Functions of Tocopherol Transfer Protein

描述(申请人提供)：维生素E(生育酚)是人体内主要的脂溶抗氧化剂。然而，令人惊讶的是，人们对这种分子进入靶细胞后的命运知之甚少，也不知道它的储存、代谢和生物功能受到哪些分子事件的调控。由于生育酚在水中很难溶解，人们认为特定的结合蛋白是维生素E功能的关键媒介，因为它们能够在细胞的水环境中溶解和运输它们的配体。在这里，我们建议研究生育酚转移蛋白(ATTP)以深入了解其生物学作用(S)，并进而深入了解维生素E的作用模式。我们将结合我们在合成化学、稳定同位素分析、蛋白质生物化学和细胞内信号转导方面的专业知识来阐明aTTP在调节维生素E生物活性中的功能。具体地说，我们建议解决三个具体目标：1)体外表征aTTP中的结构/功能关系。利用新的光学活性分析和定点突变，我们将在分子水平上了解aTTP的配体结合和转移活性，以及这种蛋白质中自然发生的突变。2)-研究体内aTTP的结构/功能关系。我们将研究自然发生的aTTP突变对培养的肝细胞分泌生育酚的影响。3)-鉴定以生育酚依赖的方式与aTTP相互作用的蛋白质，并确定蛋白质的亚细胞定位。这项申请中提出的研究将提供关键信息，这些信息可能会改善我们对维生素E作为人类健康介体的功能的理解。

DESCRIPTION (provided by applicant): Vitamin E (tocopherol) is the major fat-soluble antioxidant in humans. However, surprisingly little is known about the fate of this molecule once it enters target cells, or about the molecular events by which its storage, metabolism, and biological functions are regulated. As tocopherols are poorly soluble in water, it is believed that specific binding proteins are critical mediators of vitamin E function by virtue of their ability to solubilize and transport their ligand in the aqueous environment of the cell. Here, we propose to study the tocopherol transfer protein (aTTP) in order to gain insights into its biological role(s) and, in turn, into the mode of action of vitamin E. We will use our combined expertise in synthetic chemistry, stable-isotope analysis, protein biochemistry, and intra-cellular signaling to elucidate the functions of aTTP in mediating the biological activities of vitamin E. Specifically, we propose to address three specific aims: 1)- to characterize the structure/function relationship in aTTP in vitro. Using novel optical activity assays and site-directed mutagenesis, we will obtain molecular- level understanding of ligand binding and transfer activities of aTTP, and of the naturally occurring mutations in this protein. 2)- To characterize the structure/function relationship in aTTP in vivo. We will examine the effects of naturally occurring mutations in aTTP on tocopherol secretion from cultured hepatocytes. 3)- To identify proteins that interact with aTTP in a tocopherol dependent manner, and to determine the sub-cellular localization of the protein. Studies proposed in this application will provide critical information that is likely to improve our understanding of vitamin E function as a mediator of human health.