STRUCTURE OF THE VACUOLAR ATPase

液泡ATP酶的结构

• 批准号: 8000056
• 负责人: Stephan Wilkens
• 金额: $ 9.91万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-29 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8000056
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; ATPase Domain; Acquired Immunodeficiency Syndrome; Archaea; Architecture; Binding; Biochemical; Biological Assay; Biological Models; Cattle; Cell membrane; Cell physiology; Cells; Complex; Crystallization; Cytoplasmic Tail; DNA Sequence Rearrangement; Data; Diabetes Mellitus; Dialysis procedure; Disease; Dissociation; Electron Microscopy; Enzymes; Eukaryota; Freezing; Funding; Goals; Human; Knowledge; Label; Lipids; Maleimides; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Membrane; Methods; Modeling; Molecular Conformation; Molecular Motors; NMR Spectroscopy; Nature; Organism; Osteoporosis; Peptide Mapping; Peptides; Peripheral; Play; Positioning Attribute; Protein Complex Subunit; Proton Pump; Protons; Regulation; Relative (related person); Research Personnel; Resolution; Roentgen Rays; Role; Rotation; Solutions; Specimen; Staining method; Stains; Structural Models; Structure; System; Techniques; Testing; Work; X-Ray Crystallography; Yeasts; base; electron crystallography; enzyme activity; enzyme mechanism; improved; in vivo; monolayer; neurotransmitter release; novel; pH Homeostasis; polarized cell; prevent; programs; protein purification; protein transport; receptor mediated endocytosis; stoichiometry; vacuolar H+-ATPase

DESCRIPTION (provided by applicant): Vacuolar ATPases (V-ATPases; V1VO-ATPases) are large, multi subunit protein complexes found in the endomembrane system of eukaryotic organisms where they function to acidify the interior of subcellular compartments. In polarized cells of higher eukaryotes, the vacuolar ATPase can also function in the plasma membrane in order to pump protons to the outside of the cell. The proton pumping action of the vacuolar ATPase is involved in a large number of intra- and inter cellular processes such as receptor mediated endocytosis, protein trafficking, pH homeostasis, storage of metabolites and neurotransmitter release. Given its widespread nature, it is not surprising that more and more diseases as fundamental as diabetes, cancer, osteoporosis and AIDS are found to be associated with a defective human vacuolar ATPase. We are studying the structure of this important enzyme by electron microscopy, X-ray crystallography and solution nuclear magnetic resonance spectroscopy as well as other biophysical techniques. In the first funding period for this project, we have generated three-dimensional structural models of the mammalian- and yeast vacuolar ATPase. Furthermore, by using difference mapping, immuno labeling and fitting of X-ray crystal structures, we have been able to determine the subunit architecture of the V-ATPase complex. We are now proposing to extend our structural studies and to use a variety of independent techniques to obtain high resolution structural data for the mammalian- and yeast vacuolar ATPase and for the vacuolar like ATPase from Archaea. Furthermore, we propose to test a number of hypotheses regarding the mechanism by which the ATPase driven proton pumping activity of the vacuolar ATPase is regulated in vivo. The specific aims for this competing continuation proposal are: 1) to investigate the structure and function of the peripheral stalk(s), 2) to elucidate the mechanism of activity silencing in the V1-ATPase domain 3) to determine the subunit architecture of the yeast VO domain by electron crystallography. From the high resolution structural data for the vacuolar ATPase will hope to better understand the catalytic mechanism of ATP hydrolysis powered proton pumping and the mechanism of reversible dissociation/reassociation by which the enzyme's activity is regulated in vivo.

描述（由申请人提供）：液泡 ATP 酶（V-ATP 酶；V1VO-ATP 酶）是在真核生物内膜系统中发现的大型多亚基蛋白质复合物，其功能是酸化亚细胞区室的内部。在高等真核生物的极化细胞中，液泡 ATP 酶也可以在质膜中发挥作用，将质子泵送到细胞外。液泡 ATP 酶的质子泵作用参与大量细胞内和细胞间过程，例如受体介导的内吞作用、蛋白质运输、pH 稳态、代谢物储存和神经递质释放。鉴于其广泛的性质，越来越多的糖尿病、癌症、骨质疏松症和艾滋病等基本疾病被发现与人类液泡 ATP 酶缺陷有关，这并不奇怪。我们正在通过电子显微镜、X射线晶体学和溶液核磁共振波谱以及其他生物物理技术研究这种重要酶的结构。在该项目的第一个资助期间，我们生成了哺乳动物和酵母液泡 ATP 酶的三维结构模型。此外，通过使用差异图谱、免疫标记和 X 射线晶体结构拟合，我们已经能够确定 V-ATP 酶复合物的亚基结构。我们现在提议扩展我们的结构研究，并使用各种独立技术来获得哺乳动物和酵母液泡 ATP 酶以及来自古细菌的液泡类似 ATP 酶的高分辨率结构数据。此外，我们建议测试一些关于体内调节液泡 ATP 酶驱动的 ATP 酶质子泵活性的机制的假设。这一竞争性延续提案的具体目标是：1) 研究外周茎的结构和功能，2) 阐明 V1-ATPase 结构域中活性沉默的机制，3) 确定 V1-ATPase 结构域的亚基结构。通过电子晶体学分析酵母 VO 结构域。通过液泡 ATP 酶的高分辨率结构数据，我们将希望更好地了解 ATP 水解驱动的质子泵的催化机制以及体内调节酶活性的可逆解离/重结合机制。