STRUCTURAL AND FUNCTIONAL ANALYSIS OF GLUCOSE TRANSPORTERS

描述（由申请人提供）：本提案的总体目标是在原子分辨率上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及其在糖尿病、癌症和其他疾病病理中的作用。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通过细胞膜携带碳水化合物（葡萄糖、果糖、半乳糖等），这些是活细胞的主要能量来源。到目前为止，已经鉴定和鉴定了14个人类glut。它们在功能和组织表达方面表现出显著的可变性，尽管它们的序列相似性相对较高。确定任何GLUT的三维结构都将是研究这一基本输运系统的一个重大突破，将开辟新的研究途径。结构信息与定点诱变和功能研究相结合，将使我们能够确定GLUT成员功能变异性的分子基础，从而可以合理地为特定的GLUT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解决方案的机会，目标包括：人类葡萄糖转运蛋白（hGLUTs），以及几种与hGLUTs氨基酸序列同源性大于25%和同源性为45%的细菌转运蛋白。生产了7种细菌和3种人类GLUTs的毫克级纯化功能蛋白。到目前为止，四种细菌GLUTs的结晶工作已经成功，其中两种产生了衍射晶体；一个细菌的GLUT晶体衍射到3.1埃的分辨率。我们的假设是：1)细菌GLUTs的三维结构将代表人类GLUTs的三维结构，因为活性位点残基从原核生物到真核生物都是保守的；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，在运输机制中发生构象变化。我们的两个具体目标是：1a)确定那些产生良好衍射晶体的细菌GLUTs的3D结构。1b)制备至少一种纯化的人GLUT的衍射晶体。2a)研究纯化GLUTs的转运。2b)开发GLUTs的高通量结合试验，以发现新的抑制剂。我们提出的研究结果将影响我们对GLUTs的基本理解。此外，它们将从两个方面为寻找治疗涉及GLUTs的疾病的新药提供支持：1)GLUTs的结构确定将指导合理的药物设计；2)通过小分子的高通量筛选来鉴定新的配体将为新药的开发提供先导化合物。

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.