Hydrolysis of vitamin A esters in liver

描述(申请人提供)：肝脏中视黄酸酯(RE)的水解在体内维生素A的新陈代谢中起着关键作用。饮食中的维生素A以乳糜粒残留物中的RE形式进入肝脏，经过水解和再酯化储存。在动员之前，RE被水解，游离视黄醇与视黄醇结合蛋白分泌到血浆中。从大鼠肝脏中分离纯化了中性和酸性视黄酯水解酶(REHS)，序列分析表明其为羧酸酯酶，Es-2和Es-10。这个项目的目标是确定ES-2、ES-10以及相关的ES-3和ES-4在维生素A代谢中的作用。人们已经用其他底物研究了羧酸酯酶，但尚不清楚它们是否在维生素A代谢中起重要作用。与其他酯相比，它们在催化稀土水解方面的效率也是未知的。将探讨这些知识差距，具体目标如下：目标1：REHS的酶学。酶动力学将被用来研究羧酸酯酶的底物专一性，并检验这些酶催化RE和其他脂酯的内在能力不同的假设。目的2：羧酸酯酶的组织分布。实时定量聚合酶链式反应(MRNA)、免疫检测(蛋白质)和酶分析将被用来确定各种组织中酶的水平和分布，并验证外周组织和肝脏中存在羧酸酯酶的假设。目的3：细胞类型在肝脏的分布。激光捕获显微切割和细胞分离结合实时定量PCR、免疫检测和酶分析将被用来确定羧酸酯酶在星状细胞和肝细胞之间的分布，并检验羧酸酯酶ES-2仅定位于肝细胞和羧酸酯酶ES-10定位于肝细胞和星形细胞的假说。目的：探讨羧酸酯酶在培养细胞稀土代谢中的生理作用，通过基因转染和RNA干扰来调节羧酸酯酶的水平，以验证各种羧酸酯酶在肝脏摄取和/或代谢乳杆菌稀土中起代谢作用的假说，以及羧酸酯酶在储存它们的细胞内稀土和胆固醇酯代谢中起代谢作用的假说。

DESCRIPTION (provided by applicant): Hydrolysis of retinyl esters (RE) in the liver plays a key role in the body's metabolism of vitamin A. Dietary vitamin A enters the liver as RE in chylomicron remnants, which undergo hydrolysis and reesterification for storage. Prior to mobilization, RE are hydrolyzed and free retinol binds to retinol-binding protein for secretion into the plasma. Neutral and acid retinyl ester hydrolases (REHs) have been purified from rat liver and shown by sequence analysis to be carboxylesterases, ES-2 and ES-10. The goal of this project is to define the role of ES-2, ES-10, and the related ES-3 and ES-4, in the metabolism of vitamin A. Carboxylesterases have been studied with other substrates, but it is not known if they are important in vitamin A metabolism. Their efficiencies in catalyzing the hydrolysis of RE compared with other esters are also unknown. These gaps in knowledge will be explored with the following specific aims: AIM 1: Enzymoloqy of REHs. Enzyme kinetics will be used to study the substrate specificity of the carboxylesterases and to test the hypothesis that these enzymes differ in their intrinsic ability to catalyze the hydrolysis of RE and other lipid esters. AIM 2: Tissue distribution of carboxylesterases. Real-time PCR (for mRNA), immunodetection (for protein) and enzyme assay will be used to define the levels and distributions of enzymes in various tissues and to test the hypothesis that the carboxylesterases are present in peripheral tissues as well as in liver. AIM 3: Cell type distribution in liver. Laser Capture Microdissection and cell separations coupled with real-time PCR, immunodetection, and enzyme assays will be used to define the distribution of carboxylesterases between stellate cells & hepatocytes and to test the hypotheses that carboxylesterase ES-2 is localized exclusively in hepatocytes and that carboxylesterase ES-10 is localized in hepatocytes and stellate cells. AIM 4: Physiologic roles of the carboxylesterases in the metabolism of RE in cultured cells, cDNA transfection and RNA interference will be used to modulate carboxylesterase enzyme levels to test the hypothesis that the various carboxylesterases play a metabolic role in the hepatic uptake and/or metabolism of chylomicron RE, and to test the hypothesis that the carboxylesterases play a metabolic role in the turnover of intracellular RE and cholesteryl esters in cells that store them.