance to hypoxia, acidic tumor microenvironment and chemotherapeutics, 2) H6PD inhibition suppresses tumor promoting properties of breast cancer cells, 3) H6PD loss impairs carbohydrate metabolism and ER-mitochondrial cross-talk, disturbing mitochondrial function, calcium signaling and UPR activation, ultimately causing skeletal myopathy in mice, 4) H6PD plays a role in hepatic metabolic adaptation by impacting carbohydrate and lipid metabolism and acting as a metabolic sensor to mediate hormonal responses, and 5) gastric surgery reverses impaired hepatic carbohydrate metabolism and dysregulated corticosteroid action due to altered H6PD and 11b-HSD1 activity. Therefore, we propose to: •study the impact of H6PD on breast cancer cell properties and resistance to chemotherapeutics,•purify human H6PD and attempt to solve its structure,•establish an optimized H6PD activity assay, evaluate inhibitors, and test them in cancer cells,•study the molecular mechanisms underlying H6PD loss-induced skeletal myopathy in mice,•assess whether nicotinamide riboside (NR) reverses effects of H6PD loss,•investigate mechanisms underlying the metabolic changes in liver of H6PD-KO mice,•study the impact of gastric surgery on H6PD/11b-HSD1 and on corticosteroid action in mice and humans.The impact of H6PD and luminal NADPH on metabolic pathways, hormonal responses and redox regulation will be studied in cell-based models by lentiviral overexpression, CRISPR/Cas9 KO, downregulation by siRNA or pharmacological inhibition. H6PD protein purification and solving its structure should help to elucidate the mechanism of interaction with other proteins and the design of potent inhibitors. Additionally, H6PD-KO mice will be employed to study mechanisms of skeletal myopathy and hepatic metabolic changes. The impact of gastric surgery on H6PD/11b-HSD1 and on corticosteroid action will be assessed by analyses of samples from mice and clinical studies before/after surgery and with/without therapeutic interventions. The proposed research should significantly enhance our knowledge on H6PD, the ER PPP and luminal NADPH, potentially leading to the identification of novel functions essential for metabolic and redox regulation. The expected findings are relevant to understand the coupling between cellular energy state, hormonal regulation, ER redox regulation, and oxidative stress-induced damage. Moreover, this work provides a basis for the design of potential therapeutic H6PD inhibitors, and it should unravel the contribution of H6PD/11b-HSD1 to the beneficial metabolic effects of gastric surgery. |
