Cell Biology of Human Vit C Transporters in Liver Cells

这项研究职业发展计划的主要目的是让首席调查员学习 成像和分子生物学技术的最新进展，推动了他成为一名 细胞生物学领域的独立学术研究者，侧重于细胞内的转运 以及上皮和非上皮细胞系统中不同转运体的膜靶向性与 为人类的健康和疾病工作。抗坏血酸(维生素C)是人体必需的微量营养素 正常的人类健康和福祉。近年来人类钠依赖维生素C的两种异构体 转运蛋白(hSVCTI和hSVCT2)已被克隆，其丰富的表达已在 人体肝脏和其他组织。关于决定hSVCTI和hSVCTI的机制我们一无所知 HSVCT2靶向人肝细胞表面。膜靶向与极化表达 膜转运蛋白的研究不仅对建立上皮细胞的极性很重要，而且是至关重要的 在研究通过膜转运蛋白的插入/恢复进行调控时。此外，最近的研究表明， 运输事件中的损害可以通过运输的有缺陷的贩运/错误瞄准来调节 载体进入细胞膜。因此，膜靶向和细胞内转运的研究 从细胞生物学/生理学的角度来看，运输载体很重要。将一种蛋白质靶向血浆 膜已被证明涉及特定的靶向信号，如酪氨酸，二亮氨酸，二酸，二 在多肽中嵌入碱性、富含Pro和PDZ结构域。以HepG2和WiF-B9细胞为研究对象 模型，我们的具体目标是：1)确定介导靶向的分子机制 通过共聚焦成像将hSVCTI和hSVCT2蛋白结合到质膜上，2)确定 微管网络和肌动蛋白微丝在hSVCTI和hSVCTI细胞内转运中的作用 HSVCT2，调查这些转运事件是否涉及囊泡和马达蛋白，以及它们是否受到调控 通过特定的蛋白激酶介导的途径。细胞/生化/分子生物学和 在这些调查中将使用成像方法。这项提案中描述的研究不仅将 使申请者能够实现他的职业目标，但也有助于更好地了解该单元 特别是维生素C转运体的生物学和调控，以及一般其他营养物质的转运体。

The main purpose of this research career development plan is to allow the Principle Investigator to learn recent advances in imaging and molecular biology techniques, boosting his career goal to become an independent academic investigator in the area of cell biology with an emphasis on the intracellular traficking and membrane targeting of different transporters in epithelial and non-epithelial cellular systems relating the work to human health and disease. Ascorbic acid (vitamin C) is an essential micronutrient required for normal human health and well being. Recently two isoforms of the human sodium-dependent vitamin C transporters (hSVCTI and hSVCT2) have been cloned and their abundant expression has been reported in human liver and other tissues. Nothing is known about the mechanisms that dictate the hSVCTI and hSVCT2 targeting to the human liver hepatocyte cell surface. Membrane targeting and polarized expression of membrane transporters is not only important in order to establish polarity in epithelial cells, but is also vital when studying regulation via insertion/retrival of membrane transporters. Further, recent studies have shown that impairment in a transport event could be mediated via defective trafficking/mis-targeting of the transport carriers to the cell membrane. Therefore, research on the membrane targeting and intracellular trafficking of transport carriers is important from cell biology/physiology standpoints. Targeting of a protein to the plasma membrane has been shown to involve specific targeting signals such as tyrosine, di-leucine, di-acidic, di- basic, proline rich and PDZ domains embedded in the polypeptide. Using HepG2 and Wif-B9 cells as models, our specific aims are:1) To identify the molecular mechanisms that mediate the targeting of the hSVCTI and hSVCT2 proteins to the plasma membrane by employing confocal imaging, 2) Determine what role the microtubule network and the actin microfilaments play in intracellular trafficking of hSVCTI and hSVCT2, investigate if these trafficking events involve vesicles and motor proteins, and if they are regulated by specific protein kinase-mediated pathways. A combination of cell/biochemical/molecular biological and imaging methods will be used in these investigations. The research described in this proposal will not only enable the applicant to achieve his career goals, but will also contribute to a better understanding of the cell biology and regulation of vitamin C transporters in particular, and transporters of other nutrients in general.