Functions of Tocopherol Transfer Protein

说明(申请人提供)：维生素E(生育酚)是人类体内主要的脂溶抗氧化剂，在保护人体免受过多的氧化应激相关疾病的侵害方面起着至关重要的作用。然而，令人惊讶的是，人们对调节体内维生素水平和活动的分子机制知之甚少。肝脏α-生育酚转移蛋白(TTP)是维生素E状态的关键调节因子；携带突变的TTP等位基因的人会出现严重的维生素E缺乏症，并伴有神经元退化。TTP被认为是肝脏中的“守门人”，它选择性地保留最具生物活性的维生素E(RRR-1-生育酚)，并将其“引导”到血浆脂蛋白中，将维生素输送到外周细胞。在这里，我们建议继续我们的体内和体外对TTP的研究，总体目标是了解该蛋白在调节维生素E在健康和疾病中的作用的分子基础。我们将利用我们在合成化学、生物物理学、生物化学和细胞生物学方面的综合专业知识来解决四个具体目标：1)-表征肝细胞中维生素E运输所涉及的步骤，以及TTP在刺激这一过程中的确切作用。使用维生素E的新型荧光类似物，我们将监测培养的肝细胞中维生素E的细胞内转运。然后，我们将使用分子和细胞生物学方法来定义TTP催化的步骤，以及这些事件背后的分子机制。2)-确定调节TTP表达和降解的因素和机制，以及维生素E状态和/或氧化应激在这些过程中所起的作用。3)-研究TTP在神经元中的功能，TTP蛋白在神经元中有显著水平的表达，并且是维生素E缺乏造成损害的主要部位。4)-了解TTP体外催化维生素E膜间转移的分子机制。这项申请中提出的研究将提供关键信息，提高我们对维生素E作为人类健康介体的功能的理解。公共卫生相关性：维生素E是保护所有细胞免受氧化损伤所必需的基本营养素。TTP是已知的唯一一种特定调节体内维生素E水平的蛋白质。我们建议确定TTP在肝细胞中的确切作用，以及在人类中发现的TTP突变如何影响其活性。

DESCRIPTION (provided by applicant): Vitamin E (tocopherol) is the major fat-soluble antioxidant in humans, and serves a critical function in protection against a plethora of oxidative-stress-related pathologies. However, surprisingly little is known about the molecular mechanisms that regulate the levels and activities of the vitamin in the body. The hepatic alpha tocopherol transfer protein (TTP) is a critical regulator of vitamin E status; humans carrying mutated TTP alleles develop severe vitamin E deficiency, accompanied by neuronal degeneration. TTP is thought to function as a 'gatekeeper' in the liver, where it selectively retains the most biologically active form of vitamin E (RRR-1-tocopherol), and 'directs' it to plasma lipoproteins that deliver the vitamin to peripheral cells. Here, we propose to continue our in vivo and in vitro studies on TTP with the overall goal of understanding the molecular bases for the function of the protein in mediating the effects of vitamin E, both in health and in disease. We will use our combined expertise in synthetic chemistry, biophysics, biochemistry and cell-biology to address four specific aims: 1)- to characterize the steps involved in the transport of vitamin E in hepatocytes, and the exact role of TTP in stimulating this process. Using novel fluorescent analogs of vitamin E, we will monitor the intracellular trafficking of the vitamin in cultured hepatocytes. We will then use molecular- and cell-biology approaches to define the steps that TTP catalyzes, and the molecular mechanisms that underlie these events. 2)- To determine the factors and mechanisms that regulate the expression and degradation of TTP, and the roles that vitamin E status and / or oxidative stress play in these processes. 3)- To examine the functions of TTP in neurons, where the protein is expressed in significant levels, and which are the primary site of damage due to vitamin E deficiency. 4)- To understand the molecular mechanisms by which TTP catalyzes the inter-membrane transfer of vitamin E in vitro. Studies proposed in this application will provide critical information that will improve our understanding of vitamin E function as a mediator of human health. PUBLIC HEALTH RELEVANCE: Vitamin E is an essential nutrient that is required for protection of all cells from oxidative damage. TTP is the only known protein that specifically regulates the levels of vitamin E in the body. We propose to determine what exactly TTP does in liver cells, and how mutations in TTP that are found in humans affect its activity.