Direct protein-protein interaction of 11beta-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase in the endoplasmic reticulum lumen.
Hexose-6-phosphate dehydrogenase (H6PDH) has been shown to stimulate 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1)-dependent local regeneration of active glucocorticoids. Here, we show that coexpression with H6PDH results in a dramatic shift from 11beta-HSD1 oxidase to reductase activity without affecting the activity of the endoplasmic reticular enzyme 17beta-HSD2. Immunoprecipitation experiments revealed coprecipitation of H6PDH with 11beta-HSD1 but not with the related enzymes 11beta-HSD2 and 17beta-HSD2, suggesting a specific interaction between H6PDH and 11beta-HSD1. The use of the 11beta-HSD1/11beta-HSD2 chimera indicates that the N-terminal 39 residues of 11beta-HSD1 are sufficient for interaction with H6PDH. An important role of the N-terminus was indicated further by the significantly stronger interaction of 11beta-HSD1 mutant Y18-21A with H6PDH compared to wild-type 11beta-HSD1. The protein-protein interaction and the involvement of the N-terminus of 11beta-HSD1 were confirmed by Far-Western blotting. Finally, fluorescence resonance energy transfer (FRET) measurements of HEK-293 cells expressing fluorescently labeled proteins provided evidence for an interaction between 11beta-HSD1 and H6PDH in intact cells. Thus, using three different methods, we provide strong evidence that the functional coupling between 11beta-HSD1 and H6PDH involves a direct physical interaction of the two proteins.
Atanasov
Atanas G
AG
Institute of Pathology, University of Berne, Murtenstrasse 31, 3010 Berne, Switzerland.
Nashev
Lyubomir G
LG
Gelman
Laurent
L
Legeza
Balazs
B
Sack
Ragna
R
Portmann
Reto
R
Odermatt
Alex
A
D043205:11-beta-Hydroxysteroid Dehydrogenase Type 1 / Q000737:chemistry / Q000378:metabolism*; D026481:Blotting, Far-Western; D002237:Carbohydrate Dehydrogenases / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D047468:Immunoprecipitation; D009249:NADP / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs
D009249:NADP; D002237:Carbohydrate Dehydrogenases; C020552:galactose-6-phosphate dehydrogenase; D043205:11-beta-Hydroxysteroid Dehydrogenase Type 1