MOLECULAR MECHANISMS OF V-ATPASES: ASSEMBLY, BIOGENESIS, REGULATION, AND FUNCTION

V-ATP酶的分子机制：组装、生物发生、调节和功能

• 批准号: 10501202
• 负责人: Tianmin Fu
• 金额: $ 39.38万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10501202
• 关键词: ATP phosphohydrolase; Acquired Immunodeficiency Syndrome; Address; Biochemical; Biogenesis; Biological Assay; Biophysics; Cells; Complement; Complex; Cryoelectron Microscopy; Diabetes Mellitus; Disease; Embryo; Endosomes; Eukaryotic Cell; Glycolipids; Golgi Apparatus; Human; Infection; Lipids; Lysosomes; Malignant Neoplasms; Mammals; Mass Spectrum Analysis; Mediating; Membrane; Methods; Modeling; Molecular; Neurodegenerative Disorders; Organelles; Organism; Osteoporosis; Physiological Processes; Play; Polysaccharides; Proteins; Proton Pump; Protons; Regulation; Renal tubular acidosis; Resolution; Role; Sensorineural Hearing Loss; Signal Transduction; Structure; Vesicle; Work; base; experimental study; extracellular; human disease; innovation; insight; microbial; novel therapeutic intervention; pH Homeostasis; vacuolar H+-ATPase; yeast genetics

Summary Vesicular- or vacuolar-type adenosine triphosphatases (V-ATPases) are multi-component, ATP-driven proton pumps consisting of a V1 complex that possesses ATPase activity and a Vo complex for proton transfer across the membrane. V-ATPases play important roles in the acidification of intracellular vesicles, organelles, and the extracellular milieu, and are essential for maintaining the pH homeostasis of endosomes, lysosomes, and the Golgi apparatus in all eukaryotic cells. V-ATPase deficiency in mammals is embryonic lethal, and malfunction of V-ATPases is associated with numerous diseases, including microbial infection, renal tubular acidosis, osteoporosis, sensorineural deafness, neurodegenerative diseases, and cancer. Despite the critical functions of V-ATPases, we have limited understanding on the biogenesis, assembly, regulation, and signaling of mammalian V-ATPases. A major challenge in studying mammalian V-ATPases is that the pure complexes are difficult to obtain for biochemical and biophysical experiments. We developed an innovative method to purify large amounts of human V-ATPase to homogeneity directly from cells. Our preliminary cryo-electron microscopy (cryo-EM) structures of human V-ATPases show three functional states at up to 3.1 Å resolution and with all known subunits, which together represent the most complete mechanistic model of V-ATPase to date. Our study revealed that mammalian V-ATPases are composed of proteins, glycans, glycolipids, and lipids. Therefore, we defined the V- ATPase as a glycoproteolipid complex. Our study opened up the field for comprehensively understanding the biogenesis, assembly, regulation, and signaling of V-ATPases. Based on our prior work, we will complement cryo-EM structure determination with biochemical and functional assays, yeast genetics, and mass spectrometry analysis to address fundamental questions in the field, including the roles of glycolipids in the V-ATPase assembly and function, the regulation of V-ATPases by reversible assembly, the detailed mechanism of proton transfer, and the mechanisms of V-ATPase mediated cell signaling. The completion of this project will not only provide conceptual innovations regarding the V-ATPases assembly, regulation, and signaling, but also inspire new therapeutic strategies for treating V-ATPase-related diseases.

概括 囊泡或真空型腺苷三磷酸酶（V-ATPases）是多组分，ATP驱动的质子 由V1复合物组成的泵具有ATPase活性和质子转移的VO复合物 膜。 V-ATP酶在细胞内蔬菜，细胞器和细胞内蔬菜和 细胞外环境，对于维持内体，溶酶体和pH稳态至关重要 所有真核细胞中的高尔基体。哺乳动物的V-ATPase缺乏症是胚胎致死的，并且故障 V-ATP酶与许多疾病有关，包括微生物感染，肾小管酸中毒， 骨质疏松，感觉神经性死亡，神经退行性疾病和癌症。尽管功能的关键功能 V-ATP酶，我们对哺乳动物的生物发生，组装，调节和信号的了解有限 V-ATPases。研究哺乳动物V-ATP酶的主要挑战是，纯络合物很难 获得生化和生物物理实验。我们开发了一种创新的方法来净化大量 直接来自细胞的人类V-ATPase到同质性。我们的初步冷冻电子显微镜（Cryo-EM） 人类V-ATP酶的结构以最高3.1Å的分辨率显示三个功能状态，并且所有已知亚基， 迄今为止，它共同代表了V-ATPase的最完整的机械模型。我们的研究表明 哺乳动物V-ATP酶由蛋白质，糖，糖脂和脂质组成。因此，我们定义了V- ATPase作为糖蛋白胆脂配合物。我们的研究开放了该领域，以全面了解 V-ATP酶的生物发生，组装，调节和信号传导。根据我们先前的工作，我们将完成 用生化和功能测定，酵母遗传学和质谱法测定冷冻EM结构 分析以解决该领域的基本问题，包括糖脂在V-ATPase中的作用 组装和功能，通过可逆组装对V-ATP酶的调节，质子的详细机制 转移以及V-ATPase介导的细胞信号传导的机制。该项目的完成不仅将 提供有关V-ATPases组件，调节和信号的概念创新，但也启发了 治疗V-ATPase相关疾病的新理论策略。