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: 8708887
• 负责人: RICHARD W VACHET
• 金额: $ 23.2万
• 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 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8708887
• 关键词: Alzheimer' s Disease; Amino Acids; Amyloid; Amyloid Fibrils; Amyloidosis; Binding; Chemistry; Copper; Data; Detection; Deuterium; Dialysis procedure; Disease; Doxycycline; Epigallocatechin Gallate; Event; Fluorescence; Goals; Grant; Human; Hydrogen; Ions; Label; Mass Spectrum Analysis; Measurement; Mediating; Metals; Methods; Molecular; Non-Insulin-Dependent Diabetes Mellitus; Outcomes Research; Parkinson Disease; Play; Process; Protein Region; Proteins; Reaction; Research; Resolution; Rifamycins; Role; Staging; Structure; System; Techniques; Testing; Therapeutic; Transition Elements; Transmission Electron Microscopy; Work; amyloid fibril formation; amyloid formation; base; beta-2 Microglobulin; design; dimer; human disease; inhibitor/antagonist; insight; novel strategies; oxidation; public health relevance; research study; therapeutic development; tool

DESCRIPTION (provided by applicant): Several human proteins are known to form amyloid fibrils, and these fibrils are associated with several devastating diseases, including Alzheimer's, Parkinson's, type II diabetes, and dialysis-related amyloidosis (DRA). One of these proteins, human beta-2- microglobulin (beta2m), can form amyloid fibrils in the presence of Cu(II). The fibrils formed by beta2m are the main pathogenic process underlying DRA. Like other amyloid systems, beta2m fibril formation proceeds by partial protein unfolding, subsequent oligomerization, and eventual elongation to form mature fibrils. While many general aspects of amyloid formation are understood, molecular-level information for the early stages of almost all amyloid forming reactions is only starting to emerge. This information, though, 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 beta2m that precedes its fibril formation, with a particular emphasis on the unique role that Cu(II) plays in inducing this reaction. We will also investigate the molecular basis of several inhibitors that show promise for disrupting beta2m amyloid formation. To obtain the desired insight, we have three main aims. First, we will explore a combination of covalent labeling and hydrogen/deuterium exchange with mass spectrometric detection to obtain a detailed picture of the structural changes that betam and its oligomers undergo prior to amyloid formation. Second, we will develop and apply new labeling methods in conjunction with mass spectrometry to study metal-protein interactions with the goal of understanding the unique role that Cu(II) plays in beta2m amyloid formation. Third, our new labeling approaches will be applied to understand the molecular basis of three inhibitors of beta2m amyloid formation. Our prior work has revealed that Cu(II) destabilizes beta2m, allowing it to form oligomers prior to amyloid fibrils. We hypothesize that Cu(II) initiate the amyloid reaction by repositioning several regions of the protein and that Cu(II)'s coordination chemistry enables these structural and oligomeric changes in a way that is unique among transition metals. Our mass spectrometry-based methods will allow us to test this hypothesis, and along with the proposed inhibitor studies, we will obtain a deeper understanding of beta2m amyloid formation, which will facilitate the design of better therapeutics against DRA. Moreover, we expect that the techniques developed here will be applicable to other amyloid systems.

描述(申请人提供)：已知几种人类蛋白质形成淀粉样纤维，这些纤维与几种毁灭性的疾病有关，包括阿尔茨海默氏症， 帕金森氏症、II型糖尿病和透析相关淀粉样变性(DRA)。其中一种蛋白质，人β-2-微球蛋白(Beta2m)，在铜(II)存在的情况下可以形成淀粉样纤维。β2m形成的纤维是DRA的主要致病过程。像其他淀粉样蛋白系统一样，β2m纤维的形成是通过部分蛋白质的展开，随后的寡聚，最终延伸形成成熟的纤维来进行的。虽然人们已经了解了淀粉样蛋白形成的许多一般方面，但几乎所有淀粉样蛋白形成反应的早期阶段的分子水平信息才刚刚开始出现。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药物。我们打算获得关于β2m在其纤维形成之前的展开和寡聚的氨基酸水平信息，特别强调铜(II)在诱导这一反应中所起的独特作用。我们还将研究几种有望破坏β2M淀粉样蛋白形成的抑制剂的分子基础。为了获得想要的洞察力，我们有三个主要目标。首先，我们将探索共价标记和氢/氚交换与质谱学检测相结合的方法，以获得Betam及其低聚物在淀粉样蛋白形成之前经历的结构变化的详细图像。其次，我们将开发和应用新的标记方法与质谱学相结合来研究金属-蛋白质相互作用，目的是了解铜(II)在β2M淀粉样蛋白形成中所起的独特作用。第三，我们的新标记方法将被应用于了解三种β2m淀粉样蛋白形成抑制剂的分子基础。我们先前的工作表明，铜(II)破坏了β2M的稳定，使其能够在淀粉样纤维之前形成低聚物。我们假设，铜(II)通过重新定位蛋白质的几个区域来启动淀粉样蛋白反应，并且铜(II)的S配位 化学使这些结构和低聚物发生变化，这在过渡金属中是独一无二的。我们基于质谱学的方法将使我们能够检验这一假说，随着拟议的抑制剂研究，我们将对β2m淀粉样蛋白的形成有更深入的了解，这将有助于设计出更好的治疗DRA的方法。此外，我们预计这里开发的技术将适用于其他淀粉样蛋白系统。