Structure and Regulatory Mechanisms of the Vacuolar ATPase

项目摘要 我们的实验室有一个长期的兴趣，了解催化和调节机制， 质子泵液泡ATP酶（V-ATPase，V1 Vo-ATPase）是一种动态的多亚基膜整合酶 在所有真核细胞中发现的旋转马达酶。V-ATP酶酸化细胞器内腔， 专业的酸分泌细胞，细胞外空间。酶的功能是基本细胞 例如内吞作用、骨重塑、蛋白质运输、酸碱平衡、精子成熟， 和神经递质的释放虽然V-ATP酶功能的完全丧失是胚胎致死的，但V-ATP酶功能的部分丧失或完全丧失是胚胎致死的。 活动过度与许多人类疾病如骨硬化症、糖尿病、男性不育症 神经退化和癌症。此外，一些病毒，如流感，依赖于酸性环境 由V-ATP酶产生的病毒。在分子水平上对抗这些疾病将需要一个详细的 了解真核生物V-ATP酶的结构、催化机制和调控。在细胞中，V- ATP酶活性由称为“可逆分解”的独特机制调节，其中ATP酶活性的变化是可逆的。 复合物可逆地解离成V1-ATP酶和Vo质子通道，两个亚复合物成为 自我抑制的尽管其在V-ATP酶生理学中的重要作用，但可逆性ATP酶的分子机制仍有待进一步研究。 对拆卸的理解很少。这种知识上的差距很大程度上是由于缺乏高分辨率 结构信息和体外模型系统，以在规定的条件下研究该过程， 我们正在努力解决。哺乳动物V-ATP酶的一个有趣且具有技术挑战性的特征是 它的大多数亚基都以多种亚型表达。然而，这样的同种型显示出差异， 组织富集，它们可以为靶向治疗提供机会。事实上，许多疾病 与含有V-ATP酶的特定同种型的功能障碍或上调有关。然而， 同种型组合决定组织定位，以及这些同种型特异性复合物是否具有独特的 生物化学或调节性质，目前尚不清楚。我们已经开始开发一个系统， 型和突变形式的人V-ATP酶的亚型特异性的方式，为生化和结构 分析。此外，我们正在开发针对特定亚单位同种型的单域抗体（纳米抗体） 作为研究工具，并探索疾病中V-ATP酶活性的亚型特异性调节。我们 研究计划采用结构生物学，细胞生物学，生物化学和生物物理学的工具，以解决 V-ATP酶催化和调节机制的广泛问题。对于V的一些基本方面- ATP酶的结构和调节，我们研究了来自酵母的酶，酵母是ATP酶的一个有据可查的模型系统。 人V-ATP酶。我们使用人体组织培养来解决酵母菌无法解决的问题，例如 含有酶的特定异构体的结构和生化性质。我们的长期目标 研究的目的是找到以同种型特异性方式调节致病V-ATP酶活性的方法。

Project Summary Our laboratory has a long standing interest in understanding the catalytic and regulatory mechanism of the proton pumping vacuolar ATPase (V-ATPase, V1Vo-ATPase), a dynamic multisubunit membrane integral rotary motor enzyme found in all eukaryotic cells. The V-ATPase acidifies the lumen of organelles and, in professional acid secreting cells, the extracellular space. Enzyme function is required for fundamental cellular processes such as endocytosis, bone remodeling, protein trafficking, acid-base balance, sperm maturation, and neurotransmitter release. While complete loss of V-ATPase function is embryonic lethal, partial loss or hyperactivity is associated with numerous human diseases such as osteopetrosis, diabetes, male infertility, neurodegeneration, and cancer. Moreover, some viruses such as influenza rely on the acidic environment created by the V-ATPase for infection. Fighting these diseases on a molecular level will require a detailed understanding of the structure, catalytic mechanism and regulation of the eukaryotic V-ATPase. In cells, V- ATPase activity is regulated by a unique mechanism referred to as “reversible disassembly”, wherein the complex reversibly dissociates into V1-ATPase and Vo proton channel, with both sub-complexes becoming autoinhibited. Despite its important role in V-ATPase physiology, the molecular mechanism of reversible disassembly is poorly understood. This gap in knowledge is largely due to a lack of both high-resolution structural information and an in vitro model system to study the process under defined conditions, aspects that we are working to address. An interesting, and technically challenging feature of the mammalian V-ATPase is that most of its subunits are expressed as multiple isoforms. However, as such isoforms display differential tissue enrichment, they may provide opportunities for targeted therapeutics. Indeed, several diseases have been linked to malfunction or upregulation of specific isoform containing V-ATPase. However, how different isoform combinations determine tissue localization, and whether these isoform specific complexes have unique biochemical or regulatory properties, is currently unknown. We have started to develop a system to purify wild type and mutant forms of human V-ATPase in an isoform specific fashion for biochemical and structural analyses. Further, we are developing single-domain antibodies (Nanobodies) against specific subunit isoforms to serve as research tools, and to explore isoform specific modulation of V-ATPase activity in disease. Our research program employs the tools of structural biology, cell biology, biochemistry and biophysics to address broad questions of V-ATPase catalytic and regulatory mechanisms. For some fundamental aspects of V- ATPase structure and regulation, we study the enzyme from yeast, a well documented model system for the human V-ATPase. We use human tissue culture for questions that cannot be addressed in yeast, such as structure and biochemical properties of specific isoform containing enzymes. The long term goal of our research is to find ways to modulate the activity of disease causing V-ATPases in an isoform specific way.