Glucose transporter structure and function

葡萄糖转运蛋白的结构和功能

• 批准号: 8064653
• 负责人: ANTHONY CARRUTHERS
• 金额: $ 28.21万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-05-01 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8064653
• 关键词: Acceleration; Architecture; Behavior; Binding Sites; Biochemical; Biological Assay; Blood Glucose; Carbohydrates; Carrier Proteins; Cells; Chemicals; Chimera organism; Clinical; Co-Immunoprecipitations; Coma; Complex; Computer Simulation; Data; Deoxyglucose; Development; Dimerization; Disease; Drug Delivery Systems; Elements; Endocrine; Enzyme-Linked Immunosorbent Assay; Epitopes; Equilibrium; Factor Xa; Family; Focal Neurologic Deficits; Fructose; GLUT-3 protein; GLUT2 gene; GLUT4 gene; Genetic; Glucose; Glucose Transporter; Goals; Health; Homeostasis; Homo; Homology Modeling; Human; Inositol; Intervention; Ion Cotransport; Kinetics; Laboratories; Learning; Ligand Binding; Ligands; Link; Location; Maps; Mediating; Membrane; Metabolism; Molecular; Mutagenesis; Organ; Phase; Protein Family; Proteins; Proteolysis; Resistance; Resolution; SLC2A1 gene; Scanning; Seizures; Sequence Analysis; Series; Site; Site-Directed Mutagenesis; Solid; Structure; Substrate Specificity; Surface; Techniques; Testing; Tissues; Translating; Uniport; antiport; base; crosslink; gain of function; glucose transport; insight; member; mutant; novel; scaffold; small molecule; solute; sugar; uptake

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.

描述（由申请人提供）：十三种“葡萄糖转运蛋白”（GLUTs 1-12和HMIT）催化人体中的平衡糖转运。GLUT是主要易化因子超家族（MFS）转运蛋白的更广泛家族的成员，其催化多种分子的转运，但共享具有细胞质N-和C-末端的12个跨膜螺旋（TM）的共同结构。GLUT显示出独特的底物特异性和对小分子抑制的敏感性，以及一系列转运行为，包括单向转运、共向转运、反向转运和寡聚化依赖性协同性。该提案描述了我们通过确定GLUT介导的底物转运的分子基础来了解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）底物特异性的决定因素及其在GLUT结构中的位置; 2）一些转运蛋白如何催化单转运，而另一些转运蛋白如何催化同向转运或反向转运; 3)哪些转运体元件促进转运体寡聚化和协同性。GLUT非常适合于此类研究，适合于生物化学，分子和动力学分析。该提案利用GLUT蛋白质家族的结构相似性来回答这些问题。我们对GLUT 1/GLUT 3嵌合体的研究表明，GLUT 1 TM 9是GLUT 1寡聚化所必需的。特定目的1通过使用新型免疫共沉淀、化学交联和TOXCAT测定研究wt-TM 9和TM 9二聚化表面突变体促进嵌合体寡聚化的能力，检验TM 9向相邻GLUT 1亚基呈现二聚化表面的假设。我们还询问其他GLUT是否形成同源寡聚体和异源寡聚体。认识到GLUT 5是一种细胞松弛素B（CB）不敏感的果糖转运蛋白，不能转运2-脱氧-D-葡萄糖（2DOG），GLUT 1是一种CB抑制的2DOG转运蛋白，不转运果糖，特异性目的2检验了GLUT序列的特定簇形成外表面和内表面底物结合位点的假设。将测定GLUT 1/GLUT 5嵌合体的GLUT 5序列依赖性CB-转运损失、2DOG转运损失和果糖转运增加。使用GLUT 5/GLUT 1嵌合体进行的功能相互增益研究将验证结果。一种新的，扫描因子Xa蛋白水解技术也将定义GLUT外和内面配体结合位点。GLUT 1和3显示“反式加速”-在预先装载糖的细胞中加速糖摄取。GLUT 2和4不需要。特定目的3检验了GLUT序列的特定簇通过扫描GLUT 1/GLUT 4或GLUT 1/GLUT 2嵌合突变决定反向转运（反式加速）和同向转运功能的假设。将测定嵌合体的GLUT 2/4序列依赖性反式加速损失和功能倒数增益研究，使用GLUT 4/GLUT 1嵌合体验证结果。扫描GLUT 1/HMIT嵌合体将用于暴露H+：肌醇共转运所需的HMIT结构域。我们的研究结果将映射到GLUT 1结构上，为GLUT结构和功能提供新的见解，并将对介导生物体能量稳态，溶质平衡和药物递送/抗性的MFS蛋白具有广泛的意义。公共卫生相关性：血糖减少症（组织葡萄糖缺乏）可能有遗传、内分泌和药理学原因，导致癫痫发作、局灶性神经功能缺损、昏迷，如果不纠正，还会损害发育。这项提议继续我们的努力，以了解人类葡萄糖转运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更广泛的主要促进剂超家族转运蛋白家族的理解。我们的长期目标是将这些见解转化为临床低血糖症的实际干预。