Glucose transporter structure and function

描述(由申请人提供)：13种“葡萄糖转运蛋白”(Gluts 1-12和HMIT)催化人体内平衡的糖转运。GLUT是一个更广泛的主要促进因子超家族(MFS)转运蛋白家族的成员，这些转运蛋白催化一系列不同分子的运输，但与细胞质N-末端和C-末端共享12个跨膜螺旋(TM)的共同结构。Glug显示出独特的底物特异性和对小分子抑制的敏感性，以及一系列运输行为，包括Uniport、symport、antiport和寡聚依赖的协作性。这项提案描述了我们通过确定GLUT介导的底物运输的分子基础来理解GLUT在健康和疾病中的功能的持续努力。要做到这一点，我们必须解决：1)底物专一性的决定因素及其在GLUT结构中的位置；2)一些转运体如何催化Uniport，而另一些转运体如何催化共转运蛋白或反转运蛋白； 3)哪些转运蛋白元件促进转运蛋白寡聚化和协同性。谷氨酸非常适合于这样的研究，这些研究服从生化、分子和动力学分析。这一建议利用过剩蛋白质家族的结构相似性来回答这些问题。我们对GLUT1/GLUT3嵌合体的研究表明，GLUT1 TM9是GLUT1齐聚所必需的。特定目的1通过使用新的免疫共沉淀、化学交联和TOXCAT分析来研究wt-TM9和TM9二聚表面突变体促进嵌合体齐聚的能力，以检验TM9向相邻GLUT1亚基呈现二聚表面的假设。我们还询问其他谷氨酸是否形成同系低聚物和杂系低聚物。认识到GLUT5是细胞松弛素B(CB)不敏感的果糖转运体，不能转运2-脱氧-D-葡萄糖(2DOG)，而GLUT1是CB抑制的2DOG转运体，不转运果糖，因此特定目标2测试了GLUT序列的特定簇形成表面外和内表面底物结合位点的假设。Glut1/GLUT5嵌合体将检测GLUT5序列依赖的CB抑制丧失、2DOG转运丧失和果糖转运获得。用GLUT5/GLUT1嵌合体进行的功能研究的互易增益将验证这一结果。一种新的扫描因子Xa蛋白分解技术也将定义过剩的表面外和内表面配体结合位点。Gluts 1和Glut 3显示了“反式加速”--预先装入糖的细胞对糖的吸收加速。Glut 2和Glut 4并非如此。特异靶3通过扫描GLUT1/GLUT4或GLUT1/GLUT2嵌合体突变，验证了GLUT序列的特定簇通过扫描GLUT1/GLUT4或GLUT1/GLUT2嵌合体突变来决定反转运(反式加速)和联合转运功能的假设。将用GLUT4/GLUT1嵌合体验证结果，对嵌合体进行GLUT2/4序列依赖的反式加速损失和功能互易增益研究。扫描GLUT1/HMIT嵌合体将用于揭示H+：Myo-inositol符号所需的HMIT结构域。我们的结果将被映射到GLUT1结构上，以提供对过剩结构和功能的新见解，并将对介导生物能量稳态、溶质平衡和药物传递/耐药的MFS蛋白具有广泛的意义。与公共卫生相关：低血糖(组织葡萄糖缺乏)可由遗传、内分泌和药理学原因引起，导致癫痫发作、局灶性神经功能障碍、昏迷，如果不纠正，还会损害发育。这项提议继续我们的努力，以了解人类葡萄糖运输蛋白家族如何允许器官和细胞从血液中吸收葡萄糖和其他糖。我们从这些研究中获得的见解将影响我们对主要促进剂超家族运输蛋白这个更广泛的家族的理解。我们的长期目标是将这些见解转化为对临床低血糖的实际干预。

DESCRIPTION (provided by applicant): Thirteen "glucose transporters" (GLUTs 1-12 & HMIT) catalyze equilibrative sugar transport in humans. The GLUTs are members of a wider family of Major Facilitator Superfamily (MFS) transporters that catalyze transport of a diverse array of molecules but share a common architecture of 12 membrane-spanning helices (TMs) with cytoplasmic N- and C-termini. The GLUTs display distinctive substrate specificities and sensitivity to inhibition by small molecules and a range of transport behaviors including uniport, symport, antiport and oligomerization-dependent cooperativity. This proposal describes our continuing efforts to understand GLUT function in health and disease by determining the molecular basis of GLUT-mediated substrate transport. To do this, we must resolve: 1) Determinants of substrate specificity and their locations within GLUT architecture; 2) How some transporters catalyze uniport while others catalyze symport or antiport; 3) Which transporter elements promote transporter oligomerization and cooperativity. The GLUTs are well-suited to such studies being amenable to biochemical, molecular and kinetic analysis. This proposal exploits the structural similarities of the GLUT family of proteins to answer these questions. Our studies of GLUT1/GLUT3 chimerae show that GLUT1 TM9 is essential for GLUT1 oligomerization. Specific Aim 1 tests the hypothesis that TM9 presents a dimerization surface to adjacent GLUT1 subunits by investigating the ability of wt-TM9 and TM9 dimerization surface mutants to promote chimerae oligomerization by using novel co-immunoprecipitation, chemical crosslinking and TOXCAT assays. We also ask whether other GLUTs form homo- and hetero- oligomers. Recognizing that GLUT5 is a cytochalasn B (CB) insensitive fructose transporter that cannot transport 2-deoxy-D-glucose (2DOG) and GLUT1 is a CB-inhibited 2DOG transporter that does not transport fructose, Specific Aim 2 tests the hypothesis that specific clusters of GLUT sequence form exo- and endofacial substrate binding sites. GLUT1/GLUT5 chimerae will be assayed for GLUT5-sequence dependent loss of CB- inhibtion, loss of 2DOG transport and gain of fructose transport. Reciprocal gain of function studies with GLUT5/GLUT1 chimerae will verify the results. A novel, scanning factor Xa proteolysis technique will also define GLUT exo- and endofacial ligand binding sites. GLUTs 1 and 3 show "trans-acceleration" - accelerated sugar uptake in cells preloaded with sugar. GLUTs 2 and 4 do not. Specific Aim 3 tests the hypothesis that specific clusters of GLUT sequence determine antiport (trans-acceleration) and symport functions by scanning GLUT1/GLUT4 or GLUT1/GLUT2 chimerae mutagenesis. Chimerae will be assayed for GLUT2/4-sequence dependent loss of trans-acceleration and reciprocal gain of function studies with GLUT4/GLUT1 chimerae used to verify the results. Scanning GLUT1/HMIT chimerae will be used to expose HMIT domains required for H+:myo-inositol symport. Our results will be mapped onto GLUT1 structure to provide new insights into GLUT structure and function and will have broad significance to the MFS proteins that mediate organismal energy homeostasis, solute equilibrium and drug delivery/resistance. PUBLIC HEALTH RELEVANCE: Glycopenia (tissue glucose shortage) can have genetic, endocrine and pharmacologic origins, results in seizures, focal neurologic deficits, coma and, if uncorrected, impairs development. This proposal continues our efforts to understand how the family of human glucose transport proteins allows organs and cells to absorb glucose and other sugars from the blood. The insights we gain from these studies will impact our understanding of the wider family of Major Facilitator Superfamily transport proteins. Our long-term goal is to translate these insights into practical intervention in clinical glycopenia.