SHORT-CHAIN DEHYDROGENASES IN RETINOL/STEROL METABOLISM

描述（由申请人提供）：维生素A衍生物全反式维甲酸调节超过530种不同基因的表达。因此，在胚胎发生过程中视黄酸的水平以空间和时间精确的方式控制。然而，这种调节的分子机制尚未完全了解。该项目的长期目标是确定短链淀粉酶/还原酶（SDR）在健康和疾病中调节视黄酸生物合成的作用。最近，我们已经确定了一个新成员的SDR超家族的蛋白质在青蛙，rdhe 2，这是高度活跃的全反式视黄醇脱氢酶，是非洲爪蟾胚胎发育的关键。重要的是，在哺乳动物中似乎有一种功能等同于青蛙rdhe 2的蛋白，它具有全反式视黄醇脱氢酶活性，并在早期胚胎发育过程中表达。我们建议，这种新的酶，命名为RDH-E2 S，是必不可少的视黄酸的生物合成在哺乳动物胚胎发育过程中，并可能在成年期。为了验证这一假设，我们将表征哺乳动物RDH-E2 S的催化特性，并使用遗传修饰的小鼠模型（特定目的1）确定其对体内视黄酸生物合成的贡献。 我们的初步研究表明，视网膜短链脱氢酶/还原酶1（retSDR 1）基因在人类细胞中的表达沉默导致视黄酸和视黄醇水平的显着增加，这转化为视黄酸响应基因的显着上调。这一发现表明，视黄酸生物合成的速率是由视黄醇脱氢酶和视黄醇醛还原酶的相对活性决定的，它们共同控制视黄酸前体，视黄醇的水平。为了验证这一假设，我们建议表征retSDR 1的催化特性，并确定其在体内使用转基因小鼠，人类皮肤器官培养，非洲爪蟾在体外模型的早期胚胎发育（具体目标2）的视黄酸水平的调节作用。 这些研究将填补我们对维甲酸稳态维持机制的认识空白，为维甲酸调节系统的潜在重要组成部分的作用提供新的信息。这些研究的结果将是重要的了解与维甲酸稳态的破坏，如胎儿酒精综合征，酒精性肝病，致癌和糖尿病相关的疾病的病理生理学。

DESCRIPTION (provided by applicant): Vitamin A derivative all-trans-retinoic acid regulates the expression of over 530 different genes. Consequently, the levels of retinoic acid during embryogenesis are controlled in a spatially and temporally precise manner. However, the molecular mechanisms underlying this regulation are not yet fully understood. The long-term objective of this project is to determine the role of short-chain dehydrogenases/reductases (SDRs) in the regulation of retinoic acid biosynthesis in health and disease. Recently, we have identified a new member of the SDR superfamily of proteins in frogs, rdhe 2, that is highly active as an all-trans-retinol dehydrogenase and is critical for embryonic development in Xenopus laevis. Importantly, there appears to be a functional equivalent of the frog rdhe2 in mammals, which exhibits an all-trans-retinol dehydrogenase activity and is expressed during early embryonic development. We propose that this novel enzyme, named RDH-E2S, is essential for retinoic acid biosynthesis in mammals during embryogenesis and, possibly, in adulthood. To test this hypothesis, we will characterize the catalytic properties of mammalian RDH-E2S and determine its contribution to retinoic acid biosynthesis in vivo using genetically modified mouse model (Specific Aim 1). Our preliminary studies indicate that silencing of retina short-chain dehydrogenase/reductase 1 (retSDR1) gene expression in human cells results in significant increase in the levels of both retinoic acid and retinaldehyde, which translates into dramatic upregulation of retinoic acid-responsive genes. This finding suggests that the rate of retinoic acid biosynthesis is determined by the relative activities of retinol dehydrogenases and retinaldehyde reductases, which together control the levels of retinoic acid precursor, retinaldehyde. To test this hypothesis, we propose to characterize the catalytic properties of retSDR1 and to determine its role in the regulation of retinoic acid levels in vivo using genetically modified mice, human skin organ culture, and Xenopus laevis in vitro model of early embryonic development (Specific Aim 2). These studies will fill the gaps in our understanding of the mechanisms responsible for the maintenance of retinoic acid homeostasis by providing new information regarding the roles of potentially important components of the retinoid regulatory system. The results of these studies will be important for understanding the pathophysiology of disorders associated with disruptions of retinoid homeostasis, such as fetal alcohol syndrome, alcoholic liver disease, carcinogenesis, and diabetes.