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REGULATION OF BBB GLUT1 GLUCOSE TRANSPORTER

BBB GLUT1 葡萄糖转运蛋白的调节

基本信息

批准号：
6540113
负责人：
WILLIAM M PARDRIDGE
金额：
$ 22.16万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-08-01 至 2004-04-30
项目状态：
已结题

项目摘要

DESCRIPTION: (Verbatim from the Applicant's Abstract) The principal glucose transporter at the brain capillary endothelium, which forms the blood-brain barrier (BBB) in vivo, is the Glut1 isoform of the sodium independent glucose transporter gene family. Previous studies have demonstrated an important mode for regulation of BBB Glut1 gene expression is at the post-transcriptional level in pathophysiologic states such as human brain tumors, glucose deprivation/hypoglycemia, development, and cerebral ischemia. Post-transcriptional regulation of mRNA function is mediated via the interaction of cytosolic or polysome proteins with specific cis-elements located within either the 5' - or 3' -untranslated region (UTR) of the Glut1 mRNA. Previous studies have described a polysome protein, designated p44, and a cytosolic protein, designated p88, which interact with two different cis-elements within the 3'-UTR of the glout1 mRNA. The p88 cis-element is associated with an increase in Glut1 gene expression and mediates the stabilization of the Glut1mRNA. The p44 cis-element causes destabilization of the Glut1 transcript, and is associated with an endonuclease/exonuclease activity. The proposed work is comprised of 3 specific aims. Fist, the mechanism of post-transcriptional regulation of Glut1 gene expression will be further defined using a luciferase reporter gene, and cells will also be stably transfected with the Glut1 gene containing selective deletions of th4 putative cis-elements within 3'-UTR. The stably transfected cells will be subjected to hypoxia and glucose deprivation in tissue culture. In parallel, in vivo studies of regional cerebral ischemia using the middle cerebral artery occlusion (MCAO) model as well as global cereal ischemia, using the transient forebrain ischemia (TFI) model, will be performed and correlations will be made between polysome p44, cytosol p88, and Glut1 mRNA levels in whole brain and polysomes, immunoreacitve Glut1 protein, both in brain and at the BBB in vivo. Second, cDNAs encoding the glut1 mRNA UTR-binding proteins will be cloned, sequenced, and analyzed, in parallel with cloning of cDNAs encoding non-glut1 transcripts expressing the cis-acting elements found in the glut1 mRNA 3'-UTR. Third, the secondary structure of the Glut1 mRNA both with and without binding to the p44 and p88 proteins will be determined experimentally using both chemical and enzymatic approaches. These studies will provide insight into molecular mechanisms of regulation of a step crucial to the maintenance of cerebral intermediary metabolism, i.e., the continuous transport of glucose across the blood-brain barrier in vivo.

描述：(逐字摘自申请人的摘要)主要的葡萄糖脑毛细血管内皮细胞的转运蛋白，它构成了血脑体内屏障(BBB)是钠非依赖性葡萄糖的Glut1亚型转运蛋白基因家族。以往的研究已经证明了一种重要的模式对于血脑屏障Glut1基因表达的调节是在转录后人脑肿瘤、血糖等病理生理状态的水平剥夺/低血糖、发育和脑缺血。转录后调节mRNA的功能是通过胞质或多聚体蛋白与特定顺式元件的相互作用位于Glut1的5‘或3’非翻译区(UTR)内 MRNA.以前的研究已经描述了一种多聚体蛋白，命名为p44，以及一种胞浆蛋白，命名为P88，它与两个不同的 Glout1基因3‘端非编码区中的顺式元件。P88顺式元件是与Glut1基因表达增加相关，并介导 Glut1mRNA的稳定化。P44顺式元件导致不稳定 Glut1转录本，与核酸内切酶/核酸外切酶相关活动。拟议的工作由三个具体目标组成。第一，拳头转录后调节Glut1基因表达的机制将是进一步定义使用荧光素酶报告基因，细胞也将稳定含有TH4选择性缺失的Glut1基因的转染 3‘-非编码区内的顺式元件。稳定转染的细胞将受到组织培养中的低氧和缺糖。同时，活体研究大脑中动脉阻塞(MCAO)对局灶性脑缺血的诊断价值利用短暂性前脑缺血模型和全脑缺血模型 (TFI)模型，并将在多聚体之间进行关联全脑和多聚体中P44、胞浆P88和Glut1的mRNA水平， Glut1蛋白在脑内和体内的血脑屏障中均呈免疫反应。第二, 将克隆编码Glut1 mRNA UTR结合蛋白的cDNA，对其进行测序，并在克隆编码非Glut1转录本的cDNA的同时进行了分析表达Glut1mRNA3‘-UTR中的顺式作用元件。第三，与p44结合和不结合的Glut1基因的二级结构和P88蛋白将通过实验确定使用化学和酶促方法。这些研究将为我们深入了解分子对维持脑功能至关重要的一步的调节机制中间代谢，即葡萄糖在体内的持续运输。体内的血脑屏障。