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ATPases（V-ATPases； V1VO-ATPases）是大型的多亚基蛋白复合物，在真核生物的内膜系统中发现，它们在其中酸化亚细胞隔室的内部。在较高的真核生物的极化细胞中，液泡ATPase也可以在质膜中起作用，以便将质子泵入细胞外部。液泡ATPase的质子泵送作用与大量细胞内和细胞间过程有关，例如受体介导的内吞作用，蛋白质运输，pH稳态，代谢物的储存和神经递质的释放。鉴于其广泛的性质，发现越来越多的糖尿病，癌症，骨质疏松症和艾滋病的疾病与人类液泡ATPase有缺陷有关。我们正在通过电子显微镜，X射线晶体学和溶液核磁共振光谱以及其他生物物理技术来研究这种重要酶的结构。在该项目的第一个资金期间，我们生成了哺乳动物和酵母液泡ATPase的三维结构模型。此外，通过使用差异映射，X射线晶体结构的免疫标记和拟合，我们能够确定V-ATPase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取哺乳动物和酵母菌液泡ATPase以及古细菌的ATPase的高分辨率结构数据。此外，我们建议检验许多关于ATPase驱动的液泡ATPase质子泵送活性的假设，并在体内调节。该竞争性持续建议的具体目的是：1）研究周围茎（S），2）的结构和功能，以阐明V1-ATPase域中活性沉默的机理，以确定通过电子结晶的酵母菌VOON域的亚基体系结构。从液泡ATPase的高分辨率结构数据中，希望更好地了解ATP水解供电质子泵送的催化机制以及可逆解离/放置的机制，通过该酶的活性在体内受到调节。