Copper-Induced Amyloidosis Studied by Mass Spectrometry

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有关（例如阿尔茨海默病、帕金森病）。其中之一 这些蛋白质，人类 ¿-2-微球蛋白 (¿2m)，可以在存在以下物质的情况下形成淀粉样原纤维 Cu(II)，这些原纤维被认为是透析的主要致病过程- 相关淀粉样变性（DRA）。与其他淀粉样蛋白系统一样，2m 原纤维的形成是通过部分 蛋白质解折叠，随后寡聚化，最终伸长形成成熟原纤维。 虽然一般淀粉样蛋白形成的许多方面已被了解，但分子水平的信息仍不清楚 缺乏几乎所有淀粉样蛋白形成反应的早期阶段；然而，这个信息是 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 的去折叠和寡聚化的氨基酸水平信息 在其原纤维形成之前。为此，我们将开发、优化和应用三个质量 基于光谱测定 (MS) 的方法，具有必要的时间和空间分辨率。 (1) 一种新的金属催化氧化（MCO）诱导氘标记策略将被 研究以补充我们现有的 MCO/MS 方法。建议的 MCO 引发 氘标记策略将提供更容易解释的 Cu-¿2m 结合数据， 包含有关所有铜结合氨基酸的更多信息，并且不太容易出现错误 积极的一面。 (2) 共价标记和 MS 检测将用于研究 ¿2m 的变化 Cu(II) 结合、解折叠和寡聚作用下的结构。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作为该蛋白质 从单体发展到低聚物。 (3)自上而下的电喷雾电离(ESI) 将探索测序作为分离和表征形成的蛋白质寡聚体的一种手段 由 ¿2m。蛋白质低聚物 ESI 期间发生的差异充电将用于 快速分离蛋白质寡聚体，并使用自上而下的测序来识别溶剂 在单个寡聚物中标记的可接近的氨基酸残基。 我们关于 ¿2m 原纤维形成的初步数据支持需要 Cu(II) 的模型 组织二聚体和初始四聚体，但在形成第二个四聚体时释放 和六聚体。我们将使用这三种基于 MS 的方法来研究该模型并确定 与每个寡聚状态的形成相关的结构变化。

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.