Structual Characterization of the Na+/Glucose Cotransporter Family

Na/葡萄糖协同转运蛋白家族的结构表征

• 批准号: 7636904
• 负责人: Jeffrey S Abramson
• 金额: $ 28.5万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7636904
• 关键词: Affinity; Amino Acids; Area; Binding; Binding Sites; Biochemical; Biological Assay; Brain; Cells; Child; Clinical; Coupled; Coupling; Crystallization; Data; Development; Diabetes Mellitus; Diarrhea; Disease; Drug Design; Drug Prescriptions; Essential Amino Acids; Family; Fluorescence; Fluoxetine; Galactose; Genes; Glucose; Glucose Transporter; Goals; Health; Heart; Human; Hyperthyroidism; Influentials; Investigation; Iodides; Ions; Kidney; Life; Malignant Neoplasms; Malignant neoplasm of lung; Malignant neoplasm of thyroid; Measures; Mediating; Membrane Proteins; Membrane Transport Proteins; Metabolic Diseases; Methods; Modeling; Molecular; Molecular Conformation; Muscle; Mutation; Neoplasm Metastasis; Omeprazole; Oral Rehydration Therapy; Physiology; Play; Protein Isoforms; Proteins; Radioactive; Research Personnel; Resolution; Robot; Role; Screening procedure; Site; Small Intestines; Specificity; Structure; Targeted Radiotherapy; Thyroid Gland; Tissues; Vertebral column; Vibrio parahaemolyticus; Work; base; cancer therapy; cancer type; design; family structure; glucose uptake; inhibitor/antagonist; member; mutant; nanolitre; overexpression; programs; sugar; symporter; three-dimensional modeling; tool; uptake

DESCRIPTION (provided by applicant): Membrane transport proteins govern energy transduction, modify ion concentrations, and actively import metabolites into the cell. Moreover, two of the most widely prescribed drugs internationally-fluoxetine and omeprazole-target membrane transport proteins. One important class of transporters is the Na+/Glucose cotransporter family (SGLT), which contains over 240 members and is present in all kingdoms of life. There are eleven human isoforms of SGLT, expressed in a variety of tissues. Mutations in at least three of these genes, encoding the Na+/sugar symporters SGLT1 and SGLT2, as well as the Na+/iodide cotransporter (NIS), are known to result in metabolic disorders. These proteins perform essential roles in physiology and are implicated in a number of diseases, most notably infectious diarrhea, diabetes and some cancers. Given the functional and pharmacological relevance of these proteins, a 3D crystal structure from this family of cotransporters, would greatly advance our understanding of how these cotransporters work and enable us to understand their role in health, disease and therapy. We have preliminary crystallographic data on the Na+/galactose cotransporter from Vibrio parahaemolyticus (vSGLT). These crystals are grown in the presence of Na+ and galactose and are currently diffracting to 4.5Angstroms, and optimization of these crystals is in progress. Using information from other members of the SGLT family and signature sequences for sugar- and Na+-binding sites, we have identified residues that should be influential in transport. We will construct mutants of vSGLT at these residues and analyze them using radioactive uptake and fluorescence assays. Using these methods we will be able to measure transport rates, apparent affinities for Na+ and galactose, and alterations in sugar specificity. Ultimately, this biochemical and biophysical data will be analyzed in conjunction with the atomic resolution structure of vSGLT. After obtaining the structure of vSGLT and identifying important mutants, we will use the backbone coordinates for molecular threading of SGLT1, SGLT2 and NIS symporters and refine the models by using available biochemical data. These models may be able to explain some key questions about these multifunctional proteins with a diverse range of substrates and possibly facilitate drug design.

描述(由申请人提供)：膜转运蛋白控制能量转导，改变离子浓度，并主动将代谢产物输入细胞。此外，国际上最广泛使用的两种处方药--氟西汀和奥美拉唑--以膜转运蛋白为靶标。一类重要的转运蛋白是钠/葡萄糖共转运蛋白家族(SGLT)，它包含240多个成员，存在于所有生命王国中。有11种人类SGLT亚型，在各种组织中表达。这些基因中至少有三个基因突变，编码Na+/糖共转运体SGLT1和SGLT2，以及Na+/碘转运体(NIS)，已知会导致代谢紊乱。这些蛋白质在生理学上发挥着重要作用，并与许多疾病有关，最明显的是感染性腹泻、糖尿病和一些癌症。鉴于这些蛋白的功能和药理相关性，从这个共转运蛋白家族获得3D晶体结构将极大地促进我们对这些共转运蛋白如何工作的理解，并使我们能够了解它们在健康、疾病和治疗中的作用。我们已经获得了副溶血性弧菌Na+/半乳糖共转运蛋白(VSGLT)的初步结晶学数据。这些晶体是在Na+和半乳糖存在下生长的，目前衍射率为4.5埃，这些晶体的优化正在进行中。利用来自SGLT家族其他成员的信息和糖和Na+结合位点的签名序列，我们已经确定了应该在运输中有影响的残基。我们将在这些残基上构建vSGLT的突变体，并使用放射性摄取和荧光分析来分析它们。使用这些方法，我们将能够测量转运速率、Na+和半乳糖的表观亲和力以及糖专一性的变化。最终，这些生化和生物物理数据将结合vSGLT的原子分辨结构进行分析。在获得vSGLT的结构并鉴定出重要的突变体后，我们将使用SGLT1、SGLT2和NIS共转运蛋白的主干坐标来进行分子线索分析，并利用现有的生化数据来改进模型。这些模型可能能够解释这些具有不同底物的多功能蛋白质的一些关键问题，并可能有助于药物设计。