Protein Amyloid Formation and Inhibition Studied by Mass Spectrometry

项目摘要 已知有几种人类蛋白质可以形成淀粉样纤维，而这些纤维与 患有毁灭性的疾病，包括阿尔茨海默氏症、帕金森氏症、II型糖尿病和透析- 相关性淀粉样变性(DRA)。蛋白质β-2-微球蛋白(β2M)在关节中形成淀粉样纤维 进行血液透析的患者，导致DRA。而蛋白质淀粉样蛋白的形成 经过广泛研究，关于淀粉样蛋白形成早期阶段的分子水平信息是 对少数几种蛋白质的研究才刚刚开始。然而，这些信息对于Rational 针对淀粉样蛋白疾病的治疗药物的发展，特别是对没有治疗方法的DRA。 我们团队一直在开发和应用新的质谱学(MS)方法来揭示 β2M在形成淀粉样纤维之前经历的结构和低聚物变化。是这样的 使用传统的生物物理方法获取结构信息非常具有挑战性，并且 我们新方法的结构性洞察力导致了关于以下第一步的令人兴奋的发现 蛋白质淀粉样蛋白的形成。我们开始利用这一洞察力来寻找DRA的抑制剂。 这个Mira项目的研究寻求(I)开发新的方法，以深化我们的 对β2M淀粉样蛋白形成的第一步的机理洞察和(Ii)利用这一新的 发现β2M淀粉样蛋白形成的强大抑制剂的机制信息。这些目标将 通过创建依赖于共价标记、离子迁移率和 计算建模。这些新方法将使我们能够探索 启动β2M淀粉样蛋白形成的结构转换，提供前所未有的数量 洞察导致这种通常稳定的蛋白质变成淀粉样蛋白的因素。我们会 也探索新的方法来表征路径上和路径外的异构体齐聚物 存在于β2M淀粉样蛋白形成的早期阶段，使我们能够揭示特定的 导致淀粉样蛋白的蛋白质-蛋白质相互作用。总而言之，我们获得的结构性见解 然后将被用于发现β2M淀粉样蛋白形成的新抑制剂。这一行动的具体结果 这项研究将成为研究蛋白质淀粉样蛋白形成和鉴定的新的生物物理工具 可能导致治疗DRA的抑制剂。技术的普适性 这项研究还将使它们适用于其他淀粉样蛋白系统和疾病。

Project Summary Several human proteins are known to form amyloid fibrils, and these fibrils are associated with devastating diseases, including Alzheimer's, Parkinson's, type II diabetes, and dialysis- related amyloidosis (DRA). The protein β-2-microglobulin (β2m) forms amyloid fibrils in the joints of patients undergoing hemodialysis, leading to DRA. While protein amyloid formation has been extensively studied, molecular-level information about the early stages of amyloid formation is only beginning to be revealed for a few proteins. This information, though, is critical for the rational development of therapeutics against amyloid diseases, particularly for DRA that has no treatment. Our group has been developing and applying new mass spectrometry (MS) methods to reveal structural and oligomeric changes that β2m undergoes before forming amyloid fibrils. Such structural information is very challenging to obtain using traditional biophysical methods, and the structural insight from our new methods has led to exciting discoveries about the first steps of protein amyloid formation. We are beginning to utilize this insight to find inhibitors of DRA. The research in this MIRA project seeks to (i) develop new methods that will deepen our mechanistic insight into the first steps of β2m amyloid formation and (ii) leverage this new mechanistic information to discover robust inhibitors of β2m amyloid formation. These goals will be accomplished by creating new MS methods that rely on covalent labeling, ion mobility, and computational modeling. These new approaches will allow us to probe the energy landscape of the structural switch that initiates β2m amyloid formation, providing unprecedented quantitative insight into the factors that cause this normally stable protein to become amyloidogenic. We will also explore new methods to characterize the on- and off-pathway isomeric oligomers that are present during the early stages of β2m amyloid formation, allowing us to reveal the specific protein-protein interactions that lead to amyloids. Altogether, the structural insights that we obtain will then be utilized to discover new inhibitors of β2m amyloid formation. Specific outcomes of this research will be new biophysical tools to study protein amyloid formation and the identification of inhibitors that could lead to therapeutics against DRA. The universality of the techniques developed in this work will also make them applicable to other amyloid systems and diseases.