title: H6PDH interacts directly with 11beta-HSD1: implications for determining the directionality of glucocorticoid catalysis.
abstract: Tissue specific amplification of glucocorticoid action through NADPH-dependent reduction of inactive glucocorticoid precursors by 11beta-hydroxysteroid dehydrogenase (11beta-HSD1) contributes to the development of visceral obesity, insulin resistance and Type 2 Diabetes. Hexose-6-phosphate dehydrogenase (H6PDH) is believed to supply NADPH for the reductase activity of 11beta-HSD1 in the lumen of the endoplasmic reticulum (ER), where the two enzymes are co-localized. We report here expression and purification of full-length and truncated N-terminal domain (NTD) of H6PDH in a mammalian expression system. Interestingly, both full-length H6PDH and the truncated NTD are secreted into the culture medium in the absence of 11beta-HSD1. Purified full-length H6PDH is a bi-functional enzyme with glucose-6-phosphate dehydrogenase (G6PDH) activity as well as 6-phosphogluconolactonase (6PGL) activity. Using co-immunoprecipitation experiments with purified H6PDH and 11beta-HSD1, and with cell lysates expressing H6PDH and 11beta-HSD1, we observe direct physical interaction between the two enzymes. We also show the modulation of 11beta-HSD1 directionality by H6PDH using overexpression and siRNA knockdown systems. The NTD retains the ability to interact with 11beta-HSD1 physically as well as modulate 11beta-HSD1 directionality indicating that the NTD of H6PDH is sufficient for the regulation of the 11beta-HSD1 activity.
authors.affiliation: Cardiovascular and Metabolic Diseases, Wyeth Research, Cambridge, MA 02140, USA.
authors.forename: Yan-ling
authors.initials: YL
authors.lastname: Zhang
authors.forename: Xiaotian
authors.initials: X
authors.lastname: Zhong
authors.forename: Zheni
authors.initials: Z
authors.lastname: Gjoka
authors.forename: Yuanhong
authors.initials: Y
authors.lastname: Li
authors.forename: Wayne
authors.initials: W
authors.lastname: Stochaj
authors.forename: Mark
authors.initials: M
authors.lastname: Stahl
authors.forename: Ron
authors.initials: R
authors.lastname: Kriz
authors.forename: James F
authors.initials: JF
authors.lastname: Tobin
authors.forename: David
authors.initials: D
authors.lastname: Erbe
authors.forename: Vipin
authors.initials: V
authors.lastname: Suri
mesh_terms: D043205:11-beta-Hydroxysteroid Dehydrogenase Type 1 / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002237:Carbohydrate Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D005938:Glucocorticoids / Q000378:metabolism*; D005942:Gluconates / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection
chemical_list: C114004:6-phosphogluconolactone; D017931:DNA Primers; D005938:Glucocorticoids; D005942:Gluconates; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D002237:Carbohydrate Dehydrogenases; C020552:galactose-6-phosphate dehydrogenase; D043205:11-beta-Hydroxysteroid Dehydrogenase Type 1