STRUCTURAL AND FUNCTIONAL ANALYSIS OF GLUCOSE TRANSPORTERS

描述(由申请人提供)：本提案的首要目标是在原子分辨率上确定葡萄糖转运体(GLUT)功能的结构基础。这将在分子水平上揭示其转运机制和参与糖尿病、癌症等疾病的病理过程。它还将为开发新药和医学检测方法提供一个合理的平台。Glut携带碳水化合物(葡萄糖、果糖、半乳糖和其他)，活细胞的主要能量来源，跨膜。到目前为止，已经鉴定和表征了14种人类谷氨酸。尽管它们具有相对较高的序列相似性，但它们在功能和组织表达上表现出显著的多样性。确定任何供过于求的三维(3D)结构将是这一基本运输系统研究的重大突破，将开辟新的研究途径。结构信息与定点突变和功能研究相结合，将使我们能够准确定位GLUT成员功能可变性的分子基础，从而能够合理地寻找特定GLUT的设计配体。为了了解葡萄糖转运蛋白的功能多样性并增加获得结构性解决方案的机会，目标包括：人葡萄糖转运蛋白(HGLUT)，以及与hGLUTS氨基酸序列具有25%以上同源性和45%同源性的几种细菌。生产了7种细菌和3种人臀部的纯化功能蛋白。到目前为止，四种细菌的结晶努力已经成功，其中两种产生了衍射晶体；一种细菌过剩的晶体的衍射分辨率高达3.1埃。我们的假设是：1)细菌Glut的三维结构将代表人类Glug的三维结构，因为活性部位残基从原核到真核物种是保守的；2)作为MFS成员，Glut具有两个对称的6-螺旋束和一个亲水性内腔，在运输机制中经历构象变化。我们的两个具体目标是：1)确定那些产生良好衍射晶体的细菌葡萄糖的3D结构。1b)产生至少一种提纯的人类过剩的衍射晶体。2a)研究纯化谷氨酸的运输。2b)建立谷氨酸的高通量结合试验，以寻找新的抑制剂。我们提出的研究结果将影响我们对Gluts的基本理解。此外，它们将通过两种方式为治疗谷氨酸相关疾病的新药的寻找提供动力：1)谷氨酸结构的确定将指导合理的药物设计；2)通过高通量筛选小分子来鉴定新的配体将为新药提供先导化合物。

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.