NMR Structure of the A-beta Peptide Inside Living Cells

活细胞内 A-β 肽的 NMR 结构

• 批准号: 9271867
• 负责人: MICHAEL Gerard ZAGORSKI
• 金额: $ 18.84万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-15 至 2020-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9271867
• 关键词: Adopted; Alzheimer' s Disease; Amino Acids; Amyloid; Amyloid Fibrils; Amyloid beta-Protein; Amyloidosis; Area; Brain; Brain Diseases; Cell Nucleus; Cells; Chemicals; Complex; Coupled; Crowding; Data; Deposition; Detection; Development; Diffusion; Disease; Disease Progression; Drug Design; Environment; Escherichia coli; Eukaryotic Cell; Event; Goals; Human; In Vitro; Isotopes; Kinetics; Label; Laboratories; Lead; Lewy Bodies; Link; Measures; Methods; Mitochondria; Monitor; NMR Spectroscopy; Neurofibrillary Tangles; Nuclear Magnetic Resonance; Parkinson Disease; Pathogenicity; Patients; Peptides; Physiological; Principal Investigator; Process; Proteins; Relaxation; Reporting; Research; Resolution; Senile Plaques; Signal Transduction; Site; Solvents; Structure; Subcellular structure; Techniques; Transgenic Mice; Work; abeta accumulation; abeta deposition; alpha synuclein; amyloid formation; aqueous; base; beta pleated sheet; experience; experimental study; extracellular; flexibility; in vivo; inhibitor/antagonist; monomer; neuron loss; neurotoxic; neurotoxicity; novel; prevent; programs; protein structure; public health relevance; recombinant peptide; three dimensional structure; tool

 DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is characterized by the abundance of intraneuronal neurofibrillary tangles and the extracellular deposition of the Aβ peptide (hereafter referred to as Aβ) into amyloid. In AD brains, the Aβ undergoes a conversion (monomer → aggregate) and eventually precipitates as β-sheet like structures that have been linked to neuronal death. The chemical mechanisms associated with these conversions remains largely unknown. Due to its high propensity to aggregate, the Aβ is extremely difficult to handle in the laboratory. Despite these difficulties, our research group was one of the first to perform solution NMR studies of the Aβ, which along with subsequent work from other labs established that the Aβ adopts a predominantly random, unfolded structure in aqueous solution at neutral pH. A major advantage of the NMR approach is that it can provide atomic level aspects of the structures and dynamics in solution that are not available with other low-resolution techniques. In this application, we will take the next important and novel step, and will use NMR to study the structural ensembles of the Aβ peptide inside living eukaryotic cells. This work will establish whether or not the Aβ structures in vitro are the same as those in living cells in vivo. This is extremely important, in that the Aβ is an "intrinsically disordered" protein, which is a major clas of proteins that have a very flexible three- dimensional structure and are particularly sensitive t environmental perturbations, and that the complex, crowded environment of the cell could potentially alter the structure and function. We will perform two Specific Aims and attempt to determine the intracellular structure and dynamics in living cells using an array of NMR experiments. We will also explore the interactions between the Aβ and mitochondria. Furthermore, the classical view is that the Aβ is deposited extracellularly, although new data from transgenic mice and human patients demonstrates that the Aβ can likewise accumulate intraneuronally and contribute to AD progression, further emphasizing the importance of in-cell NMR studies.

 描述（申请人提供）：阿尔茨海默氏病（AD）的特征是神经元内神经原纤维缠结丰富，并且 Aβ 肽（以下简称 Aβ）在细胞外沉积为淀粉样蛋白。在 AD 大脑中，Aβ 发生转化（单体）。 → 聚集）并最终沉淀为与神经死亡相关的β-折叠结构，但与这些转化相关的化学机制仍然很大程度上未知。由于 Aβ 易于聚集，因此在实验室中处理起来极其困难，尽管存在这些困难，我们的研究小组是最早对 Aβ 进行溶液核磁共振研究的研究小组之一，该研究连同其他实验室的后续工作证实了 Aβ。 NMR 方法的一个主要优点是，它可以在溶液中提供其他低分辨率技术无法提供的原子水平方面的结构和动力学。 ，我们将取下一个重要且新颖的步骤是，将使用核磁共振来研究活体真核细胞内的 Aβ 肽的结构整体，这项工作将确定体外的 Aβ 结构是否与体内活细胞中的相同。极其重要的是，Aβ是一种“本质上无序”的蛋白质，它是一类主要的蛋白质，具有非常灵活的三维结构，对环境扰动特别敏感，并且细胞内复杂、拥挤的环境我们将执行两个特定目标，并尝试使用一系列 NMR 实验来确定 Aβ 和线粒体之间的相互作用。尽管来自转基因小鼠和人类患者的新数据表明 Aβ 同样可以在神经元内积累并促进 AD 进展，但 Aβ 沉积在细胞外，这进一步强调了细胞内 NMR 研究的重要性。