Structure of the Vacuolar ATPase

描述(申请人提供)：液泡型H+ATPase(V1Vo-或V-ATPase)是所有真核细胞的基本成分。该复合体存在于多种细胞内小泡、嗜铬颗粒、内小体、溶酶体、突触小泡、高尔基体衍生小泡和酵母空泡的细胞膜中。在高等真核生物中，V型ATPase也存在于破骨细胞和肾上皮细胞等极化细胞的质膜中。在古生菌和细菌的质膜中也发现了结构相似的ATPase，分别称为A-ATPase和细菌A/V-ATPase。液泡ATPase的质子泵作用在许多细胞内和细胞间的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、代谢产物的储存和神经递质的释放。在高等真核生物的极化细胞中，液泡型ATPase将质子泵过质膜，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着重要作用。人类空泡ATPase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。在分子水平上抗击这些疾病将需要详细了解真核细胞V-ATPase复合体的结构和机制，这是该项目的长期目标。目前提出的关于液泡ATPase的工作的具体目标是：(1)液泡ATPase质子通道结构域的分子结构和功能；(2)V1-Vo界面的分子结构和功能。在第一个目标中，我们计划确定酵母液泡ATPase质子通道结构域的原子分辨X射线晶体结构。此外，我们建议通过实验来阐明跨分离的V-ATPase膜结构域的质子转移机制的一些方面。在第二个目标中，我们建议确定EGChead亚基外围柄复合体的原子分辨晶体结构，并确定定义V1-ATPase与Vo质子通道结构域的界面的分子相互作用。这些研究结果将为质子转运机制以及催化的V1ATPase部分和膜结合的Vo质子通道结构域如何相互作用形成偶联酶复合体提供重要的分子信息。这项拟议的工作也将阐明目前尚不清楚的通过调节可逆酶解离和重新结合来调节V-ATPase活性的机制，这一机制现在被发现参与了包括人类在内的高等动物的细胞的发育和成熟。 与公共健康相关：液泡ATPase是一种大的、多亚单位的酶复合体，参与许多基本的细胞过程。空泡ATPase缺陷或过度活跃可能与毁灭性的人类疾病有关，如肾小管性酸中毒、骨质疏松症、糖尿病和癌症。了解这些疾病的分子起源需要详细了解引起生物大分子的疾病的分子结构。这项提案要求为研究真核细胞质子泵液泡ATPase的结构和机制提供资金，目的是从分子上了解这种酶在人类疾病中的作用。

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. PUBLIC HEALTH RELEVANCE: The vacuolar ATPase is a large, multi subunit enzyme complex that is involved in numerous fundamental cellular processes. A defective or hyper active vacuolar ATPase can be associated with devastating human diseases such as renal tubular acidosis, osteoporosis, diabetes and cancer. Understanding the molecular origin of these diseases requires detailed knowledge of the molecular structure of the disease causing bio macromolecules. This proposal requests funds for studying the structure and mechanism of the eukaryotic proton pumping vacuolar ATPase with the goal of gaining a molecular understanding of the enzyme's role in human disease.