Placental 3B-Hydroxysteroid Dehydrogenase/Isomerase

描述(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，后者参与分娩前的一系列事件。HSD1也选择性地在乳腺和乳腺肿瘤中表达，是脱氢表雄酮(DHEA)产生雌二醇的关键酶。在目前的资助期间，我们确定纯化的3？-HSD1利用底物并结合了一种抑制物(Epostane)，其亲和力比3？-HSD2高14倍，3？-HSD2是人类肾上腺和性腺中表达的同工酶。赠款的第一个目标是利用突变来确定两种异构体中负责催化、底物和辅酶利用的氨基酸的特征，以确定可利用的差异。我们的酶结构同源模型针对执行这些功能的潜在关键残基，并确定了一种突变体，该突变体可以稳定地结合底物，而不需要对结晶学进行催化。由于3？-HSD1和3？-HSD2之间的差异似乎也与亚基的相互作用有关，因此同源二聚体预测的亚基界面上的靶向残基被突变，从而表达、纯化和鉴定了3？-HSD1和3？-HSD2的单体形式，以确定亚基相互作用的作用。以人乳腺肿瘤MCF-7Tet-off细胞为研究对象，用3？-HSD1或3？-HSD2编码载体，进行了动力学研究，以确定膜结合的3？-HSD1能否在不影响3？-HSD2活性的情况下被选择性地抑制。我们的细胞质形式的微粒体3？-HSD 1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，以获得衍射数据和三/四级蛋白质结构。还提出了在我们的模型预测的膜区中含有取代的疏水残基的第二种胞质形式的3？-HSD1，随后产生了一种胞质形式的3？-HSD2酶。在获得3？-HSD1和3？-HSD2的衍射数据后，比较了这两种同工酶在3β-HSD(结合底物)和异构酶(结合NADH)构象下的结构。这些研究可能最终会产生预防早产和治疗激素敏感型乳腺癌的新疗法，同时保持肾上腺类固醇激素的完整。

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.