Nutritional Biochemistry of beta-Carotene Dioxygenase
β-胡萝卜素双加氧酶的营养生物化学
基本信息
- 批准号:6885744
- 负责人:
- 金额:$ 19.37万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2003
- 资助国家:美国
- 起止时间:2003-05-01 至 2007-03-31
- 项目状态:已结题
- 来源:
- 关键词:biochemistrycarotenecarotenoidsclinical researchenzyme activityenzyme mechanismexpression cloninggene expressiongene mutationhuman subjectimmunologic techniqueslaboratory mousemass spectrometrynucleic acid hybridizationnutrition related tagoxygenasespatient oriented researchposttranslational modificationsprotein localizationprotein structure functionproteomicsrecombinant proteinsretinoidssite directed mutagenesisvitamin A deficiencyvitamin biosynthesis
项目摘要
DESCRIPTION (provided by applicant): The overall objective of the proposed research is to gain insight into the biochemistry and physiological role of human beta-carotene 15,15'-dioxygenase (BCDO), an enzyme that catalyzes the first step in the synthesis of vitamin A from dietary provitamin A carotenoids. This pathway is especially crucial for persons on a vegetarian diet lacking preformed vitamin A. Derivatives of vitamin A serve as ligands for the retinoic acid nuclear hormone receptors that are essential for development and normal physiological functions. We have isolated and characterized the cDNA and gene that encode the human BCDO enzyme, as well as initiated biochemical characterizations of the purified recombinant enzyme. Our findings raise a number of questions relating to the physiological roles of BCDO in different tissues, and how the enzyme's biochemical properties influence these roles. Our initial goal will be a detailed analysis of tissue distribution and cell type-specific expression of the BCDO enzyme. These analyses will be accomplished using immunoblotting, immunohistochemistry, RNA blotting, in situ mRNA hybridization, and BCDO enzyme assays. Our second objective is to identify potential subjects with vitamin A deficiency-like symptoms. If these symptoms are a result of mutations in the BCDO gene, we will analyze how the mutations cause biochemical abnormalities in the enzyme function. These studies will help to define important structural domains of the protein. The third goal is to expand the structure-function information derived from naturally occurring mutations by creating, and expressing additional site-directed mutations and domain chimeras in the BCDO using the baculovirus system. We will focus on identifying amino acids that form substrate and nonheme iron binding domains of the protein. Detailed kinetic, pharmacological, and structural properties of the purified mutant enzymes will be compared to those of the wild-type enzyme. The final objective is to identify accessory/activating proteins of BCDO by use of two distinct expression cloning strategies, along with a proteomics approach that involves mass spectrometry of immunoprecipitated BCDO from human tissue extracts. Identified proteins will be cloned, expressed, purified, and tested in a reconstituted system together with recombinant BCDO. The Michaelis-Menten constants of the BCDO enzyme, alone or complexed with selected proteins, will be determined, as well as the mechanism by which the accessory/activating protein(s) act. The studies described in this proposal will further our knowledge on vitamin A synthesis from dietary provitamin A carotenoids in man.
描述(由申请人提供):拟议研究的总体目标是深入了解人β-胡萝卜素15,15 '-双加氧酶(BCDO)的生物化学和生理作用,BCDO是一种催化从膳食维生素A原类胡萝卜素合成维生素A的第一步的酶。这一途径对于缺乏预制维生素A的素食者尤其重要。维生素A的衍生物作为视黄酸核激素受体的配体,视黄酸核激素受体对发育和正常生理功能至关重要。我们已经分离和表征了编码人BCDO酶的cDNA和基因,以及开始纯化的重组酶的生化表征。我们的研究结果提出了一些问题,涉及BCDO在不同组织中的生理作用,以及酶的生化特性如何影响这些作用。我们最初的目标是详细分析BCDO酶的组织分布和细胞类型特异性表达。将使用免疫印迹、免疫组织化学、RNA印迹、原位mRNA杂交和BCDO酶测定完成这些分析。我们的第二个目标是确定潜在的受试者与维生素A缺乏样症状。如果这些症状是BCDO基因突变的结果,我们将分析突变如何导致酶功能的生化异常。这些研究将有助于确定蛋白质的重要结构域。第三个目标是通过使用杆状病毒系统在BCDO中产生和表达额外的定点突变和结构域嵌合体来扩展源自天然存在的突变的结构-功能信息。我们将重点确定形成底物和非血红素铁结合域的蛋白质的氨基酸。详细的动力学,药理学和结构特性的纯化的突变体酶将进行比较,野生型酶。最终的目标是通过使用两种不同的表达克隆策略,沿着蛋白质组学方法,包括从人组织提取物中免疫沉淀的BCDO的质谱分析,来鉴定BCDO的辅助/活化蛋白。将对鉴定的蛋白质进行克隆、表达、纯化,并与重组BCDO一起在复溶系统中进行检测。将确定单独或与选定蛋白质复合的BCDO酶的Michaelis-Menten常数,以及辅助/活化蛋白作用的机制。本建议中描述的研究将进一步加深我们对人类从膳食维生素A原类胡萝卜素合成维生素A的认识。
项目成果
期刊论文数量(3)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Stromal-epithelial interactions regulate cervical function during pregnancy
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$ 19.37万 - 项目类别:
Nutritional Biochemistry of beta-Carotene Dioxygenase
β-胡萝卜素双加氧酶的营养生物化学
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6618832 - 财政年份:2003
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$ 19.37万 - 项目类别:
Nutritional Biochemistry of beta-Carotene Dioxygenase
β-胡萝卜素双加氧酶的营养生物化学
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6740155 - 财政年份:2003
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ENDOCRINOLOGY OF 17BETA-HYDROXYSTEROID DEHYDROGENASES
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2749615 - 财政年份:1997
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$ 19.37万 - 项目类别:
ENDOCRINOLOGY OF 17BETA-HYDROXYSTEROID DEHYDROGENASES
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$ 19.37万 - 项目类别:
ENDOCRINOLOGY OF 17BETA-HYDROXYSTEROID DEHYDROGENASES
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