Project

Discipline

inhibitor; redox; 11beta-hydroxysteroid dehydrogenase; endoplasmic reticulum; glucose-6-phosphate; pentose-phosphate pathway; NADPH; bile acid

Lead
Lay summary
The reduced pyridine nucleotide NADPH acts as cofactor for a multitude of reduction reactions and is essential for major physiological processes. NADPH cannot freely move across cellular membranes, and distinct pools exist in the cytoplasm, the mitochondria and the endoplasmic reticulum (ER). In contrast to the cytoplasm, much less is known on NADPH generation and utilization in the ER. To date, only one enzyme has been identified in the ER that generates NADPH. It uses glucose-6-phosphate as substrate and its activity is dependent on the cellular energy state. The reactions within the ER that utilize NADPH are not well studied. To date only one enzyme within the ER is known to utilize NADPH. It interconverts inactive and active glucocorticoids and therefore represents a hormonal response mechanism to alterations in energy status of cells that express this enzyme. There are, however, several important questions that need to be addressed: 1) what is the role of the NADPH generating enzyme in cells that do not express the glucocorticoid activating enzyme? 2) which other enzymes require NADPH in the ER for their function? 3) how can NADPH be generated in the absence of the glucose-6-phosphate dependent enzyme, are there other mechanisms to generate NADPH in the ER? 4) How do pyridine nucleotides reach the ER and how is the pool in the ER initially formed? 5) does the product of the NADPH generating enzyme in the ER react further, i.e. is there a pentose-phosphate cycle in the ER? 6) what are the consequences of impaired redox control and NADPH homeostasis in the ER for metabolic functions? By employing biochemical and cell biological methods we attempt to identify additional enzymes within the ER that are dependent on NADPH. Upon identification, expression plasmids will be constructed, followed by expression of proteins in cells, functional characterization and investigation of their physiological roles. Moreover, we will characterize the role of the luminal glucocorticoid activating enzyme in the metabolism of other substrates, including bile acids, oxysterols and xenobiotics, with respect to NADPH dependence.   The proposed research should significantly enhance the current knowledge on NADPH generation and utilization in the ER. The expected findings are relevant regarding the understanding of the coupling between cellular energy state, hormonal regulation, ER redox regulation, and oxidative stress-induced damage. Disturbed functions of the enzymes investigated are likely to be associated with metabolic diseases.

Direct link to Lay Summary

Last update: 21.02.2013

Active participation

Self-organised

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Purpose and aim of the projectThe reduced pyridine nucleotide NADPH acts as cofactor for a multitude of essential reduction reactions. The enzymes involved in NADPH synthesis and regeneration, and the metabolic reactions utilizing NADPH in the cytoplasm, have been extensively investigated. In contrast, much less is known on NADPH generation and utilization in the endoplasmic reticulum (ER). The recent recognition of the role of hexose-6-phosphate dehydrogenase (H6PDH) in generating luminal NADPH provided an explanation for the maintenance of the NADP+/NADPH pool in the ER. Biochemical evidence further indicated the existence of all enzymes of the pentose-phosphate pathway in the ER. However, so far only H6PDH has been characterized at the molecular level, and further research is required to elucidate this pathway in the ER. Recent cell-based experiments and studies with transgenic mice lacking H6PDH demonstrated the importance of luminal NADPH for glucocorticoid activation. Moreover, H6PDH-deficient mice suffered from myopathy and activation of the unfolded-protein response (UPR), whereby glucocorticoid-independent impairment of ER redox regulation was suggested as underlying mechanism. H6PDH activity depends on glucose-6-phosphate (G6P) availability. G6P can be provided from the cytoplasm by the G6P-transporter (G6PT) or, alternatively, by a luminal isomerase converting fructose-6-phosphate (F6P) to G6P that we characterized recently. Nevertheless, the gene encoding this enzyme remains to be identified. We have previously shown that H6PDH determines the reaction direction of 11beta-hydroxysteroid dehydrogenase 1 (11b-HSD1) to catalyze the reduction of inactive 11-keto- to active 11b-hydroxyglucocorticoids. Also, we found that 11b-HSD1 metabolizes several other substrates, including oxysterols, bile acids and carbonyl containing xenobiotics. The physiological relevance of these additional functions is poorly understood and requires further investigation.So far, 11b-HSD1 is the only NADPH-dependent enzyme in the ER that has been thoroughly characterized. Evidence from H6PDH-deficient transgenic mice and cell-based experiments emphasizes the existence of other NADPH-dependent luminal enzymes. Therefore, we aim to further characterize the mechanisms of NADPH generation in the ER and to search for enzymes that utilize NADPH in this compartment. We propose to:I.elucidate the mechanisms involved in the regulation of NADPH generation in the ER by:a) an attempt to identify the gene for the luminal hexose-6-phosphate isomerase;b) characterization of the luminal 6-phosphogluconate dehydrogenase (6PGDH) activity;c) trying to identify enzymes other than H6PDH that generate NADPH in the ER; andII.characterize NADPH-dependent reactions in the ER by:a) determination of the impact of 11b-HSD1 on bile acid metabolism and profiles;b) determination of 17b-HSD3 membrane topology and functional impact of H6PDH;c) performing a search for substrate(s) of 11b-HSD3 and elucidating its function; andd) trying to identify additional ER luminal reductases that utilize NADPH.For the identification of luminal enzymes we will use rat liver microsomes and apply an activity-guided purification strategy. A combination of fractionation, affinity purification, gel-electrophoretic separation and identification by mass spectrometry will be applied. Upon identification, expression plasmids will be constructed, followed by functional characterization. The proposed research should significantly enhance the current knowledge on NADPH generation and utilization in the ER. The expected findings are relevant regarding the understanding of the coupling between cellular energy state, hormonal regulation, ER redox regulation, and oxidative stress-induced damage. Disturbed functions of the enzymes investigated are likely to be associated with metabolic disturbances.