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

总结 囊泡型或空泡型腺苷三磷酸酶（V-ATP酶）是一种多组分、ATP驱动的质子酶 由具有ATP酶活性的V1复合物和用于质子转移的Vo复合物组成的泵 膜。V-ATP酶在细胞内囊泡、细胞器和细胞外基质的酸化中起重要作用。 细胞外环境，并为维持内体，溶酶体和细胞外环境的pH稳态所必需。 所有真核细胞中的高尔基体。哺乳动物的V-ATP酶缺乏是胚胎致死性的， V-ATP酶与许多疾病有关，包括微生物感染、肾小管酸中毒、 骨质疏松症、感觉神经性耳聋、神经变性疾病和癌症。尽管有关键的功能， V-ATP酶，我们对哺乳动物细胞的生物发生、组装、调节和信号传导的了解有限， V-ATP酶研究哺乳动物V-ATP酶的一个主要挑战是，纯复合物很难被纯化。 用于生物化学和生物物理实验。我们发明了一种新方法 将人V-ATP酶直接从细胞中纯化至均一。我们初步的冷冻电镜（cryo-EM） 人V-ATP酶的结构在高达3.1 μ m的分辨率下显示出三种功能状态并且具有所有已知的亚基， 它们一起代表了迄今为止V-ATP酶的最完整的机理模型。我们的研究显示， 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酶作为糖蛋白脂质复合物。我们的研究为全面了解 V-ATP酶的生物发生、组装、调节和信号传导。基于我们之前的工作，我们将补充 cryo-EM结构测定与生化和功能测定、酵母遗传学和质谱分析 分析以解决该领域的基本问题，包括糖脂在V-ATPase中的作用 V-ATP酶的组装与功能，V-ATP酶的可逆组装调控，质子转运的详细机制， 以及V-ATP酶介导的细胞信号转导机制。该项目的完成不仅将 提供有关V-ATP酶组装，调节和信号传导的概念创新，但也启发了 治疗V-ATP酶相关疾病的新治疗策略。

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.