Hexose-6-phosphate dehydrogenase: novel interactors and role in lipid metabolism

Hexose-6-phosphate dehydrogenase (H6PD) is the endoplasmic reticulum (ER) luminal counterpart of the cytosolic glucose-6-phosphate dehydrogenase (G6PD) and it catalyzes the first two steps of the pentose-phosphate pathway (PPP) in the ER, generating nicotinamide adenine dinucleotide phosphate (NADPH) in this process (Beutler & Morrison, 1967; Bublitz, 1981; Takahashi & Hori, 1978; Watanabe, 2017). To date, the only well-described interactor of H6PD is 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1), which utilizes luminal NADPH during the activation of glucocorticoids: cortisol and corticosterone in humans and rodents, respectively (Gathercole et al., 2013; Mziaut et al., 1999; Odermatt et al., 1999; Odermatt & Kratschmar, 2012). Increasing evidence suggests that H6PD plays a role in lipid metabolism. Lipolysis is impaired in adipose tissue and adipocyte-specific H6PD knock-out (KO) (Bujalska et al., 2008; Wang et al., 2019). It has also been demonstrated that upon fasting, no changes occur in the mobilization of fat and serum-free fatty acid (FA; pl. FAs) levels in H6PD KO mice, indicating the contribution of H6PD to lipolysis in adipose tissue (Bujalska et al., 2008). Therefore, H6PD appears to act as a bridge between the production of glucocorticoids and the metabolism of carbohydrates and lipids. However, despite increasing evidence of the function of H6PD in lipid metabolism of adipose tissue, the role of H6PD in the liver, which is a central hub for lipid metabolism, is poorly understood, and the exact mechanisms and pathways in which this protein participates remain to be discovered.
The first part of the thesis consists of a comprehensive review of H6PD, which discusses the role of this protein in health and disease. This review summarizes the molecular and biochemical properties of H6PD. Moreover, it provides a broad overview of the known functions of H6PD and its involvement in pathological processes. Particularly, the current knowledge regarding H6PD involvement in the luminal PPP and glucocorticoid activation is discussed. Moreover, H6PD role in skeletal muscle myopathy, metabolic disorders and cancer pathogenesis is reviewed in detail, presenting all available studies.
The second part of this thesis aims to elucidate novel interactors of H6PD in the ER lumen. To accomplish this aim, we took advantage of the BioID approach, which is a cutting-edge technique for identifying protein-protein interactions through proximity-based biotinylation. By employing BioID we identified 50 potential H6PD interactors. Among the identified H6PD interactome, protein disulfide isomerase (PDI) family member anterior gradient protein 2 (AGR2) was found. AGR2, as well as H6PD, has been previously indicated to contribute to breast cancer progression. Therefore, we validated the interaction by co-immunoprecipitation. Additionally, gain of function and loss of function studies provided evidence that AGR2 regulates H6PD activity. Finally, mRNA expression analysis of AGR2 and H6PD using the Cancer Genome Atlas (TCGA) database was performed. Analysis of H6PD expression in breast cancer tumors compared to the normal tissue indicated that H6PD expression is lower in malignant tissue. Further analysis provided evidence that H6PD expression positively correlates with the survival of triple-negative patients. Additionally, AGR2 expression in estrogen receptor-expressing tumors correlated with better survival. Overall, this study confirmed the applicability and reliability of the BioID approach in the ER compartment and helped to identify novel H6PD interactors.
In the final part of this thesis, we aimed to understand the role of H6PD in hepatic lipid metabolism. Increasing evidence suggests H6PD function in lipid metabolism of adipose tissue. However, the role of H6PD in the liver, which is a central hub for lipid metabolism, is poorly understood, and the exact mechanisms and pathways in which this protein participates remain to be discovered. Therefore, we aimed to understand the function of H6PD in lipid metabolism pathways in the liver. First, we employed lipidomics approach to compare lipid profiles of wild type (WT) and H6PD KO mouse liver tissue. Lipidomics analysis indicated increased accumulation of long chain, low saturation triacylglycerol (TG) when H6PD is lacking. Next, we validated increased TG accumulation in H6PD KO mouse liver tissue sections and AML12 mouse hepatocytes. To reveal lipid metabolism pathways that are affected by H6PD KO, we employed proteomics-based pathway analysis of WT and H6PD KO mice liver tissue. Pathway analysis revealed that H6PD KO influences multiple aspects of lipid metabolism. Subsequently, based on multi-omics findings, we formulated a hypothesis and experimentally investigated changes that could lead to increased accumulation of TG in H6PD KO liver tissue and cells. We observed that H6PD KO AML12 cells shift from glucose to FA dependency, accompanied by increased extracellular uptake of FAs and increased palmitic acid PA-induced cytotoxicity. Our analysis revealed a downregulation of key enzymes involved in mitochondrial FA β-oxidation (mtFAO), including carnitine palmitoyltransferase 1 A and B (CPT1A; CPT1B), and carnitine palmitoyltransferase 2 (CPT2) in both H6PD KO liver and AML12 cells. A notable decrease in mtFAO in H6PD KO AML12 cells was observed. Furthermore, we demonstrated that CPT1 downregulation and the subsequent decrease in mtFAO occurs through the adenosine monophosphate (AMP)-activated protein kinase alpha subunit (AMPKα) and acetyl-CoA carboxylase 1 (ACC1) axis. Therefore, apart from playing a critical role in glucocorticoid production, H6PD is also involved in lipid homeostasis, regulating FA import and metabolic processes.
In summary, the studies described in this thesis provided valuable insights regarding the H6PD interactome and the involvement of this protein in lipid metabolism pathways. The combination of complex techniques, such as BioID, lipidomics, and proteomics analysis combined with functional assays, were useful in achieving the goals of this thesis. However, many aspects regarding H6PD interactions and functional roles still remain to be answered.