Cell Biology of Human Vit C Transporters in Liver Cells

描述(由候选人提供)： 这项研究职业发展计划的主要目的是让首席研究员学习成像和分子生物学技术的最新进展，促进他的职业目标，成为细胞生物学领域的独立学术研究员，重点是上皮和非上皮细胞系统中不同转运蛋白的细胞内转运和膜靶向，这项工作与人类健康和疾病有关。抗坏血酸(维生素C)是人体正常健康所必需的微量营养素。最近，人类钠依赖的维生素C转运蛋白的两种亚型(hSVCT1和hSVCT2)被克隆出来，并在人的肝脏和其他组织中大量表达。关于hSVCT1和hSVCT2靶向于人肝细胞表面的机制尚不清楚。膜转运蛋白的膜靶向和极化表达不仅对建立上皮细胞的极性很重要，而且在研究膜转运蛋白插入/恢复的调控时也是至关重要的。此外，最近的研究表明，运输事件中的损害可以通过运输载体到细胞膜的有缺陷的运输/错误靶向来调节。因此，从细胞生物学和生理学的角度研究转运载体的膜靶向和胞内转运具有重要意义。蛋白质在质膜上的靶向涉及到特定的靶向信号，如嵌入在多肽中的酪氨酸、二亮氨酸、二酸、二碱、富含脯氨酸和PDZ结构域。以HepG2和WiF-B9细胞为模型，我们的具体目标是：1)通过共聚焦成像确定hSVCT1和hSVCT2蛋白靶向质膜的分子机制；2)确定微管网络和肌动蛋白微丝在hSVCT1和hSVCT2细胞内转运中所起的作用，研究这些转运事件是否涉及囊泡和马达蛋白，以及它们是否受到特定的蛋白激酶介导的途径的调控。在这些研究中将使用细胞/生化/分子生物学和成像方法的组合。这项建议中描述的研究不仅将使申请者实现其职业目标，还将有助于更好地了解细胞生物学和维生素C转运体的调节，特别是其他营养物质的转运体。

DESCRIPTION (provided by candidate): 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 nonepithelial 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 (hSVCT1 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 hSVCT1 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, dibasic, 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 hSVCT1 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 hSVCT1 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.