Placental 3B-Hydroxysteroid Dehydrogenase/Isomerase

描述（由申请人提供）：本研究的目的是确定人1型和2型3 <$-羟基类固醇脱氢酶/异构酶（3 <$-HSD 1和3 <$-HSD 2）的结构/功能关系。在胎盘中，-HSD 1催化双烯醇酮转化为孕酮，并利用胎儿脱氢表雄酮（DHEA）产生雄烯二酮，雄烯二酮进一步代谢为17 <$-雌二醇，雌二醇参与分娩前的级联反应。3 ²-HSA 1也在乳腺和乳腺肿瘤中选择性表达，作为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 <$-HSD 1利用底物并结合抑制剂（epostane），其亲和力比3 <$-HSD 2高14倍，3 <$-HSD 2是人类肾上腺和性腺中表达的同工酶。该补助金的第一个目的是表征两种异构体中负责催化，底物和辅酶利用的氨基酸，使用诱变来确定可利用的差异。我们的同源模型的酶结构有针对性的潜在的关键残基，执行这些功能，并确定了一个突变体，可以稳定地结合底物，而不执行催化结晶。因为亚基的相互作用似乎也涉及3 <$-HSD 1和3 <$-HSD 2之间的差异，所以同源二聚体的预测亚基界面中的靶向残基被突变，使得单体形式的3 <$-HSD 1和3 <$-HSD 2被表达、纯化和表征以确定亚基相互作用的作用。使用我们已经用编码3 <$-HSD 1或3 <$-HSD 2的载体转染的人乳腺肿瘤MCF-7 Tet-Off细胞，进行动力学研究以确定膜结合的3 <$-HSD 1是否可以被选择性抑制而不影响3 <$-HSD 2活性。我们的细胞溶质形式的微粒体3 <$-HSD 1与删除的膜结构域已产生酶晶体，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。还提出了创建第二种胞质形式的3 <$-HSD 1，其包含由我们的模型预测的膜结构域中的取代的疏水残基，随后产生胞质形式的3 <$-HSD 2酶。在获得3 <$-HSD 1和3 <$-HSD 2的衍射数据后，当蛋白质处于3 β-HSD（具有结合底物）和异构酶（具有结合NADH）构象时，比较两种同工酶的结构。这些研究可能最终产生新的治疗方法，用于预防早产和治疗对激素敏感的乳腺癌，同时保持肾上腺类固醇合成完整。

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.