The objective of this research is to define the structure/function relationships of human type 1 and type 2 3?-hydroxysteroid dehydrogenase/isomerase (3?-HSD 1 and 3?-HSD2). In placenta, 3?-HSD 1 catalyzes the conversion of pregnenolone to progesterone and utilizes fetal dehydroepiandrosterone (DHEA) to produce androstenedione that is further metabolized to 17?-estradiol, which participates in the cascade of events that precede labor. 3?- HSD1 is also selectively expressed in the mammary gland and breast tumors as a key enzyme in the production of estradiol from DHEA. During the current grant period, we determined that purified 3?-HSD1 utilizes substrates and binds an inhibitor (epostane) with 14-fold higher affinity than 3 ?-HSD2, the isoenzyme expressed in human adrenals and gonads.
The first aim of the grant is to characterize the amino acids responsible for catalysis, substrate and coenzyme utilization in two isoforms using mutagenesis to identify exploitable differences. Our homology model of enzyme structure has targeted potentially critical residues that perform these functions and identified a mutant that may stably bind substrate without performing catalysis for crystallography. Because the interaction, of subunits also appears to be involved in the differences between 3?-HSD 1 and 3?-HSD2, targeted residues in the predicted subunit interface of the homodimer are mutated so that monomeric forms of 3 ?-HSD 1 and 3 ?-HSD2 are expressed, purified and characterized to determine the role of subunit interactions. Using human breast tumor MCF-7 Tet-Off cells that we have transfected with vectors encoding either 3 ?-HSD 1 or 3 ?-HSD2, kinetic studies are performed to determine if membrane-bound 3 ?-HSD 1 can be selectively inhibited without affecting 3 ?-HSD2 activity. Our cytosolic form of microsomal 3?-HSD 1 with a deleted membrane-domain has produced enzyme crystals, and the second aim is to produce diffraction-quality crystals to obtain diffraction data and a tertiary/quaternary protein structure. Creation of a second cytosolic form of 3 ?-HSD1 that contains substituted hydrophobic residues in membrane domains predicted by our model is also proposed, followed by production of a cytosolic form of the 3 ?-HSD2 enzyme. After diffraction data are obtained of 3?-HSD 1 and 3 ?-HSD2, the structures of the two isoenzymes are compared when the proteins are in the 3beta-HSD (with bound substrate) and isomerase (with bound NADH) conformations. These studies may ultimately produce new treatments for the prevention of premature birth and the treatment of hormone-sensitive breast cancer while leaving adrenal steroidogenesis intact. ? ?

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA114717-20
Application #
7176164
Study Section
Molecular and Cellular Endocrinology Study Section (MCE)
Program Officer
Knowlton, John R
Project Start
1985-08-01
Project End
2009-01-31
Budget Start
2007-02-01
Budget End
2008-01-31
Support Year
20
Fiscal Year
2007
Total Cost
$219,991
Indirect Cost
Name
Mercer University Macon
Department
Other Basic Sciences
Type
Schools of Medicine
DUNS #
065365041
City
Macon
State
GA
Country
United States
Zip Code
31207
Thomas, James L; Bucholtz, Kevin M; Kacsoh, Balint (2011) Selective inhibition of human 3?-hydroxysteroid dehydrogenase type 1 as a potential treatment for breast cancer. J Steroid Biochem Mol Biol 125:57-65
Thomas, James L; Mack, Vance L; Sun, Jingping et al. (2010) The functions of key residues in the inhibitor, substrate and cofactor sites of human 3beta-hydroxysteroid dehydrogenase type 1 are validated by mutagenesis. J Steroid Biochem Mol Biol 120:192-9
Thomas, James L; Bucholtz, Kevin M; Sun, Jingping et al. (2009) Structural basis for the selective inhibition of human 3beta-hydroxysteroid dehydrogenase 1 in human breast tumor MCF-7 cells. Mol Cell Endocrinol 301:174-82
Thomas, James L; Mack, Vance L; Glow, Jason A et al. (2008) Structure/function of the inhibition of human 3beta-hydroxysteroid dehydrogenase type 1 and type 2 by trilostane. J Steroid Biochem Mol Biol 111:66-73
Thomas, James L; Huether, Robert; Mack, Vance L et al. (2007) Structure/function of human type 1 3beta-hydroxysteroid dehydrogenase: An intrasubunit disulfide bond in the Rossmann-fold domain and a Cys residue in the active site are critical for substrate and coenzyme utilization. J Steroid Biochem Mol Biol 107:80-7
Pletnev, Vladimir Z; Thomas, James L; Rhaney, Felicia L et al. (2006) Rational proteomics V: structure-based mutagenesis has revealed key residues responsible for substrate recognition and catalysis by the dehydrogenase and isomerase activities in human 3beta-hydroxysteroid dehydrogenase/isomerase type 1. J Steroid Biochem Mol Biol 101:50-60
Duax, William L; Thomas, James; Pletnev, Vladimir et al. (2005) Determining structure and function of steroid dehydrogenase enzymes by sequence analysis, homology modeling, and rational mutational analysis. Ann N Y Acad Sci 1061:135-48
Thomas, James L; Boswell, Elizabeth L; Scaccia, Launa A et al. (2005) Identification of key amino acids responsible for the substantially higher affinities of human type 1 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD1) for substrates, coenzymes, and inhibitors relative to human 3beta-HSD2. J Biol Chem 280:21321-8
Thomas, James L; Umland, Timothy C; Scaccia, Launa A et al. (2004) The higher affinity of human type 1 3beta-hydroxysteroid dehydrogenase (3beta-HSD1) for substrate and inhibitor steroids relative to human 3beta-HSD2 is validated in MCF-7 tumor cells and related to subunit interactions. Endocr Res 30:935-41