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(申请人提供)：已知越来越多的蛋白质在体内形成淀粉样纤维，这些纤维的形成与几种疾病(如阿尔茨海默氏症、帕金森氏症)有关。其中，人β-2-微球蛋白(？2M)可在铜(II)存在下形成淀粉样纤维，这些纤维被认为是透析相关淀粉样变性(DRA)的主要致病过程。像其他淀粉样蛋白系统一样，2M原纤维的形成是通过部分蛋白质的展开、随后的寡聚和最终的伸长形成成熟的原纤维。虽然一般淀粉样蛋白形成的许多方面都已被了解，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，这些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在其原纤维形成之前的展开和寡聚的氨基酸水平的信息。为此，我们将开发、优化和应用三种基于质谱学(MS)的方法，并提供必要的时间和空间分辨率。(1)我们将研究一种新的金属催化氧化(MCO)诱导的氚标记方法，以补充我们现有的MCO/MS方法。所提出的MCO诱导的氚标记策略将提供更容易解释的铜-？2M结合数据，包含更多关于所有铜结合氨基酸的信息，并且不容易出现假阳性。(2)共价标记和MS检测将用于研究铜(II)结合、去折叠和齐聚过程中β2M结构的变化。共价标记反应将识别不同氨基酸在？2M内从单体到低聚物过程中溶剂可及性的变化。(3)电喷雾电离(ESI)和自上而下测序将作为分离和表征~2M形成的蛋白质低聚体的一种手段。在蛋白质低聚物的ESI过程中发生的不同电荷将被用于快速分离蛋白质低聚物，并将使用自上而下的测序来识别在单个低聚物中标记的可溶氨基酸残基。我们关于2M纤维形成的初步数据支持这样一个模型，在该模型中，铜(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释放。我们将使用这三种基于MS的方法来研究这一模型，并确定与每个低聚态的形成相关的结构变化。公共卫生相关性：蛋白质淀粉样纤维的形成与大约20种人类疾病有关，包括阿尔茨海默氏症、帕金森氏症和透析相关的淀粉样变性(DRA)，但这些淀粉样蛋白如何开始形成的分子细节大多尚不清楚。我们打算开发新的测量工具，提供必要的分子细节，以了解β-2-微球蛋白(？2M)的淀粉样蛋白的形成，这是与DRA有关的蛋白质。这些关于2M的分子信息应该有助于设计治疗DRA的药物，但也将使人们对蛋白质淀粉样蛋白的形成有更深入的了解。