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

基本信息

批准号：
7448704
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
项目类别：
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/7448704
关键词：
Affinity Amino Acids Area Binding Binding Sites Biochemical Biological Assay Brain Cells Child Class Clinical Condition 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 Numbers Omeprazole Oral Rehydration Therapy Physiology Play Protein Isoforms Protein Overexpression Proteins Radioactive Range Rate Research Personnel Resolution Robot Role Screening procedure Site Small Intestines Specificity Structure Targeted Radiotherapy Thyroid Gland Thyroid carcinoma Tissues Vertebral column Vibrio parahaemolyticus Work base cancer therapy cancer type design family structure glucose uptake inhibitor/antagonist member mutant nanolitre programs sugar symporter three-dimensional modeling tool uptake

项目摘要

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，并主动将代谢物导入细胞。此外，国际上最广泛使用的两种药物——氟西汀和奥美拉唑——靶向膜转运蛋白。 Na+/葡萄糖协同转运蛋白家族 (SGLT) 是一类重要的转运蛋白，它包含 240 多个成员，存在于所有生命领域。 SGLT 有 11 种人类亚型，在多种组织中表达。已知这些基因中至少三个编码 Na+/糖同向转运蛋白 SGLT1 和 SGLT2 以及 Na+/碘协同转运蛋白 (NIS) 的突变会导致代谢紊乱。这些蛋白质在生理学中发挥着重要作用，并与许多疾病有关，尤其是感染性腹泻、糖尿病和一些癌症。鉴于这些蛋白质的功能和药理学相关性，该协同转运蛋白家族的 3D 晶体结构将极大地促进我们对这些协同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。我们拥有副溶血弧菌 (vSGLT) 的 Na+/半乳糖协同转运蛋白的初步晶体学数据。这些晶体在 Na+ 和半乳糖存在下生长，目前衍射度达到 4.5 埃，这些晶体的优化正在进行中。利用 SGLT 家族其他成员的信息以及糖和 Na+ 结合位点的特征序列，我们已经确定了对运输有影响的残基。我们将在这些残基处构建 vSGLT 突变体，并使用放射性吸收和荧光测定法对其进行分析。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特异性的变化。最终，这些生化和生物物理数据将结合 vSGLT 的原子分辨率结构进行分析。在获得 vSGLT 的结构并识别重要突变体后，我们将使用 SGLT1、SGLT2 和 NIS 同向转运蛋白的分子线索的骨架坐标，并利用现有的生化数据完善模型。这些模型可能能够解释有关这些具有多种底物的多功能蛋白质的一些关键问题，并可能促进药物设计。