Carotene Cleavage Enzyme: Properties and Physiology

胡萝卜素裂解酶：特性和生理学

• 批准号: 7174667
• 负责人: WILLIAM S BLANER
• 金额: $ 36.43万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7174667
• 关键词: Adult; Animals; Beta Carotene; Biochemical; Biochemistry; Carotene; Carotenoids; Cells; Chimeric Proteins; Chinese Hamster Ovary Cell; Classification; Cleaved cell; Cloning; Complementary DNA; Data; Diet; Dietary Carotenoid; Dioxygenases; Drug or chemical Tissue Distribution; Embryo; Embryonic Development; Enzymes; Escherichia coli; Future; Gel; Genes; Glutathione S-Transferase; Health; Hormonal; Hormones; Human; Human Cloning; In Situ Hybridization; Intestines; Knockout Mice; Life; Literature; Messenger RNA; Metabolism; Mus; Mutant Strains Mice; Normal Cell; Nutrient; Nutritional; Object Attachment; Organism; Pattern; Physiological; Physiology; Plants; Play; Property; Proteins; Provitamin A Carotenoid; Recombinants; Reporting; Reproduction; Retinaldehyde; Retinoids; Role; Techniques; Testis; Thinking; Tissues; Transfection; Vision; Vitamin A; Whole Organism; base; cell growth; expression vector; immune function; in vivo; interest; man; microorganism; prevent; uptake

DESCRIPTION (provided by applicant): All vitamin A that is present in higher animals must be derived from the diet. Provitamin A carotenoids like beta-carotene, act essentially in man and other species as precursors for vitamin A synthesis. In addition, dietary carotenoids may have other beneficial actions that are independent of their provitamin A activity. Although carotenoid uptake and carotenoid actions have been systematically studied for over 40 years, very little detailed biochemical information regarding the uptake and metabolism of these substance is presently available. This lack of biochemical information has in large part been due to technical difficulties in purifying and cloning proteins that are importantly involved in carotenoid uptake and metabolism. In the last year, we reported the cloning and characterization of a mudne cDNA that encodes an enzyme that when expressed in either E. coli or CHO cells confers the ability to cleave beta-carotene to vitamin A aldehyde. We now propose to investigate the biochemistry and physiology of mouse and human carotene cleavage enzyme (CCE) and to explore the uptake and metabolism of carotenoids in mutant mice where levels of this enzyme are manipulated genetically. Our proposal consists of 4 Specific Aims. In Aim 1, we will identify proteins present in tissues that express CCE that interact physiologically with this enzyme. Here we are interested in understanding how a soluble enzyme like CCE obtains and cleaves extremely insoluble carotenoids to insoluble retinoid products. Based on our data, we hypothesize that other as yet unidentified cellular protein(s) must play a role in metabolically channeling carotene cleavage towards vitamin A formation. Aim 2 proposes a systematic study of the tissue and cellular distribution of CCE protein and mRNA levels in the adult, post-natal and embryonic mouse. In addition, we will investigate how nutritional and hormonal factors influence enzyme expression in the mouse. In Aim 3, we propose to generate and study tissue-specific knockout mice for CCE. These studies will allow us to identify and characterize the essential and nonessential actions of this enzyme in living organisms. In Aim 4, we propose to use a cDNA clone for human CCE to characterize the biochemical properties of the recombinant enzyme.

描述（由申请人提供）：高等动物中存在的所有维生素A必须来自饮食。维生素A原类胡萝卜素像β-胡萝卜素一样，基本上在人类和其他物种中作为维生素A合成的前体。此外，膳食类胡萝卜素可能有其他有益的行动，是独立的维生素A原的活动。虽然类胡萝卜素的吸收和类胡萝卜素的作用已经系统地研究了40多年，但目前关于这些物质的吸收和代谢的详细生物化学信息很少。这种缺乏生化信息在很大程度上是由于技术上的困难，在纯化和克隆蛋白质，是重要的类胡萝卜素的吸收和代谢。去年，我们报道了一个mudne cDNA的克隆和鉴定，该cDNA编码一种酶，当在大肠杆菌中表达时，大肠杆菌或CHO细胞赋予将β-胡萝卜素裂解为维生素A醛的能力。我们现在建议调查的生物化学和生理学的小鼠和人类胡萝卜素裂解酶（CCE），并探讨在突变小鼠的这种酶的水平进行遗传操纵的类胡萝卜素的摄取和代谢。我们的计划有四个具体目标。在目标1中，我们将鉴定存在于表达CCE的组织中的蛋白质，所述蛋白质在生理上与该酶相互作用。在这里，我们有兴趣了解如何像CCE获得可溶性酶和切割极不溶性类胡萝卜素不溶性类维生素A产品。根据我们的数据，我们假设其他尚未鉴定的细胞蛋白质必须在代谢引导胡萝卜素裂解形成维生素A中发挥作用。目的2系统研究CCE蛋白和mRNA在成年、出生后和胚胎小鼠体内的组织和细胞分布。此外，我们还将研究营养和激素因素如何影响小鼠中的酶表达。在目标3中，我们建议产生和研究CCE的组织特异性敲除小鼠。这些研究将使我们能够识别和表征这种酶在生物体中的必要和非必要作用。在目的4中，我们建议使用人CCE的cDNA克隆来表征重组酶的生化特性。