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 囊泡或液泡型腺苷三磷酸酶 (V-ATPase) 是多组分、ATP 驱动的质子 泵由具有 ATP 酶活性的 V1 复合物和用于质子转移的 Vo 复合物组成 V-ATP酶在细胞内囊泡、细胞器和细胞膜的酸化中发挥重要作用。 细胞外环境，对于维持内体、溶酶体和细胞的 pH 稳态至关重要 所有真核细胞中的高尔基体缺乏 V-ATP 酶都会导致胚胎死亡，并且出现功能障碍。 V-ATP酶与许多疾病相关，包括微生物感染、肾小管酸中毒、 尽管具有重要功能，但骨质疏松症、感音神经性耳聋、神经退行性疾病和癌症。 V-ATP酶，我们对哺乳动物的生物发生、组装、调节和信号传导了解有限 研究哺乳动物 V-ATP 酶的一个主要挑战是很难获得纯复合物。 我们开发了一种创新方法来纯化大量物质。 我们的初步冷冻电子显微镜 (cryo-EM) 直接从细胞中检测人类 V-ATP 酶的同质性。 人类 V-ATP 酶的结构以高达 3.1 Å 的分辨率显示出三种功能状态，并且具有所有已知的亚基， 我们的研究表明，它们共同代表了迄今为止最完整的 V-ATP 酶机制模型。 哺乳动物V-ATP酶由蛋白质、聚糖、糖脂和脂质组成，因此，我们定义了V-ATP酶。 我们的研究开辟了全面了解 ATP 酶的领域。 基于我们之前的工作，我们将补充 V-ATP 酶的生物发生、组装、调节和信号传导。 通过生化和功能测定、酵母遗传学和质谱法测定冷冻电镜结构 分析以解决该领域的基本问题，包括糖脂在 V-ATP 酶中的作用 组装和功能、可逆组装对 V-ATP 酶的调节、质子的详细机制 该项目的完成不仅将有助于研究V-ATP酶介导的细胞信号传导机制。 提供有关 V-ATPase 组装、调节和信号传导的概念创新，同时也启发 治疗 V-ATP 酶相关疾病的新治疗策略。