Towards the Understanding of the Molecular and Physiological Role of Hexose-6-Phosphate Dehydrogenase

The presented thesis is split into four major parts, aiming to improve the comprehension of luminal processes in mammalian cells. The main focus was the examination of the physiological and molecular role of the only known source of NADPH in the ER, hexose-6-phosphate dehydrogenase (H6PD). NADPH,generated by luminal H6PD, is needed as cofactor for reduction reactions such as the interconversion of inactive glucocorticoid cortisone to active cortisol by luminal 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). However, besides its interaction with 11β-HSD1 and its role as a NADPH generating enzyme, little is known about H6PD. Therefore, the first project of this thesis was aimed at the elucidation of the interactome of luminal H6PD. We applied the BioID approach, a recently introduced method to screen for protein-protein interactions (PPI) by proximity biotinylation. We were able to demonstrate the functionality of this new tool for luminal applications and to reveal potential novel interactors of H6PD. Anterior gradient 2 protein homolog (AGR2), a member of the protein disulfide isomerase (PDI) family, seemed to directly interact with H6PD in MCF7 cells. Our results further indicated that AGR2 has an impact on H6PD activity and H6PD protein expression, which directly affects the luminal pool of reduced phosphorylated pyridine nucleotides.
The second project was dedicated to develop a better understanding of ER stress (ERS) in the context of parasitic infections. Since the ER represents the major site of synthesis and folding of proteins in eukaryotic cells, it is essential that the luminal folding machinery and protein transport systems, which facilitate crossing the ER membrane, function efficiently. Disturbances regarding folding of polypeptides can lead to an accumulation of misfolded proteins in the ER lumen and therefore induce ERS and, as consequence, the activation of the unfolded protein response (UPR). Various diseases have been shown to be associated with ERS, and therefore the potential to identify druggable targets of UPR-related process would be of interest to the pharmaceutical industry. In the second project, we examined whether an infection by the fox tapeworm Echinococcus multilocularis causes ERS in mice. We could demonstrate that the expression levels of several proteins of the activating transcription factor 6 alpha(ATF6) branch of the UPR were significantly upregulated upon E. multilocularis infection in mice compared to non-infected animals. Protein expression levels of inositol-requiring protein 1 alpha (IRE1α), a major sensor of ERS and regulator of the UPR, and activating transcription factor-4 (ATF4)were found to be significantly decreased in mice upon infection compared to the non-infected group. In contrast, increased protein expression levels of H6PD and ER resident chaperone calreticulin (CRT) were detected after E. multilocularis infection. Furthermore, we demonstrate that treatment of infected animals using the anthelmintic drug albendazole (ABZ) (partially) restored protein expression levels back to the baseline of noninfected
animals.
In the third project, we focused on alterations of bile acid (BA) profiles upon E.multilocularis infection in mice. Since selected BA and their corresponding ratios are considered as potential biomarkers for several diseases, we were interested whether parasitic infection, caused by E. multilocularis, alters the BA concentrations in infected animals. In the third project of the presented thesis, we showed that infected animals exhibited significantly lower unconjugated primary and secondary BA levels in serum compared to non-infected animals. Moreover, taurocholic acid (TCA) and tauro-β-muricholic acid (TβMCA), two taurine-conjugated BA were found at significantly higher serum concentrations when compared to the non-infected group. Regarding the expression of hepatic BA transporters, we found decreased mRNA and protein expression levels for the bile salt export pump (BSEP) and Na+-taurocholate co-transporting polypeptide (NTCP) upon E. multilocularis infection, which could be a result of inflammation due to the parasite proliferation in the liver. Finally, restored BA and protein expression levels were observed following ABZ treatment of infected animals, showing the effectiveness of this drug.
The fourth project aimed to identify novel biomarker(s) to assess 11β-HSD1 activity. By the examination of different BA (-ratios) we found that the ratio of ursodeoxycholyltaurine (UDC-Tau) to 7-oxolithocholyltaurine (7oxo-Tau) represents a suitable biomarker to detect decreased 11β-HSD1 activity. The applicability of the presented biomarker was confirmed in four independent mouse models and the application of pharmacological 11β-HSD1 inhibitor carbenoxolone (CBX). Through the projects described in this thesis we improved the understanding of ER-related processes by different aspects. The study of the interactome of H6PD revealed novel insights in in the maintenance of luminal NADPH-generation. Further, ERS upon E. multilocularis infection and the potential usability of BA profiles to detect this parasitic infection were examined. Finally, we presented a suitable biomarker to assess decreased enzymatic activity of luminal 11β-HSD1.