Placental 3B-Hydroxysteroid Dehydrogenase/Isomerase

描述（由申请人提供）：本研究的目的是确定人类1型和2型3¿-羟基类固醇脱氢酶/异构酶（3¿- hsd1和3¿-HSD2）的结构/功能关系。在胎盘中，3¿-HSD 1催化孕烯醇酮转化为孕酮，并利用胎儿脱氢表雄酮（DHEA）产生雄烯二酮，雄烯二酮进一步代谢为17¿-雌二醇，参与分娩前的级联反应。3¿- HSD1也在乳腺和乳腺肿瘤中选择性表达，作为脱氢表雄酮生成雌二醇的关键酶。在目前的资助期间，我们确定纯化的3¿-HSD1利用底物并结合一种抑制剂（epostane），其亲和力比3¿-HSD2高14倍，3¿-HSD2在人类肾上腺和性腺中表达。该基金的第一个目的是利用诱变技术鉴定两种同工异构体中负责催化、底物和辅酶利用的氨基酸，以确定可利用的差异。我们的酶结构同源性模型针对执行这些功能的潜在关键残基，并确定了一个突变体，该突变体可以稳定地结合底物，而无需进行晶体学催化。由于亚基之间的相互作用似乎也与3¿- hsd1和3¿-HSD2之间的差异有关，因此同源二聚体预测亚基界面中的靶向残基发生突变，从而表达、纯化和表征3¿- hsd1和3¿-HSD2的单体形式，以确定亚基相互作用的作用。使用我们用编码3¿- hsd1或3¿-HSD2的载体转染的人乳腺肿瘤MCF-7 et- off细胞，进行动力学研究以确定膜结合的3¿- hsd1是否可以选择性地抑制而不影响3¿-HSD2的活性。我们的微粒体3¿-HSD 1的细胞质形式具有缺失的膜结构域，已经产生了酶晶体，第二个目标是产生衍射质量的晶体，以获得衍射数据和三级/四级蛋白质结构。在我们的模型预测的膜结构域中含有取代的疏水残基的3¿-HSD1的第二种胞质形式的创造也被提出，然后是3¿-HSD2酶的胞质形式的生产。在获得3¿- hsd1和3¿-HSD2的衍射数据后，比较了两种同工酶在3 β -HSD（结合底物）和异构酶（结合NADH）构象下的结构。这些研究可能最终产生预防早产和治疗激素敏感性乳腺癌的新疗法，同时保持肾上腺甾体生成完整。

DESCRIPTION (provided by applicant): 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.