SHORT CHAIN DEHYDROGENASES IN RETINOL/STEROL METABOLISM

类维甲酸和类固醇激素调节从形态发生到繁殖的各种生理过程。这两种激素的生物合成都涉及到将其相应的前体氧化和还原为生物活性形式。来自该实验室和其他实验室的最新数据表明，哺乳动物组织中含有一组相关酶，能够在体外利用类视黄醇和类固醇醇及醛作为底物。这些酶具有超过40%的序列同一性，属于短链脱氢酶/还原酶超家族。本研究的主要假设是：(1)这组结构相关的酶在体内参与类视黄酮和类固醇激素的生物合成；(2)个体视黄醇/甾醇脱氢酶在人体组织中表现出不同的组织特异性表达模式，这决定了它们对每种代谢途径的贡献；(3)这些微粒体脱氢酶的活性位点面向细胞质，其中依赖于NAD+的酶在氧化方向起作用，而依赖于NADP+的酶在还原方向起作用。共同酶的存在为类维生素a和类固醇信号通路的联合调控提供了手段。这一假设与维甲酸显著降低血清双氢睾酮水平和维甲酸水平在前列腺癌组织中降低的观察结果一致。本提案的总体目标是确定人类微粒体视黄醇/甾醇脱氢酶的作用，该实验室最近发现，在细胞中的类视黄醇和类固醇代谢。本提案的具体目标是：(1)开发一种制备具有催化活性的纯化重组视黄醇/甾醇脱氢酶的方法；(2)表征人视黄醇/甾醇脱氢酶的底物特异性和催化性能；(3)测定人体组织中视黄醇/甾醇脱氢酶的表达规律；4)确定人脱氢酶在微粒体膜上跨膜插入的拓扑结构。这些目标的实现将使我们了解这组新的短链脱氢酶在健康和疾病中的类视黄醇和类固醇代谢中的作用。

Retinoid and steroid hormones regulate a variety of physiological processes from morphogenesis to reproduction. Biosynthesis of both types of hormones involves oxidation and reduction of their corresponding precursors to the biologically active forms. Recent data from this and other laboratories suggest that mammalian tissues contain a group of elated enzymes capable of utilizing in vitro both retinoid and steroid alcohols and aldehydes as substrates. These enzymes share more than 40 percent sequence identity and belong to the superfamily of short-chain dehydrogenases/reductases. The main hypotheses of this proposal are that: (1) this group of structurally related enzymes is involved in biosynthesis of both retinoid and steroid hormones in vivo; (2) individual retinol/sterol dehydrogenases exhibit different tissue-specific expression patterns in human tissues, which determines their contribution to each metabolic pathway; and (3) the active site of these microsomal dehydrogenases faces the cytosol, where the NAD+-dependent enzymes function in the oxidative direction, and the NADP+-dependent enzymes function in the reductive direction. The existence of common enzymes should provide the means for joint regulation of retinoid and steroid signaling pathways. This hypothesis is consistent with the observations that retinoic acid significantly decreases serum levels of dihydrotestosterone and that the levels of retinoic acid are decreased in prostate carcinoma tissue. The overall objective of this proposal is to determine the role of the human microsomal retinol/sterol dehydrogenases, recently identified by this laboratory, in retinoid and steroid metabolism in the cells. The specific aims of this proposal are to: (1) develop a procedure for preparation of catalytically active purified recombinant retinol/sterol dehydrogenases; (2) characterize the substrate specificity and catalytic properties of the human retinol/sterol dehydrogenases; (3) determine the expression patterns of retinol/sterol dehydrogenases in human tissues; and 4) determine the topology of transmembrane insertion of human dehydrogenases in the microsomal membrane. Accomplishment of these objectives will allow us to gain understanding of the role of this new group of short-chain dehydrogenases in retinoid and steroid metabolism in health and disease.