Structure and Regulatory Mechanisms of the Vacuolar ATPase

液泡ATP酶的结构和调节机制

• 批准号: 10398935
• 负责人: Stephan Wilkens
• 金额: $ 40.5万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10398935
• 关键词: ATP phosphohydrolase; Acid-Base Equilibrium; Acids; Address; Albers-Schonberg disease; Binding; Biochemical; Biochemistry; Biological Models; Biophysics; Bone remodeling; Cell physiology; Cells; Cellular biology; Complex; Diabetes Mellitus; Disease; Embryo; Endocytosis; Environment; Enzymes; Eukaryotic Cell; Extracellular Space; Funding; Goals; Human; Hyperactivity; Infection; Influenza; Knowledge; Laboratories; Link; Lipids; Male Infertility; Malignant Neoplasms; Membrane; Molecular; Motor; Multienzyme Complexes; Nerve Degeneration; Organelles; Organism; Physiology; Process; Property; Protein Isoforms; Proton Pump; Protons; Regulation; Renal tubular acidosis; Request for Proposals; Research; Resolution; Role; Sperm Maturation; Structure; System; Tissues; Up-Regulation; Virus; Virus Diseases; Yeasts; fighting; human disease; human tissue; in vitro Model; inhibitor; interest; mRNA Differential Displays; microbial; mutant; nanobodies; nanodisk; neurotransmitter release; novel; programs; protein transport; structural biology; targeted treatment; tissue culture; tool; vacuolar H+-ATPase

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.

项目概要 我们的实验室对了解催化和调节机制有着长期的兴趣 质子泵空泡 ATP 酶（V-ATP 酶、V1Vo-ATP 酶），一种动态多亚基膜积分 所有真核细胞中都存在旋转运动酶 V-ATP 酶可酸化细胞器的内腔。 专业的酸分泌细胞，细胞外空间是细胞的基本功能所必需的。 内吞作用、骨重塑、蛋白质运输、酸碱平衡、精子成熟等过程， V-ATP酶功能完全丧失则胚胎致死，部分丧失或丧失则导致神经递质释放。 多动症与许多人类疾病有关，例如骨硬化症、糖尿病、男性不育症、 此外，某些病毒（例如流感）依赖于酸性环境。 在分子水平上对抗这些疾病需要详细的研究。 了解细胞中真核 V-ATP 酶的结构、催化机制和调节。 ATPase 活性由一种称为“可逆分解”的独特机制调节，因此 复合物可逆地解离成 V1-ATPase 和 Vo 质子通道，两个子复合物都变成 尽管其在 V-ATP 酶生理学中具有重要作用，但其分子机制是可逆的。 人们对反汇编知之甚少，这种知识差距很大程度上是由于缺乏高分辨率造成的。 结构信息和体外模型系统，用于研究特定条件下的过程， 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。 它的大多数亚基都表达为多种同工型，但是，这些同工型表现出差异。 组织富集，它们可能为靶向治疗提供机会 事实上，有几种疾病已经发生了。 与含有 V-ATP 酶的特定亚型的故障或上调有关，但是，有何不同。 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 生化或调节特性目前尚不清楚，我们已开始开发一种纯化野生系统。 人类 V-ATP 酶的类型和突变形式，以亚型特异性方式进行生化和结构分析 此外，我们正在开发针对特定亚基异构体的单域抗体（纳米抗体） 作为研究工具，并探索疾病中 V-ATP 酶活性的异构体特异性调节。 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来解决 V-ATP 酶催化和调节机制的广泛问题。 ATP 酶结构和调节，我们研究来自酵母的酶，这是一个有据可查的模型系统 我们使用人体组织培养来解决酵母无法解决的问题，例如 含有特定异构体的酶的结构和生化特性是我们的长期目标。 研究的目的是寻找以异构体特异性方式调节引起疾病的 V-ATP 酶活性的方法。