Copper-Induced Amyloidosis Studied by Mass Spectrometry

通过质谱研究铜诱导的淀粉样变性

• 批准号: 7914121
• 负责人: RICHARD W VACHET
• 金额: $ 18.76万
• 依托单位: UNIVERSITY OF MASSACHUSETTS AMHERST
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7914121
• 关键词: Alzheimer' s Disease; Amino Acids; Amyloid; Amyloid Fibrils; Amyloidosis; Binding; Charge; Complement; Copper; Data; Detection; Deuterium; Dialysis procedure; Disease; Dissociation; Electron Transport; Electrospray Ionization; Human; Individual; Label; Mass Spectrum Analysis; Measurement; Metals; Methods; Modeling; Molecular; Non-Insulin-Dependent Diabetes Mellitus; Outcomes Research; Parkinson Disease; Play; Procedures; Process; Proteins; Reaction; Reagent; Research; Resolution; Role; Solvents; Staging; Structure; Study models; System; Techniques; Therapeutic; amyloid fibril formation; amyloid formation; base; design; dimer; human disease; in vivo; insight; monomer; numb protein; oxidation; protein oligomer; public health relevance; therapeutic development; tool

DESCRIPTION (provided by applicant): An increasing number of proteins are known to form amyloid fibrils in vivo, and the formation of these fibrils is implicated in several diseases (e.g. Alzheimer's, Parkinson's). One of these proteins, human ?-2-microglobulin (?2m), can form amyloid fibrils in the presence of Cu(II), and these fibrils are presumed to be the main pathogenic process underlying dialysis- related amyloidosis (DRA). Like other amyloid systems, ?2m fibril formation proceeds by partial protein unfolding, subsequent oligomerization, and eventual elongation to form mature fibrils. While many aspects of general amyloid formation are understood, molecular-level information is lacking for the early stages of almost all amyloid forming reactions; however, this information is critical for the rational development of therapeutics against amyloid diseases like DRA. We intend to obtain amino acid-level information about the unfolding and oligomerization of ?2m that precedes its fibril formation. To do so, we will develop, optimize, and apply three mass spectrometry (MS)-based methods with the necessary temporal and spatial resolution. (1) A new metal-catalyzed oxidation (MCO) induced deuterium labeling strategy will be investigated to complement our existing MCO/MS method. The proposed MCO-induced deuterium labeling strategy will provide Cu-??2m binding data that are more easily interpreted, contain more information about all the Cu-bound amino acids, and are less prone to false positives. (2) Covalent labeling with MS detection will be used to study changes in ?2m structure upon Cu(II) binding, unfolding, and oligomerization. The covalent labeling reactions will identify changes to the solvent accessibility of different amino acids in ?2m as this protein progresses from monomer to oligomers. (3) Electrospray ionization (ESI) with top-down sequencing will be explored as a means to separate and characterize protein oligomers formed by ?2m. The differential charging that occurs during ESI of protein oligomers will be used to rapidly separate protein oligomers, and top-down sequencing will be used to identify the solvent accessible amino acid residues that are labeled in individual oligomers. Our preliminary data on ?2m fibril formation support a model in which Cu(II) is necessary to organize the dimer and an initial tetramer but is released upon formation of a second tetramer and the hexamer. We will use these three MS-based methods to study this model and identify the structural changes associated with the formation of each oligomeric state. PUBLIC HEALTH RELEVANCE: The formation of protein amyloid fibrils is associated with about 20 human diseases, including Alzheimer's, Parkinson's, and Dialysis-Related Amyloidosis (DRA), but the molecular details of how these amyloids begin to form are mostly unknown. We intend to develop new measurement tools that will provide the molecular details necessary to understand the amyloid formation of ?-2-microglobulin (?2m), which is the protein implicated in DRA. This molecular information about ?2m should inform efforts to design therapeutics against DRA but will also give insight into protein amyloid formation in general.

描述（由申请人提供）：已知越来越多的蛋白质在体内形成淀粉样原纤维，并且这些原纤维的形成与多种疾病（例如阿尔茨海默病、帕金森病）有关。这些蛋白质之一，人β-2-微球蛋白(β2m)，可以在Cu(II)存在的情况下形成淀粉样原纤维，并且这些原纤维被认为是透析相关淀粉样变性(DRA)的主要致病过程。与其他淀粉样蛋白系统一样，μ2m原纤维的形成是通过部分蛋白质解折叠、随后的寡聚化以及最终伸长形成成熟原纤维来进行的。虽然一般淀粉样蛋白形成的许多方面已被了解，但几乎所有淀粉样蛋白形成反应的早期阶段都缺乏分子水平的信息；然而，这一信息对于合理开发针对 DRA 等淀粉样蛋白疾病的治疗方法至关重要。我们打算获得有关 ?2m 在原纤维形成之前的去折叠和寡聚化的氨基酸水平信息。为此，我们将开发、优化和应用三种基于质谱 (MS) 的方法，并具有必要的时间和空间分辨率。 (1) 将研究一种新的金属催化氧化 (MCO) 诱导的氘标记策略，以补充我们现有的 MCO/MS 方法。所提出的 MCO 诱导氘标记策略将提供更容易解释的 Cu-??2m 结合数据，包含有关所有 Cu 结合氨基酸的更多信息，并且不太容易出现误报。 (2) 共价标记和 MS 检测将用于研究 Cu(II) 结合、解折叠和寡聚化时 ?2m 结构的变化。当这种蛋白质从单体发展到寡聚体时，共价标记反应将识别 ?2m 中不同氨基酸的溶剂可及性的变化。 (3) 将探索采用自上而下测序的电喷雾电离（ESI）作为分离和表征由 2m 形成的蛋白质寡聚体的方法。蛋白质寡聚物 ESI 期间发生的差异充电将用于快速分离蛋白质寡聚物，自上而下测序将用于识别单个寡聚物中标记的溶剂可及的氨基酸残基。我们关于 2m 原纤维形成的初步数据支持一个模型，其中 Cu(II) 是组织二聚体和初始四聚体所必需的，但在形成第二个四聚体和六聚体时被释放。我们将使用这三种基于 MS 的方法来研究该模型并识别与每种寡聚态形成相关的结构变化。公共健康相关性：蛋白质淀粉样原纤维的形成与大约 20 种人类疾病有关，包括阿尔茨海默病、帕金森病和透析相关淀粉样变性 (DRA)，但这些淀粉样蛋白如何开始形成的分子细节大多未知。我们打算开发新的测量工具，提供了解 β-2-微球蛋白 (β2m) 淀粉样蛋白形成所需的分子细节，β-2-微球蛋白是 DRA 中涉及的蛋白质。有关 ?2m 的分子信息将为设计针对 DRA 的疗法提供信息，同时也将有助于深入了解蛋白质淀粉样蛋白的一般形成。