Functions of Tocopherol Transfer Trotein

描述（由申请方提供）：维生素E（生育酚）是人体内主要的脂溶性抗氧化剂。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，或者对调节其储存、代谢和生物学功能的分子事件知之甚少。由于生育酚难溶于水，因此认为特异性结合蛋白是维生素E功能的关键介质，因为它们能够在细胞的水性环境中溶解和转运其配体。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，进而了解维生素E的作用模式。我们将利用我们在合成化学，稳定同位素分析，蛋白质生物化学和细胞内信号传导方面的综合专业知识来阐明aTTP在介导维生素E生物活性中的功能。具体而言，我们提出解决三个具体目标：1）-表征体外aTTP的结构/功能关系。使用新的光学活性测定和定点诱变，我们将获得对aTTP的配体结合和转移活性以及该蛋白质中天然存在的突变的分子水平的理解。2)-在体内表征aTTP的结构/功能关系。我们将研究aTTP中天然发生的突变对培养肝细胞中生育酚分泌的影响。3)-鉴定以生育酚依赖性方式与aTTP相互作用的蛋白质，并确定该蛋白质的亚细胞定位。本申请中提出的研究将提供关键信息，这些信息可能会提高我们对维生素E作为人类健康媒介的理解。

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.