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; 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 我们的实验室对理解的催化和调节机制具有很长的兴趣 质子泵送真空极性ATPase（V-ATPase，V1VO-ATPase），一种动态的多亚基膜积分 在所有真核细胞中发现的旋转运动酶。 V-ATPase会酸化细胞器的管腔，并在 专业酸分泌细胞，细胞外空间。基本细胞需要酶功能 诸如内吞作用，骨骼重塑，蛋白质运输，酸碱平衡，精子成熟等过程 和神经递质释放。虽然V-ATPase功能的完全丧失是胚胎致死，部分损失或 多动症与众多人类疾病有关，例如骨质疏松症，糖尿病，男性不育症， 神经变性和癌症。此外，某些病毒（例如影响力）依赖于酸性环境 由V-ATPase创建的感染。在分子水平上与这些疾病作斗争将需要详细 了解真核V-ATPase的结构，催化机制和调节。在细胞中，v- ATPase活性由称为“可逆拆卸”的独特机制调节，其中 复杂的可逆性分离为V1-ATPase和Vo Proton通道，两个子复合物变为 自身抑制。尽管在V-ATPase生理学中起着重要作用，但可逆的分子机制 拆卸知之甚少。知道这一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，用于研究定义条件下的过程 我们正在努力解决。哺乳动物V-ATPase的一个有趣而技术上的挑战特征是 它的大多数亚基表示为多种同工型。但是，同工型显示差异 他们的组织富集可能会提供靶向治疗的机会。确实，几种疾病有 与含有V-ATPase的特定同工型的故障或上调有关。但是，多么不同 同工型组合决定组织定位，以及这些同工型特异性复合物是否具有独特 目前尚不清楚生化特性或调节性能。我们已经开始开发一个净化野外的系统 人类V-ATPase的类型和突变形式以同工型的特异性方式用于生化和结构 分析。此外，我们正在开发针对特定亚基同工型的单域抗体（纳米型） 作为研究工具，并探索疾病中V-ATPase活性的同工型特定调节。我们的 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-ATPase催化和调节机制的广泛问题。对于V-的某些基本方面 ATPase结构和调节，我们研究了酵母中的酶，这是一个有据可查的模型系统 人V-ATPase。我们使用人类组织文化来解决酵母无法解决的问题，例如 含有酶的特定同工型的结构和生化特性。我们的长期目标 研究是找到以特定于同工型的方式调节疾病活性的方法。