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The microsomal enzyme 17β-hydroxysteroid dehydrogenase 3 faces the cytoplasm and uses NADPH generated by glucose-6-phosphate dehydrogenase

Legeza, Balázs and Balázs, Zoltán and Nashev, Lyubomir G. and Odermatt, Alex. (2013) The microsomal enzyme 17β-hydroxysteroid dehydrogenase 3 faces the cytoplasm and uses NADPH generated by glucose-6-phosphate dehydrogenase. Endocrinology, Vol. 154, H. 1. pp. 205-213.

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Official URL: http://edoc.unibas.ch/dok/A6070435

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Abstract

Recent studies proposed a functional coupling between 17β-hydroxysteroid dehydrogenase 3 (17β-HSD3)-dependent testosterone formation and 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1)-mediated interconversion of glucocorticoids through competition for the luminal pyridine nucleotide pool. To test this hypothesis, we used human embryonic kidney-293 cells transfected with 17β-HSD3 and/or 11β-HSD1, in the absence or presence of hexose-6-phosphate dehydrogenase that generates reduced nicotinamide adenine dinucleotide phosphate (NADPH) in the endoplasmic reticulum and determined enzyme activities. As an endogenous cell model, mouse MA-10 Leydig cells were used. 17β-HSD3-dependent reduction of Δ4-androstene-3,17-dione was affected by neither coexpression with 11β-HSD1 nor overexpression or knockdown of hexose-6-phosphate dehydrogenase. In contrast, knockdown of glucose-6-phosphate dehydrogenase decreased 17β-HSD3 activity, indicating dependence on cytoplasmic NADPH. Upon selective permeabilization of the plasma membrane by digitonin, 17β-HSD3 but not 11β-HSD1 was detected by antibodies against C-terminal epitope tags, suggesting a cytoplasmic orientation of 17β-HSD3. The cytoplasmic orientation was confirmed using proteinase K digestion of microsomal preparations and by analysis of glycosylation of wild-type 17β-HSD3 and chimera in which the N-terminal anchor sequences between 17β-HSD3 and 11β-HSD1 were exchanged. In conclusion, the results demonstrate a cytoplasmic orientation of 17β-HSD3 and dependence on glucose-6-phosphate dehydrogenase-generated NADPH, explaining the lack of a direct functional coupling with the luminal 11β-HSD1-mediated glucocorticoid metabolism.
Faculties and Departments:05 Faculty of Science > Departement Pharmazeutische Wissenschaften > Pharmazie > Molecular and Systems Toxicology (Odermatt)
UniBasel Contributors:Odermatt, Alex and Legeza, Balazs and Balazs, Zoltan and Nashev, Lyubomir Georgiev
Item Type:Article, refereed
Article Subtype:Research Article
Bibsysno:Link to catalogue
Publisher:Thomas
ISSN:0013-7227
Note:Publication type according to Uni Basel Research Database: Journal article
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Last Modified:24 May 2013 09:21
Deposited On:24 May 2013 08:58

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