A freezing robot for cryo-EM

用于冷冻电镜的冷冻机器人

• 批准号: 10581952
• 负责人: Stephan Wilkens
• 金额: $ 9.3万
• 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/10581952
• 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; Cryoelectron Microscopy; Diabetes Mellitus; Disease; Embryo; Endocytosis; Environment; Enzymes; Eukaryotic Cell; Extracellular Space; Freezing; 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; Robot; 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.

项目摘要 我们的实验室长期以来一直有兴趣了解 质子泵液泡ATPase(V-ATPase，V1Vo-ATPase)--一种动态的多亚单位膜积分 在所有真核细胞中发现的旋转马达酶。V-ATPase酸化细胞器的管腔，在 专业的酸性分泌细胞，胞外空间。酶功能是基本细胞所必需的 内吞作用、骨骼重塑、蛋白质运输、酸碱平衡、精子成熟、 和神经递质的释放。虽然V-ATPase功能的完全丧失是胚胎致命的，但部分丧失或 多动症与许多人类疾病有关，如骨化症、糖尿病、男性不育、 神经变性和癌症。此外，一些病毒，如流感，依赖于酸性环境 由V-ATPase产生，用于感染。在分子水平上抗击这些疾病将需要一个详细的 了解真核生物V-ATPase的结构、催化机制及其调控。在细胞中，V- ATPase活性受一种独特的机制调节，这种机制被称为“可逆分解”，其中 络合物可逆地解离成V1-ATPase和Vo质子通道，两个亚复合体都成为 自动抑制。尽管它在V-ATPase生理中起着重要的作用，但可逆的分子机制 人们对拆卸知之甚少。这一知识差距在很大程度上是由于缺乏高分辨率的 结构信息和体外模型系统，以在定义的条件下研究过程，方面 我们正在努力解决这一问题。哺乳动物V-ATPase的一个有趣的、在技术上具有挑战性的特征是 它的大部分亚基都表达为多种亚型。然而，这种异构体表现出差异性 组织浓缩，它们可能为靶向治疗提供机会。事实上，有几种疾病 与含有V-ATPase的特定异构体功能障碍或上调有关。然而，有多么不同 异构体组合决定了组织的定位，以及这些异构体特异性复合体是否具有独特的 生化或调节特性，目前尚不清楚。我们已经开始开发一种净化野生动物的系统 人类V-ATPase的类型和突变形式在生化和结构上的同工型特异性 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，探索V-ATPase活性在疾病中的异构体特异性调节。我们的 研究计划利用结构生物学、细胞生物学、生物化学和生物物理学的工具来解决 关于V-ATPase催化和调节机制的广泛问题。对于V的一些基本方面- ATPase的结构和调控，我们研究了酵母中的酶，一个有充分证据的模型系统 人类V-ATPase。我们使用人体组织培养来解决在酵母中无法解决的问题，例如 含有特定异构体的酶的结构和生化性质。我们的长期目标是 研究的目的是找到一种方法，以异构体特异性的方式调节引起V-ATPase的疾病的活动。