Direct protein-protein interaction of 11beta-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase in the endoplasmic reticulum lumen

• Published in: Biochimica et biophysica acta Vol. 1783; no. 8; pp. 1536 - 1543
• Main Authors: Atanasov, Atanas G, Nashev, Lyubomir G, Gelman, Laurent, Legeza, Balazs, Sack, Ragna, Portmann, Reto, Odermatt, Alex
• Format: Journal Article
• Language: English
• Published: Netherlands 01.08.2008
• Subjects: 11-beta-Hydroxysteroid Dehydrogenase Type 1 - chemistry; 11-beta-Hydroxysteroid Dehydrogenase Type 1 - metabolism; Blotting, Far-Western; Carbohydrate Dehydrogenases - analysis; Carbohydrate Dehydrogenases - isolation & purification; Carbohydrate Dehydrogenases - metabolism; Cell Line; Endoplasmic Reticulum - enzymology; Fluorescence Resonance Energy Transfer; Humans; Immunoprecipitation; NADP - metabolism; Protein Interaction Domains and Motifs
• ISSN: 0006-3002
• DOI: 10.1016/j.bbamcr.2008.03.001

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.