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)：蛋白质介导的促进葡萄糖转运对于细胞新陈代谢、翻译后蛋白质修饰、细胞对氧化应激的保护和机体碳水化合物的动态平衡是必不可少的。尽管有广泛的研究，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。为了实现这一点，我们建议研究人类葡萄糖转运蛋白Glut1的结构和功能。这种转运蛋白是唯一可以研究作为来自红细胞的纯化蛋白，作为来自Cos-7细胞的重组蛋白，并且能够通过在葡萄糖运输零酵母中表达来进行功能筛选的研究。具体目标1和2侧重于细胞膜中Glut1齐聚的结构决定因素和功能后果。特定目标3和4研究转运蛋白动力学和催化。特异性目标1验证了Glut1齐聚所需的Glut1特异序列的假设。在过去的资助周期中，我们发现细胞膜Glut1是一个由Glut1特异的寡聚序列稳定的同源四聚体，并被一些去污剂保存，但被另一些去污剂破坏。我们现在通过Glut1结构域交换和突变实验直接挑战这一假说，询问转运蛋白寡聚体的结构和功能是否被保留。特定目的2验证了Glut1寡聚对于葡萄糖转运协同性是必要的，但不是充分的假设。一些(但不是全部)Glut1配体促进Glut1亚基之间的协同作用。我们已经将促进协作性的配体立体化学映射到配体，但还没有映射到Glut1。我们使用特定目的1的构建来询问Glut1齐聚是否对于转运体的协同性是必要的，并详细地研究Glut1配体结合位点的协同性要求。特异性目标3验证了这样的假设，即特定的Glut1结构域是构象动态的，并且在配体存在的情况下经历可及性变化，方法是使用质谱学来绘制暴露于溶剂和与Glut1以底物依赖的方式反应的小的亲水分子的Glut1结构域。特定目标4验证了这样一种假设，即新发现的“e(S)”--一种将底物排除在水环境中的瞬时运输中间体--同时与e1和e2配体结合。这些生化和快速猝灭流动的配基结合研究对转运的四聚体和简单载体模型提出了严峻的挑战，并进一步探讨了e(S)的性质和亚基协同作用在底物封闭中的作用。