How glucose transporter structure affects its function

葡萄糖转运蛋白结构如何影响其功能

• 批准号: 8733151
• 负责人: ANTHONY CARRUTHERS
• 金额: $ 29.15万
• 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 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8733151
• 关键词: Address; Adopted; Affect; Affinity; Amino Acids; Architecture; Behavior; Binding; Biochemical Genetics; Carbohydrates; Carrier Proteins; Cell membrane; Cells; Characteristics; Clinical; Coma; Complex; Cytochalasin B; Detergents; Deuterium; Development; Dimerization; Disease; Endocrine; Erythrocytes; Extracellular Domain; Family; Fructose; GLUT-3 protein; GLUT4 gene; Genetic; Glucose; Glucose Transporter; HIV Protease Inhibitors; Health; Homeostasis; Homo; Homologous Gene; Homology Modeling; Human; Hydrogen; Insulin Resistance; Intervention; Kidney; Knowledge; Lead; Learning; Lipid Bilayers; Mass Spectrum Analysis; Mediating; Membrane; Micelles; Modeling; Molecular; Molecular Conformation; Mutagenesis; Mutate; Mutation; Neurologic Deficit; Pathway interactions; Process; Protein Binding; Protein Family; Protein Isoforms; Protein Subunits; Protein translocation; Proteins; SLC2A1 gene; Scanning; Seizures; Solvents; Specificity; Structure; Syndrome; Testing; Tissues; Tritium; Tryptophan; absorption; alpha helix; crosslink; dimer; glucose transport; human disease; inhibitor/antagonist; insight; prevent; public health relevance; research study; scaffold; screening; sugar; tandem mass spectrometry; tumor

DESCRIPTION (provided by applicant): Glucose transport proteins (GLUTs) catalyze equilibrative, cellular sugar transport, are essential for carbohydrate homeostasis in humans, contain 12 membrane-spanning alpha helices (TMs) which form a translocation pathway (amphipathic TMs 1, 2, 4, 5, 7, 8, 10 and 11) stabilized by a scaffold (hydrophobic TMs 3, 6, 9 and 12) and adopt 3 conformational states: the inward, the occluded and the outward orientations. Gaps in our understanding of the GLUTs are: 1) How substrates bind; 2) How substrates are translocated; 3) How GLUTs oligomerize. We address these gaps and ask how each process is altered in a human disease - GLUT1 deficiency syndrome (GLUT1-DS). Specific Aim 1 tests the hypothesis that substrates bind in the GLUT translocation pathway. GLUT1 transports glucose but not fructose while GLUT5 transports fructose not glucose. Mutagenesis studies suggest that TMs 2, 7 and 11 contribute to GLUT5 fructose binding and TMs 5, 7, 8, 10 and 11 to GLUT1 glucose binding. Using homology scanning mutagenesis, we ask if substitution of specific GLUT5 translocation TMs into GLUT1 converts GLUT1 from a glucose to a fructose transporter and vice versa and we ask which residues are critical for binding. Mass spectrometry of purified human GLUT1 will reveal which GLUT1 residues are modified by photo-affinity substrates/antagonists and which are substrate- protected against hydrogen-deuterium exchange (H/DX)? We then ask if critical substrate binding residues are altered in GLUT1-DS. Specific Aim 2 tests the hypothesis that transport involves translocation pathway conformational changes controlled by scaffold TM6 - pathway TM1 interactions. GLUT1 hydrogens (80%) undergo conformation-sensitive exchange with solvent tritium or deuterium but which amino acids exchange is unknown. We will identify them by H/DX-mass spectrometry of purified human GLUT1 trapped in inward, occluded and outward orientations. GLUT1-GLUT4 homology scanning mutagenesis shows that TM6 controls the rate of conformational change between inward and outward GLUT1 and GLUT4 orientations by interacting with TM1. We ask if this is a hallmark of all GLUTs, we investigate which TM6 residues interact with TM1 by tryptophan scanning and dicysteine cross-linking mutagenesis and ask if these residues are altered in GLUT1-DS. Specific Aim 3 tests the hypothesis that GLUT dimerization and tetramerization determine transport efficiency and are mediated by specific translocation and scaffold TMs. GLUT1 forms homo- dimers and homo-tetramers whereas GLUTs 2-4 form homo-dimers. The GLUTs do not hetero-oligomerize. Substituting GLUT1 scaffold TM9 into GLUT3 allows GLUT3 to tetramerize and to form heterocomplexes with GLUT1. GLUT3 TM9 causes GLUT1 to dissociate into dimers. Pathway TMs 5, 8, 2 and 11 may be responsible for isoform specific dimerization. We propose to test this hypothesis directly and outline experiments to investigate how GLUT activity is affected by oligomeric state and whether mutations that cause GLUT1-DS also affect GLUT1 quaternary structure and/or the effects of quaternary structure on function.

描述（由申请人提供）：葡萄糖转运蛋白（GLUTs）催化平衡，细胞糖转运，是人体碳水化合物稳态所必需的，包含12个跨膜α螺旋（TMs），形成易位途径（两性TMs 1, 2, 4, 5, 7, 8, 10和11），由支架稳定（疏水性TMs 3, 6, 9和12），并采用3种构象状态：向内，封闭和向外取向。我们对GLUTs的理解存在以下空白：1)底物如何结合；2)底物如何移位；3) glut是如何寡聚的。我们解决了这些差距，并询问每个过程如何在人类疾病- GLUT1缺乏症（GLUT1- ds）中改变。特异性目的1验证了底物在GLUT转运途径中结合的假设。GLUT1转运葡萄糖而不是果糖，GLUT5转运果糖而不是葡萄糖。诱变研究表明，tms2、7和11参与GLUT5果糖结合，tms5、7、8、10和11参与GLUT1葡萄糖结合。使用同源扫描诱变技术，我们询问是否将特定的GLUT5易位TMs替换为GLUT1将GLUT1从葡萄糖转运体转化为果糖转运体，反之亦然，我们询问哪些残基对结合至关重要。纯化的人GLUT1的质谱分析将揭示哪些GLUT1残基被光亲和底物/拮抗剂修饰，哪些被底物保护以防止氢-氘交换（H/DX）？然后我们问关键底物结合残基是否在GLUT1-DS中改变。特异性目的2验证了转运涉及转运途径构象改变的假设，构象改变由支架TM6 -途径TM1相互作用控制。GLUT1氢（80%）与溶剂氚或氘进行构象敏感交换，但氨基酸交换未知。我们将通过H/ dx -质谱法鉴定纯化的人GLUT1在内向、封闭和外向的方向。GLUT1-GLUT4同源扫描诱变表明，TM6通过与TM1的相互作用控制GLUT1和GLUT4向内和向外取向的构象变化速率。我们询问这是否是所有glut1的标志，我们通过色氨酸扫描和二半胱氨酸交联诱变研究TM6残基与TM1的相互作用，并询问这些残基是否在GLUT1-DS中发生改变。特异性目的3验证了GLUT二聚化和四聚化决定转运效率的假设，并由特异性易位和支架TMs介导。GLUT1形成同二聚体和同四聚体，而glut2 -4形成同二聚体。GLUTs不发生异聚。将GLUT1支架TM9替换为GLUT3可以使GLUT3四聚并与GLUT1形成异位复合物。glut3tm9导致GLUT1解离成二聚体。途径TMs 5、8、2和11可能与异构体特异性二聚化有关。我们建议直接验证这一假设，并概述实验来研究GLUT活性如何受到寡聚物状态的影响，以及导致GLUT1- ds的突变是否也影响GLUT1的四级结构和/或四级结构对功能的影响。