Structual Characterization of the Na+/Glucose Cotransporter Family

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，并主动将代谢物输入细胞。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。一类重要的转运蛋白是Na+/葡萄糖协同转运蛋白家族（SGLT），其包含超过240个成员并且存在于所有生命王国中。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有三个基因（编码Na+/糖协同转运蛋白SGLT 1和SGLT 2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。考虑到这些蛋白质的功能和药理学相关性，来自这个共转运蛋白家族的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。我们有初步的晶体学数据的Na+/半乳糖协同转运副溶血性弧菌（vSGLT）。这些晶体在Na+和半乳糖存在下生长，目前衍射至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和糖和Na+结合位点的签名序列，我们已经确定了在运输中应该有影响力的残基。我们将在这些残基上构建vSGLT突变体，并使用放射性摄取和荧光测定法对其进行分析。使用这些方法，我们将能够测量运输速率，Na+和半乳糖的表观亲和力，以及糖特异性的改变。最终，这些生化和生物物理数据将与vSGLT的原子分辨率结构一起进行分析。在获得vSGLT的结构并鉴定出重要的突变体后，我们将使用SGLT 1、SGLT 2和NIS同向转运体的分子穿线的骨架坐标，并通过使用现有的生化数据来完善模型。这些模型可能能够解释这些多功能蛋白质与各种底物的一些关键问题，并可能促进药物设计。

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.