Generation and utilization of NADPH in the endoplasmic reticulum : novel insight into the role of luminal NADPH in pathophysiological processes

Increasing evidence emphasizes the importance of the redox balance in the endoplasmic reticulum
(ER). Disturbance of redox regulation can cause ER stress and contribute to the development of
metabolic disease, cancer and neurodegenerative disorders. Nevertheless, the mechanisms underlying
the well-regulated NADPH balance, and the generation and utilization of pyridine nucleotides in the
luminal compartment are insufficiently understood. The aim of this work is to identify novel
components involved in NADPH regulation in the ER.
Due to the observation that fructose-6-phosphate stimulates luminal NADPH generation, and enhances
11ß-hydroxysteroid dehydrogenase 1 dependent glucocorticoid activation, we hypothesized the
existence of a luminal hexose-6-phosphate isomerase. Using microsomal fractions, we characterized a
novel luminal hexose-6-phosphate isomerase, which converts fructose-6-phosphate to
glucose-6-phosphate. By further purification and protein sequencing, we try to identify the
gene encoding this enzyme.
In order to identify additional genes encoding luminal enzymes involved in NAPDH
generation in the ER (potential enzymes of the luminal pentose-phosphate pathway), we decided to
apply a combination of classical activity-guided purification, mass-spectrometric analysis and
sequence analysis. Furthermore, for promising candidate proteins, we attempt to confirm their
intracellular localization and investigate their impact on luminal NADPH balance. To determine
whether ER-associated and membrane proteins are facing the cytoplasmic or luminal compartment, we
optimized the methods to determine membrane topology and intracellular localization. We used
selective semi-permeabilization analysis using digitonin, followed by immunodetection and confocal
microscopy, proteinase protection assays of microsomal preparations as well as glycosylation
assays.
Furthermore, we determined the membrane topology of 17ß-hydroxysteroid dehydrogenase
3, an enzyme responsible for the oxoreduction of androstenedione. We provide information on the
functional impact of hexose-6-phosphate dehydrogenase, as well as the nutritional state of the cell
on the formation of testosterone.
The findings are relevant regarding the understanding of the coupling between the cellular energy
state, hormonal regulation, ER redox regulation and oxidative stress-induced damage
in a cell.