Novel Intestinal Nutrient Membrane Transporters

背景：本基础研究计划旨在发现新的营养膜转运蛋白，以了解转运途径的生物学。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，我们已经为发现膜转运蛋白开发了一个“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新方法来探索。由于肠道吸收作为系统平衡的决定因素的关键作用，本研究计划的长期重点放在吸收上皮细胞肠细胞的运输途径上。由于我们对肠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目标。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。两个子项目将为未来的研究目标提供信息。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细胞中的差异基因表达来鉴定额外的候选物。2. 抗坏血酸释放：slc23a3和slc22a23作为“释放剂”的特性。3. 小肠中的脱氢抗坏血酸摄取：GLUT14在该途径中的作用表征。4. 有机阳离子的吸收：slc22a23作为多特异性“吸收者”的表征。有机阳离子释放：mfsd14b作为伯胺“释放剂”的表征。6. 肠细胞的转录组产生未来的研究目标。设计：我们的分子“研究管道”利用共同的方法来表征目标2-5中的单个膜转运蛋白。这将导致基因(a)注释，(b)主要蛋白亚型的亚克隆，(c)亚细胞定位的确定，(d)底物研究。两项转录组学研究有助于鉴定另一种难以捉摸的抗坏血酸摄取转运蛋白和肠细胞的转录组，并将为未来的靶标提供信息。意义：该项目将发现新的膜转运蛋白。其方法将成为进一步研究的模板。这些基本的发现将使翻译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.