STRUCTURAL AND FUNCTIONAL ANALYSIS OF GLUCOSE TRANSPORTERS

葡萄糖转运蛋白的结构和功能分析

• 批准号: 8496768
• 负责人: Jun-Yong Choe
• 金额: $ 32.43万
• 依托单位: ROSALIND FRANKLIN UNIV OF MEDICINE & SCI
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8496768
• 关键词: Active Sites; Adopted; Amino Acid Sequence; Binding; Biological Assay; Biological Markers; Carbohydrates; Cell membrane; Cells; Crystallization; Data; Detection; Detergents; Diabetes Mellitus; Disease; Drug Design; Energy-Generating Resources; Escherichia coli; Exhibits; Fluorescence; Fructose; Galactose; Glucose; Glucose Transporter; Heavy Metals; Heterogeneity; Homologous Gene; Human; Label; Lead; Life; Ligand Binding; Ligands; Lipids; Malignant Neoplasms; Medical; Membrane; Membrane Proteins; Methods; Modeling; Molecular; Molecular Bank; Molecular Conformation; Obesity; Pathology; Pharmaceutical Preparations; Phase; Production; Proteins; Research; Resolution; SLC2A1 gene; Sampling; Site; Site-Directed Mutagenesis; Solutions; Solvents; Specificity; Staging; Structure; System; Therapeutic Studies; Tissues; United States National Institutes of Health; analog; base; carbohydrate transport; design; drug discovery; empowered; glycosylation; high throughput screening; improved; inhibitor/antagonist; meetings; member; milligram; mutant; nervous system disorder; novel; programs; screening; small molecule; three dimensional structure

DESCRIPTION (provided by applicant): The overarching aim in this proposal is to determine at atomic resolution the structural basis of glucose transporter (GLUT) function. This will reveal, at a molecular level, its transport mechanism and involvement in pathologies of diabetes, cancer and other diseases. It will also provide a rational platform for developing both novel drugs and medical detection methods. GLUT carries carbohydrates (glucose, fructose, galactose and others), major energy sources for living cells, across membranes. So far fourteen human GLUTs have been identified and characterized. They exhibit significant variability in function and tissue expression despite their relatively high sequence similarity. Determining the three-dimensional (3D) structure of any GLUT will be a remarkable breakthrough in the study of this fundamental transport system that wil open new research avenues. Structural information in conjunction with site-directed mutagenesis and functional studies will allow us to pinpoint the molecular base of the functional variability of GLUT members, so that designed ligands for a particular GLUT can be rationally pursued. To understand the functional diversity of glucose transporters and increase the chances of obtaining a structural solution, the targets include: human glucose transporters (hGLUTs), and several bacterial counterparts with greater than 25% identity and 45% homology to hGLUTs amino-acid sequences. Milligram quantities of purified, functional protein for seven bacterial and three human GLUTs were produced. Thus far, crystallization efforts have been successful with four bacterial GLUTs among which two produced diffracting crystals; crystals of one bacterial GLUT diffracted up to 3.1 Angstroms resolution. Our hypotheses are that 1) the 3D structure of bacterial GLUTs will be representative for those of human GLUTs, as the active site residues are conserved from prokaryotic to eukaryotic species, and 2) as a MFS member, GLUT has two symmetric 6-helicies bundles and a hydrophilic internal cavity which undergo conformational changes during the transport mechanism. Our two specific aims are to: 1a) Determine the 3D structures of those bacterial GLUTs that yield well-diffracting crystals. 1b) Produce diffracting crystals of at least one purified human GLUT. 2a) Investigate the transport of purified GLUTs. 2b) Develop high-throughput binding assay for GLUTs in order to find novel inhibitors. The results of our proposed studies will impact our fundamental understanding of GLUTs. Furthermore, they will empower the search for novel drugs for the treatment of diseases involving GLUTs in two ways: 1) structure determination of GLUTs will guide rational drug design; 2) identification of novel ligands through high- throughput screening of small molecules will provide lead compounds for new drugs.

描述（由申请人提供）：该提案中的总体目的是在原子分辨率下确定葡萄糖转运蛋白（GLUT）功能的结构基础。这将在分子水平上揭示其转运机制以及参与糖尿病，癌症和其他疾病的病理学。它还将提供一个合理的平台，用于开发新型药物和医疗检测方法。 Glut携带碳水化合物（葡萄糖，果糖，半乳糖等），跨膜的主要能源。到目前为止，已经确定和表征了14个人的过剩。尽管它们的序列相似性相对较高，但它们在功能和组织表达方面表现出显着差异。在对这种基本运输系统的研究中，确定任何过滤的三维（3D）结构将是一个显着的突破，这将开放新的研究途径。结构信息与位置定向的诱变和功能研究结合使用，将使我们能够指出Glut成员功能变异性的分子基础，因此可以合理地追求特定特定Glut的配体。为了了解葡萄糖转运蛋白的功能多样性并增加了获得结构溶液的机会，目标包括：人类葡萄糖转运蛋白（HGLUTS）和几种具有大于25％同一性和45％同源性HGLUTS HGLUTS氨基酸性序列的细菌对应物。生产了千分之一的纯化的纯化的功能蛋白，用于七个细菌和三个人的过剩。到目前为止，结晶的努力已经成功地使用了四个细菌的过剩，其中两种产生了衍射晶体。一个细菌插入的晶体衍射为3.1埃的分辨率。 我们的假设是：1）细菌插槽的3D结构将代表人类glut的3D结构，因为活性位点残基是从定原性到真核物种的保守的，而2）作为MFS成员，Glut具有两个对称的6个螺旋式套件，并且在构型内部的腔内构型会发生了一种构型的构型的变化。我们的两个具体目的是：1a）确定那些产生良好裂片晶体的细菌过剩的3D结构。 1b）产生至少一个纯化的人口的衍射晶体。 2A）研究纯化的过剩的运输。 2b）为了找到新型抑制剂，开发了过淋浴的高通量结合测定法。我们提出的研究的结果将影响我们对过剩的基本理解。此外，他们将赋予寻找新型药物的能力，以通过两种方式治疗涉及过剩的疾病：1）结构确定过多的疾病将指导理性药物设计； 2）通过对小分子的高吞吐量筛选对新型配体的鉴定将为新药提供铅化合物。