Regulation of vitamin A synthesis from carotenoids

描述(申请人提供)：所有高等动物，包括人类，必须从饮食中获取维生素A，因为它们缺乏从头合成维生素A的能力。归根结底，所有的维生素A都是由维生素A原类胡萝卜素形成的。维生素A原类胡萝卜素转化为维生素A的过程已经研究了40多年，尽管负责将维生素A原类胡萝卜素裂解成维生素A的酶的克隆和纯化直到最近才完成，当时我们和其他人克隆并鉴定了鼠和人的胡萝卜素裂解酶(CCE)。CCE被发现在全身普遍表达，因此很可能在向靶组织提供维生素A方面发挥普遍作用。我们提出的研究的目的是了解肠道和肝脏中控制维生素A原转化为维生素A的调控机制。这项规定将从两个方面进行探讨。首先，我们将研究其他蛋白质对CCE活性的调节，可能是通过直接的蛋白质-蛋白质相互作用。细胞视黄醇结合蛋白(CRBP)与视网膜相互作用，视网膜是细胞内胡萝卜素裂解的产物。CRBP对CCE活性的影响将通过体外和体内技术进行检测。此外，我们还将探索CCE的直接下游酶，即视网膜还原酶。我们已经确定了两个已知的短链脱氢酶/还原酶，retSDR和RalR1，作为候选的肠道视网膜还原酶。这两种酶在从β-胡萝卜素中产生视黄醇的作用将被深入探讨。最后，我们将研究hCCE表达的转录调控。在这里，我们建议检测人CCE基因的启动子，以确定与该基因中这些调控序列结合的顺式调控元件和转录因子。这些数据将为人类CCE基因的调控提供洞察力。综上所述，这些研究将全面了解CCE在两个主要组织部位--肠道和肝脏--的活性。

DESCRIPTION (provided by applicant): All higher animals, including humans, must obtain vitamin A from the diet since they lack the ability to synthesize it de novo. Ultimately all vitamin A is formed from provitamin A carotenoids. The conversion process of provitamin A carotenoids to vitamin A has been studied for more than 4 decades although the cloning and purification of the enzyme responsible for the cleavage of provitamin A carotenoids to vitamin A were only recently accomplished when we and others cloned and characterized both mouse and human carotene cleavage enzyme (CCE). CCE was found to be ubiquitously expressed throughout the body and thus likely plays a general role in providing vitamin A to target tissues. The goal of our proposed studies is to understand the regulatory mechanisms controlling provitamin A conversion to vitamin A in intestine and liver. Two aspects of this regulation will be explored. First, we will examine the regulation of CCE activity by other proteins, possibly through direct protein-protein interactions. Cellular retinol-binding proteins (CRBPs) are known to interact with retinal, the product of carotene cleavage within cells. The effects of CRBPs on CCE activity will be examined using both in vitro and in vivo techniques. In addition, we will explore the direct down-stream enzyme to CCE, retinal reductase. We have identified two known short chain dehydrogenase/reductases, retSDR and RalR1, as candidate intestinal retinal reductases. The role of these two enzymes on retinol production from beta-carotene will be explored in depth. Finally, we will investigate the transcriptional regulation of hCCE expression. Here, we propose to examine the promoter of the human CCE gene to identify cis-regulatory elements and transcription factors that bind to these regulatory sequences in this gene. These data will provide insights into regulation of the CCE gene in humans. Taken together these studies will provide comprehensive understanding of CCE activity in two major tissue sites, intestine and liver.