Copper-Induced Amyloidosis Studied by Mass Spectrometry

描述（由申请人提供）：已知几种人类蛋白质形成淀粉样原纤维，并且这些原纤维与几种破坏性疾病相关，包括阿尔茨海默病， 帕金森氏症、II型糖尿病和透析相关淀粉样变性（dialysis-related amyloidosis，AAD）。这些蛋白质之一，人类β-2-微球蛋白（β 2m），可以在Cu（II）的存在下形成淀粉样纤维。β 2m形成的纤维是引起纤维化的主要致病过程。与其他淀粉样蛋白系统一样，β 2m原纤维的形成是通过部分蛋白质解折叠、随后的寡聚化和最终的伸长来进行的，以形成成熟的原纤维。虽然淀粉样蛋白形成的许多一般方面已经了解，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 合理开发淀粉样蛋白疾病（如糖尿病）的治疗方法。我们打算获得氨基酸水平的信息展开和寡聚化的beta2m之前，其原纤维的形成，特别强调的独特作用，铜（II）在诱导这一反应中发挥。我们还将研究几种抑制剂的分子基础，这些抑制剂有望破坏β 2m淀粉样蛋白的形成。为了获得所需的洞察力，我们有三个主要目标。 首先，我们将探索共价标记和氢/氘交换与质谱检测的组合，以获得β内酰胺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片。 其次，我们将开发和应用新的标记方法，结合质谱法来研究金属-蛋白质相互作用，目的是了解Cu（II）在β 2m淀粉样蛋白形成中的独特作用。 第三，我们的新标记方法将被应用于了解β 2m淀粉样蛋白形成的三种抑制剂的分子基础。 我们先前的工作已经揭示了Cu（II）使β 2m不稳定，使其在淀粉样蛋白原纤维之前形成低聚物。我们假设Cu（II）通过重新定位蛋白质的几个区域启动淀粉样蛋白反应，并且Cu（II）的配位 化学使得这些结构和低聚物的变化在过渡金属中是独特的。我们基于质谱的方法将使我们能够测试这一假设，并且沿着所提出的抑制剂研究，我们将获得对β 2 m淀粉样蛋白形成的更深入的理解，这将有助于设计更好的抗β 2 m的治疗方法。此外，我们希望这里开发的技术将适用于其他淀粉样蛋白系统。

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.