Structure of the Vacuolar ATPase

液泡 ATP 酶的结构

• 批准号: 8391694
• 负责人: Stephan Wilkens
• 金额: $ 32.32万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8391694
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Acquired Immunodeficiency Syndrome; Address; Affinity; Animals; Archaea; Bacteria; Binding; Biochemical; Bone Resorption; Bone Surface; Bone remodeling; Cell Maturation; Cell membrane; Cell physiology; Cells; Chromaffin granule; Clathrin-Coated Vesicles; Complex; Coupled; Crystallization; Cytoplasmic Tail; Defect; Deletion Mutation; Development; Diabetes Mellitus; Disease; Dissociation; Drug Design; Endosomes; Enzymes; Epithelial Cells; Eukaryota; Eukaryotic Cell; Extravasation; Funding; Goals; Golgi Apparatus; Human; Kidney; Knowledge; Light; Lysosomes; Maintenance; Malignant Neoplasms; Membrane; Molecular; Molecular Structure; Motor; Multienzyme Complexes; Osteoclasts; Osteoporosis; Peripheral; Play; Procedures; Process; Proteolipids; Proton Pump; Proton-Translocating ATPases; Protons; Regulation; Renal tubular acidosis; Request for Proposals; Research; Resolution; Roentgen Rays; Role; Rotation; Sensorineural Hearing Loss; Side; Signal Transduction; Structure; Synaptic Vesicles; System; Testing; Vacuole; Vesicle; Work; Yeasts; design; enzyme activity; enzyme mechanism; fight against; fighting; human disease; inhibitor/antagonist; insight; macromolecule; neurotransmitter release; pH Homeostasis; pathogen; polarized cell; prevent; protein transport; receptor mediated endocytosis; reconstitution; reconstruction; research study; response; vacuolar H+-ATPase

DESCRIPTION (provided by applicant): The vacuolar type H+ATPase (V1Vo- or V-ATPase) is a fundamental component of all eukaryotic cells. The complex is found in the membranes of a wide variety of intracellular compartments like clathrin-coated vesicles, chromaffin granules, endosomes, lysosomes, synaptic vesicles, Golgi derived vesicles and the yeast vacuole. In higher eukaryotes, V-type ATPases are also found in the plasma membrane of polarized cells such as osteoclasts and renal epithelial cells. Structurally similar ATPases have also been identified in the plasma membrane of Archaea and bacteria, where they are called A-ATPases and bacterial A/V-ATPases, respectively. The proton pumping action of the vacuolar ATPase plays a vital role in a large number of intra- and inter- cellular processes. In eukaryotic cells, these processes include receptor mediated endocytosis, protein trafficking, pH maintenance, storage of metabolites and neurotransmitter release. In polarized cells of higher eukaryotes, a vacuolar type ATPase is pumping protons across the plasma membrane leading to an extra- cellular acidification. Acidification of the enclosed space between the ruffled membrane of osteoclasts and the bone surface plays an important role in bone resorption and remodeling. Defects in the human vacuolar ATPase have been associated with a number of diseases such as renal tubular acidosis, sensorineural deafness, osteoporosis, diabetes and cancer. Fighting these diseases on a molecular level will require a detailed understanding of the structure and mechanism of the eukaryotic V-ATPase complex, which is the long term goal of this project. The Specific Aims of the now proposed work on the vacuolar ATPase are: (1) molecular structure and function of the vacuolar ATPase proton channel domain and (2) molecular structure and function of the V1 - Vo interface. In the first Aim, we plan to determine the atomic resolution x-ray crystal structure of the yeast vacuolar ATPase proton channel domain. In addition, we propose experiments to elucidate aspects of the mechanism of proton translocation across the isolated V-ATPase membrane domain. In the second Aim, we propose to determine the atomic resolution crystal structure of the subunit EGChead peripheral stalk complex and we will determine the molecular interactions that define the interface connecting V1-ATPase with the Vo proton channel domain. Results from the proposed studies will provide important molecular information on the mechanism of proton translocation and how the catalytic V1 ATPase sector and the membrane bound Vo proton channel domain interact to form a coupled enzyme complex. The proposed work will also shed light on the, as of yet poorly understood mechanism of V-ATPase activity regulation by regulated reversible enzyme dissociation and re-association, a mechanism now found to be involved in the development and maturation of cells in higher animals including human.

描述（由申请人提供）：液泡型H+ ATP酶（V1 Vo-或V-ATP酶）是所有真核细胞的基本组成部分。该复合物存在于多种细胞内隔室的膜中，如网格蛋白包被的囊泡、嗜铬颗粒、核内体、溶酶体、突触囊泡、高尔基体衍生的囊泡和酵母液泡。在高等真核生物中，V型ATP酶也存在于极化细胞如破骨细胞和肾上皮细胞的质膜中。结构相似的ATP酶也已在细菌和细菌的质膜中被鉴定，其中它们分别被称为A-ATP酶和细菌A/V-ATP酶。液泡ATP酶的质子泵作用在细胞内和细胞间的许多过程中起着至关重要的作用。在真核细胞中，这些过程包括受体介导的内吞作用、蛋白质运输、pH维持、代谢物储存和神经递质释放。在高等真核生物的极化细胞中，液泡型ATP酶将质子泵送穿过质膜，导致细胞外酸化。破骨细胞皱褶膜与骨表面之间封闭空间的酸化在骨吸收和骨重建中起重要作用。人空泡ATP酶的缺陷与许多疾病有关，如肾小管性酸中毒、感觉神经性耳聋、骨质疏松症、糖尿病和癌症。在分子水平上对抗这些疾病将需要详细了解真核V-ATP酶复合物的结构和机制，这是该项目的长期目标。目前拟对液泡ATP酶进行的工作的具体目标是：（1）液泡ATP酶质子通道结构域的分子结构和功能;（2）V_1- Vo界面的分子结构和功能。在第一个目标中，我们计划确定原子分辨率的X-射线晶体结构的酵母液泡ATP酶质子通道域。此外，我们提出的实验，以阐明质子跨隔离的V-ATP酶膜域易位的机制方面。在第二个目标中，我们建议确定亚基EGChead外周柄复合物的原子分辨率晶体结构，我们将确定定义V1-ATP酶与Vo质子通道结构域连接界面的分子相互作用。从拟议的研究结果将提供重要的分子信息的质子转运的机制，以及如何催化V1 ATP酶部门和膜结合Vo质子通道域相互作用，形成一个耦合的酶复合物。这项工作还将阐明迄今为止尚不清楚的通过调节可逆酶解离和再结合来调节V-ATP酶活性的机制，该机制现在被发现参与高等动物（包括人类）细胞的发育和成熟。