Role of Rab proteins in hSVCT1 cell biology in intestinal epithelial cells

描述(由申请人提供)： 这项提议的主要目的是使首席调查员变得更加独立，并在细胞生物学领域开展一项试点研究，重点是膜靶向和维生素C转运体的贩运，并为未来的赠款产生更多的出版物和数据。维生素C(抗坏血酸)对人类的正常生长和发育是必不可少的。人类肠道和其他组织表达两种钠依赖的维生素C转运蛋白(hSVCT1和-2)。最近的研究表明，特定的Rab蛋白参与了肠细胞膜蛋白的膜靶向/定位、转运和功能。到目前为止，Rab蛋白在hSVCT1的细胞生物学和运输生理方面的作用还不清楚，具体涉及膜的靶向/定位、运输和在人肠细胞中的功能。最近的研究表明，膜靶向和转运事件的改变可能会影响特定转运蛋白的极化表达，并导致上皮细胞整体吸收/分泌功能受损。此外，这些细胞事件被发现通过将所涉及的转运蛋白插入细胞膜和从细胞膜恢复，在调节某些运输过程中发挥重要作用。因此，针对Rab等调节蛋白在膜靶向和转运运输蛋白方面的研究具有重要的意义和细胞生物学/生理学意义。最近的研究表明，Rab8a和11a参与了肠上皮细胞转运蛋白的膜靶向和运输，并将这些蛋白与人类肠道微绒毛包裹体疾病联系在一起。我们的具体目的是利用人肠上皮细胞，在体外确定Rab8a和11a蛋白在hSVCT1极化膜靶向/定位、转运和功能中的作用，并用Rab8a基因敲除小鼠证实体内的结果。在这些研究中，我们将使用细胞/生化/分子生物学和成像方法的组合。这些研究的结果不仅将为申请者提供一个实现其职业目标的机会，还将有助于增进对hSVCT1细胞生物学以及其他微量营养素转运体的了解。 与公共健康相关：维生素C是人类福祉的基本营养素，不能合成，通过肠道吸收从饮食中获得。人体肠道在调节和维持机体维生素C动态平衡方面起着重要作用，对其转运蛋白在肠道细胞中的细胞生物学/生理学研究具有重要意义。这些研究的结果将有助于我们优化肠道维生素C水平，使其处于缺乏和次优水平。

DESCRIPTION (provided by applicant): The main aim of this proposal is to allow the principle investigator to become more independent and to perform a pilot study in the area of cell biology with an emphasis on the membrane targeting and trafficking of vitamin C transporter and to generate additional publications and data for future grants. Vitamin C (ascorbic acid) is essential for normal human growth and development. Human intestine and other tissues express two sodium-dependent vitamin C transporters (hSVCT1 and -2). Recent studies have shown the involvement of specific Rab proteins in the membrane targeting/localization, trafficking and function of membrane proteins in intestinal cells. To date, nothing is known about the role of Rab proteins in cell biology and transport physiological aspects of hSVCT1 with specific regards to membrane targeting/localization, trafficking and function in human enterocytes. Recent studies have shown that the alteration in membrane targeting and trafficking events may affect the polarized expression of the given transporters and lead to overall impaired absorption/secretion in epithelia. Further, these cellular events were found to play an important role in the regulation of certain transport processes via insertion/retrieval of the involved transporters to and from the cell membrane. Therefore, studies aimed at regulators such as Rab proteins in membrane targeting and trafficking of transport proteins are of great significance and of cell biological/physiological importance. Recent studies have shown the involvement of Rab8a and 11a in the membrane targeting and trafficking of transport proteins in intestinal epithelial cells and also linked the proteins to human intestinal microvillus inclusion disease. Our specific aim is to determine the role of Rab8a and 11a proteins in polarized membrane targeting/localization, trafficking and function of hSVCT1 in vitro using human enterocytes and confirm results in vivo using Rab8a knockout mice. In these investigations we will use a combination of cell/biochemical/molecular biological and imaging methods. Outcome of these investigations will not only provide the applicant an opportunity to reach his career goals but also contribute to enhance the understanding of the cell biology of hSVCT1 and also other micronutrient transporters. PUBLIC HEALTH RELEVANCE: Vitamin C is an essential nutrient for human well-being and cannot be synthesized, obtains it from diet via intestinal absorption. Human intestine plays a role in regulating and maintaining body vitamin C homeostasis, studies on the cell biology/physiology of its transporters in intestinal cells are important. Outcome of these investigations will assist us to optimize intestinal vitamin C levels in deficiency and sub-optimal levels.