GLUCOSE TRANSPORTER STRUCTURE AND FUNCTION

葡萄糖转运蛋白的结构和功能

• 批准号: 7244269
• 负责人: ANTHONY CARRUTHERS
• 金额: $ 21.73万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-05-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7244269
• 关键词: Behavior; Binding; Binding Sites; Biochemical; C-terminal; Carbohydrates; Carrier Proteins; Catalysis; Cell membrane; Cells; Chemicals; Chemistry; Chimera organism; Complex; Cytochalasin B; Detergents; Diffusion; Disruption; Environment; Erythrocytes; Family; Forskolin; Glucose Transporter; Goals; Grant; Homeostasis; Homo; Human; Individual; Integral Membrane Protein; Length; Ligand Binding; Ligands; Maltose; Mammalian Cell; Maps; Mass Spectrum Analysis; Measurement; Mediating; Membrane; Metabolism; Modeling; Molecular; Molecular Conformation; Monosaccharides; Mutagenesis; N-terminal; Nature; Oxidative Stress; Pathway interactions; Point Mutation; Post-Translational Protein Processing; Protein Subunits; Proteins; Range; Recombinant Proteins; Reducing Agents; Research Personnel; Role; Site; Solvents; Structure; Testing; Translations; Yeasts; aqueous; glucose transport; inhibitor/antagonist; insight; programs; research study; stereochemistry; sugar; uptake

DESCRIPTION (provided by applicant): Protein-mediated facilitative glucose transport is essential for cellular metabolism, post-translation protein modification, cellular protection against oxidative stress and organismal carbohydrate homeostasis. In spite of extensive study, the mechanism of glucose transport is unknown. Our goal is to understand the molecular mechanism of protein-mediated glucose transport. To achieve this, we propose to investigate the structure and function of the human glucose transport protein GluT1. This transport protein is uniquely amenable to study being available as a purified protein from red cells, as a recombinant protein from Cos-7 cells and being amenable to function-screens by expression in glucose-transport null yeast. Specific aims 1 and 2 focus on structural determinants and functional consequences of GluT1 oligomerization in the cell membrane. Specific Aims 3 and 4 investigate transporter dynamics and catalysis. Specific Aim 1 tests the hypothesis that GluT1-specific sequence is required for GluT1 oligomerization. During the past grant cycle we discovered that cell membrane GluT1 is a homotetramer stabilized by GluT1-specific oligomerization sequence and preserved by some detergents but destabilized by others. We now challenge this hypothesis directly by GluT1 domain swapping and mutagenesis experiments asking whether transporter oligomeric structure and function are preserved. Specific Aim 2 tests the hypothesis that GluT1 oligomerization is necessary but not sufficient for glucose transport cooperativity. Some (but not all) GluT1 ligands promote cooperativity between GluT1 subunits. We have mapped ligand stereochemistry promoting cooperativity to the ligand but not yet to GluT1. We use constructs of specific aim 1 to ask whether GluT1 oligomerization is necessary for transporter cooperativity and examine GluT1 ligand binding site requirements for cooperativity in detail. Specific Aim 3 tests the hypothesis that specific GluT1 domains are conformationally dynamic and undergo accessibility changes in the presence of ligand by using mass spectrometry to map GluT1 domains exposed to solvent and to small, hydrophilic molecules that react with GluT1 in a substrate-dependent manner. Specific Aim 4 tests the hypothesis that the newly discovered "e(S)" - a transient, transport intermediate that excludes substrate from the aqueous environment - binds e1 and e2 ligands simultaneously. These biochemical and rapid quench-flow ligand binding studies critically challenge the tetramer and simple carrier models for transport and further investigate the nature of e(S) and the role of subunit cooperativity in substrate occlusion.

描述（由申请人提供）：蛋白质介导的促进性葡萄糖转运对于细胞代谢，翻译后蛋白质修饰，抗氧化应激的细胞保护和有机碳水化合物稳态至关重要。尽管进行了广泛的研究，但葡萄糖转运的机制尚不清楚。我们的目标是了解蛋白质介导的葡萄糖转运的分子机制。为了实现这一目标，我们建议研究人葡萄糖转运蛋白glut1的结构和功能。该转运蛋白可独特地研究，可作为来自红细胞的纯化蛋白，作为COS-7细胞的重组蛋白提供的纯化蛋白，并且可以通过在葡萄糖传输Null酵母中表达来适应功能屏幕。具体目的1和2关注细胞膜中GLUT1低聚的结构决定因素和功能后果。具体目的3和4研究转运蛋白动力学和催化。特定的目标1检验了GLUT1低聚需要GLUT1特异性序列的假设。在过去的赠款周期中，我们发现细胞膜glut1是一种通过GLUT1特异性寡聚序列稳定的同倍聚体，并由某些洗涤剂保存，但其他洗涤剂不稳定。现在，我们通过GLUT1域交换和诱变实验直接挑战了这一假设，询问转运蛋白的寡聚结构和功能。具体目标2检验了GLUT1低聚是必要的，但不足以进行葡萄糖转运合作的假设。一些（但不是全部）GLUT1配体促进了GLUT1亚基之间的合作。我们已经绘制了配体立体化学促进配体的合作性，但尚未促进GLUT1。我们使用特定目标1的结构来询问GLUT1寡聚化是否对于运输蛋白的协作性是必要的，并检查GLUT1配体结合位点的需求以详细研究合作。特定的目标3检验了以下假设：特定的GLUT1结构域是构象动态的，并且通过使用质谱法来映射暴露于溶剂的GLUT1结构域，并在底物依赖于底物中与Glut1反应的小型亲水性分子，从而在配体的存在下发生可及性变化。特定目标4检验了新发现的“ E（s）”的假设，即一种瞬态的转运中间体，将底物排除在水性环境之外 - 同时结合E1和E2配体。这些生化和快速淬灭的配体结合研究严重挑战了四聚体和简单的载体模型，以进一步研究E（S）的性质以及亚基合作性在底物闭塞中的作用。