Cell Biology of Human Thiamine Transporters

人类硫胺素转运蛋白的细胞生物学

• 批准号: 6855717
• 负责人: VEEDAMALI S SUBRAMANIAN
• 金额: $ 5.65万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-02-04 至 2006-02-03
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6855717
• 关键词: cell membrane; confocal scanning microscopy; cytoskeleton; gastrointestinal absorption /transport; gastrointestinal epithelium; human tissue; intestines; intracellular transport; microtubules; postdoctoral investigator; protein transport; site directed mutagenesis; thiamine

DESCRIPTION (provided by applicant): Thiamine (vitamin B1) is an important nutrient that is essential for human health. Recently two thiamine transporter genes SLC19A2 and SLC19A3 (encoding hTHTR1 and hTHTR2, respectively) have been cloned and characterized from a number of human tissues. Nothing is known about the membrane targeting and intracellular trafficking of these thiamine transporters. Targeting of a protein to the plasma membrane has been shown to involve specific targeting signals such as tyrosine, di-leucine, diacidic, dibasic, histidine, and PDZ domains encoded in the polypeptide. The hTHTRI& 2 polypeptides have a number of such potential sorting signals. In preliminary studies, we fused hTHTR1 to EGFP and demonstrated functionality and membrane localization of the stably expressed fusion protein (hTHTR1-EGFP) in HuTu-80 cells. In order to investigate the role of specific targeting signals in directing hTHTRI& 2 to the plasma membrane of human intestinal epithelial cells, I will compare wild-type, truncated, and site-directed mutant constructs of hTHTRI& 2 fused to EGFP, and image by confocal microscopy. I aim to also determine if the expression of hTHTRI& 2 at a given cell membrane domain is cell-type specific using wild-type constructs of hTHTRI& 2 proteins fused to EGFP. To further examine the intracellular trafficking of hTHTRI& 2 proteins I will examine the role of the microtubule network and the actin-based cytoskeleton, perform kinetic analysis of the delivery of newly synthesized thiamine transporters to the plasma membrane, and investigate the role of vesicular transport of the proteins to the plasma membrane (insertion/retrival).

描述（由申请人提供）：硫胺素（维生素 B1）是一种对人类健康至关重要的重要营养素。最近，两个硫胺素转运蛋白基因 SLC19A2 和 SLC19A3（分别编码 hTHTR1 和 hTHTR2）已从许多人体组织中克隆并鉴定。关于这些硫胺素转运蛋白的膜靶向和细胞内运输尚不清楚。已证明蛋白质靶向质膜涉及特异性靶向信号，例如多肽中编码的酪氨酸、二亮氨酸、二酸、二碱基、组氨酸和PDZ结构域。 hTHTRI&2多肽具有许多这样的潜在分选信号。在初步研究中，我们将 hTHTR1 与 EGFP 融合，并证明了 HuTu-80 细胞中稳定表达的融合蛋白 (hTHTR1-EGFP) 的功能和膜定位。为了研究特异性靶向信号在将 hTHTRI&2 引导至人肠上皮细胞质膜方面的作用，我将比较与 EGFP 融合的 hTHTRI&2 的野生型、截短型和定点突变体构建体，并通过共聚焦显微镜进行成像。我的目的还在于使用与 EGFP 融合的 hTHTRI& 2 蛋白野生型构建体来确定 hTHTRI& 2 在给定细胞膜结构域的表达是否具有细胞类型特异性。为了进一步检查 hTHTRI& 2 蛋白的细胞内运输，我将检查微管网络和基于肌动蛋白的细胞骨架的作用，对新合成的硫胺素转运蛋白向质膜的递送进行动力学分析，并研究蛋白质向质膜的囊泡转运（插入/回收）的作用。