Novel Intestinal Nutrient Membrane Transporters

背景：这个基础研究项目是为了发现新的营养膜转运蛋白，以了解运输途径的生物学。我们的研究小组在识别新型转运蛋白方面有着良好的记录，我们还开发了一条用于膜转运蛋白发现的“研究管道”。我们目前有四个关于抗坏血酸和有机阳离子途径“缺失环节”的研究线索，我们建议使用我们开发的一种新的方法来探索这些线索。*由于肠道吸收作为全身内稳态的决定因素起着关键作用，本研究计划的长期重点放在吸收上皮细胞，即肠细胞的运输途径上。由于我们对肠上皮细胞中表达的所有转运蛋白没有完全的了解，我们提出了两种实验方法来确定未来的靶点。目的：探索维生素C和有机阳离子转运途径中存在的四个候选基因。两个子项目将介绍未来的研究目标。个人目标是：*1.抗坏血酸摄取：通过暴露于不同浓度抗坏血酸的Caco-2细胞中不同的基因表达来识别其他候选者。*2.抗坏血酸释放：slc23a3和slc22a23为“释放剂”。*3.小肠对脱氢抗坏血酸的摄取：GLUT14‘S在该途径中的作用*4.有机阳离子吸收：将slc22a23描述为多个特定的“吸收者”。*5.有机阳离子释放：将mfsd14b描述为伯胺的“释放者”。*6.肠细胞的转录组，以产生未来的研究靶点。*设计：我们的分子“研究管道”使用共同的方法来表征目标2-5中的单个膜转运体。这将导致基因(A)注释，(B)主要蛋白质异构体的亚克隆，(C)亚细胞定位的确定，(D)底物研究。两项转录学研究有助于确定另一种难以捉摸的抗坏血酸摄取转运体和肠细胞的转录组，并将为未来的靶点提供信息。*意义：该计划将发现新的膜转运蛋白。其方法将成为进一步研究的模板。这些基本发现将启动翻译，以提高生理性能或环境挑战。**

Background: This basic research program is set up to discover novel nutrient membrane transporters in order to understand the biology of transport pathways. Our research group has a track record of identifying novel transporters and we have developed a “Research Pipeline” for membrane transporters discovery. We have four current research leads on “missing links” in ascorbic acid and organic cation pathways, which we propose to explore using a novel methodology developed by us. ***Due to a key role of intestinal absorption as a determinant of systemic homoeostasis the long term focus of this research program lays on transport pathways in the absorbing epithelial cell, the enterocyte. Since we do not have a complete understanding of all the transporters expressed in the enterocyte, we propose two experimental approaches to identify future targets. ***Objectives: Four existing candidate genes in pathways of vitamin C and organic cation transport will be explored. Two subprojects will inform on future research targets. The individual objectives are: ***1. Ascorbic Acid Uptake: Identification of additional candidates through differential gene expression in CaCo-2 cells exposed to varying concentration of ascorbic acid. ***2. Ascorbic Acid Release: Characterization of slc23a3 and slc22a23 as “releaser”. ***3. Dehydroascorbic Acid Uptake in the Small Intestine: Characterization of GLUT14's role in the pathway. ***4. Organic Cations Uptake: Characterization of slc22a23 as multispecific “uptaker”.***5. Organic Cations Release: Characterization of mfsd14b as “releaser” of primary amines. ***6. The Transcriptome of the Enterocyte to generate future research targets. ******Design: Our molecular “Research Pipeline” utilizes common methodology to characterize individual membrane transporters in objectives 2-5. This will result in genes (a) annotation, (b) Subcloning of major protein isoforms, (c) determination of subcellular localization, (d) substrate studies. Two transcriptomics studies aid the identification of another elusive ascorbic acid uptake transporter and the transcriptome of the enterocyte and will inform on future targets. ***Significance: The program will discover novel membrane transporters. Its methodology will be a template for further studies. The basic discoveries will prime translation to enhance physiologic performance or environmental challenges. **