Copper-Induced Amyloidosis Studied by Mass Spectrometry

描述（由申请人提供）：已知几种人类蛋白质可形成淀粉样原纤维，这些原纤维与多种破坏性疾病有关，包括阿尔茨海默病、 帕金森病、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 (β2m)，可以在 Cu(II) 存在的情况下形成淀粉样原纤维。 beta2m形成的原纤维是DRA的主要致病过程。与其他淀粉样蛋白系统一样，β2m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。虽然淀粉样蛋白形成的许多一般方面已被了解，但几乎所有淀粉样蛋白形成反应早期阶段的分子水平信息才刚刚开始出现。不过，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。我们打算获得有关 beta2m 在原纤维形成之前的去折叠和寡聚化的氨基酸水平信息，特别强调 Cu(II) 在诱导该反应中发挥的独特作用。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结合，以获得βam及其寡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，我们将开发并应用新的标记方法与质谱相结合来研究金属-蛋白质相互作用，目的是了解 Cu(II) 在 β2m 淀粉样蛋白形成中发挥的独特作用。 第三，我们的新标记方法将用于了解 β2m 淀粉样蛋白形成的三种抑制剂的分子基础。 我们之前的工作表明，Cu(II) 会破坏 beta2m 的稳定性，使其在淀粉样原纤维之前形成寡聚物。我们假设 Cu(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 Cu(II) 的配位 化学以过渡金属中独特的方式实现这些结构和低聚物的变化。我们基于质谱的方法将使我们能够检验这一假设，并且随着拟议的抑制剂研究，我们将更深入地了解 β2m 淀粉样蛋白的形成，这将有助于设计更好的 DRA 治疗方法。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。

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.