Influence of Cellular Localization on Activity of Hydroxysteroid Dehydrogenases

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Title: Influence of Cellular Localization on Activity of Hydroxysteroid Dehydrogenases
Author: Wooding, Kerry Michael
Abstract: Hydroxysteroid dehydrogenases (HSDs ) catalyze the interconversion of inactive steroids and active hormones . HSDs use nicotinamide cofactors in the cytosol and endoplasmic reticulum (ER ) lumen to either reduce or oxidize their steroid substrates . Our lab has extensively studied the 17beta -HSDs types 1 , 2 and 3 of the short -chain oxidoreductase family , particularly human 17beta -HSD1 , which favors estrone reduction to estradiol . Rat AKR1C9 has also been thoroughly studied as a model HSD of the aldo -keto reductase family ; AKR1C9 catalyzes the reduction of dihydrotestosterone to androstanediol . These two enzymes provide a basis for comparative studies with 11beta -HSD1 , which catalyzes the reduction of cortisone to cortisol . Most mammalian cells supplied with adequate glucose and oxygen maintain cytoplasmic high nicotinamide concentration gradients , [NADPH] > > [NADP+] and [NAD+] > > [NADH] , and in the strongly oxidizing environment of the ER lumen , both these gradients are shifted to more oxidized cofactor . Whereas 17betaHSD types 1 , 2 , 3 and AKR1C9 catalyze their respective reactions in a thermodynamically predictable manner based on cofactor gradients , 11beta -HSD1 does the opposite . 17beta -HSD1 , 17beta -HSD3 , and AKR1C9 favor reduction in the cytosol using NADPH , and 17beta -HSD2 favors oxidation in the ER lumen using NAD+ . In contrast , 11beta -HSD1 reduces cortisone to cortisol in the highly oxidative ER lumen but requires hexose -6 -phosphate -dehydrogenase (H6PD ) to regenerate NADPH in the ER lumen . We hypothesize that H6PD directly channels NADPH to 11beta -HSD1 through specific interactions . To test this hypothesis , we have targeted 17beta -HSD1 and AKR1C9 to the ER lumen rather than the cytosol . Conversely , we have targeted 11beta -HSD1 to the cytoplasmic surface of the ER . In addition , we have engineered point mutations in 17beta -HSD1 , AKR1C9 , 11beta -HSD1 and H6PD , designed to attenuate the directional preferences by altering cofactor binding . Targeting and retaining 17beta -HSD1 in the ER lumen proved troublesome ; regardless of the transfection conditions , ER -targeted 17beta -HSD1 was always detected in the cytosol . We conclude that either 17beta -HSD1’s activity or structure causes its translocation to the cytosol .
URI: http : / /hdl .handle .net /2152 .5 /955
Date: 2011-12-14

Citation

Influence of Cellular Localization on Activity of Hydroxysteroid Dehydrogenases. Graduate School of Biomedical Sciences. Available electronically from http : / /hdl .handle .net /2152 .5 /955 .

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