Copper-Induced Amyloidosis Studied by Mass Spectrometry

描述（由申请人提供）：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几种疾病有关。这些蛋白质之一，人β B-2-微球蛋白（B2 m），在Cu（II）存在下可以形成淀粉样原纤维，并且这些原纤维被认为是透析相关淀粉样变性（AML）的主要致病过程。与其他淀粉样蛋白系统一样，p2 m原纤维的形成通过部分蛋白质解折叠、随后的寡聚化和最终的延伸来进行，以形成成熟的原纤维。虽然一般淀粉样蛋白形成的方面是了解的，分子水平的信息是缺乏大多数淀粉样蛋白系统;然而，这些信息是针对淀粉样蛋白疾病，如阿尔茨海默病的治疗的合理设计的关键。我们计划获得与B2 m纤维形成相关的展开和寡聚化过程的氨基酸水平信息。为此，我们将应用，优化和开发3个基于质谱（MS）的工具，并具有必要的时间和空间分辨率。（一）.具有MS检测的金属催化氧化（MCO）反应将提供关于在解折叠和低聚化期间Cu（II）-B2 m相互作用的演变的信息。还将对MCO/MS方法进行改进，以简化分析并提高该技术的时间分辨率。（二）.一种新的“失谐”MCO/MS方法将测量在（B2 m）解折叠和低聚化期间围绕Cu（II）发生的三级结构变化。将研究这种新方法的范围，包括其提供有关氨基酸与Cu（II）的相对距离的信息的潜力。（三）、基于自由基的蛋白质足迹法将用于研究B2 m结构在解折叠和寡聚化过程中的变化。过氧亚硝酸盐将作为一种新的蛋白质足迹试剂进行研究。我们假设Cu（II）结合使B2 m的N-末端区域不稳定。这种不稳定导致部分解折叠，随后的结构变化导致寡聚化将使用我们的3种互补结构分析工具来阐明。

DESCRIPTION (provided by applicant): An increasing number of proteins are known to form amyloid fibrils in vivo, and the formation of these fibrils is implicated in several diseases. One of these proteins, human (B-2-microglobulin (B2m), can form amyloid fibrils in the presence of Cu (ll), and these fibrils are presumed to be the main pathogenic process underlying dialysis-related amyloidosis (DRA). Like other amyloid systems, p2m fibril formation proceeds by partial protein unfolding, subsequent oligomerization, and eventual elongation to form mature fibrils. While aspects of general amyloid formation are understood, molecular-level information is lacking for most amyloid systems; however, this information is critical for the rational design of therapeutics against amyloid diseases like DRA. We plan to obtain amino acid-level information of the unfolding and oligomerization process associated with B2m fibrillogenesis. To do so, we will apply, optimize, and develop 3 mass spectrometry (MS)-based tools with the necessary temporal and spatial resolution. (1). Metal-catalyzed oxidation (MCO) reactions with MS detection will provide information about the evolution of Cu (ll)-B2m interactions during unfolding and oligomerization. Improvements will also be made to the MCO/MS method in order to simplify the analysis and increase the temporal resolution of this technique. (2). A new 'detuned' MCO/MS method will measure the tertiary structural changes that occur around Cu (ll) during (B2m unfolding and oligomerization. The scope of this new method will be studied, including its potential to provide information about the relative distances of amino acids from Cu (ll). (3). Radical-based protein footprinting will be used to study changes in B2m structure during unfolding and oligomerization. Peroxynitrite will be investigated as a new protein footprinting reagent. We hypothesize that Cu (ll) binding destabilizes the N-terminal region of B2m. This destabilization leads to partial unfolding, and the subsequent structural changes that lead to oligomerization will be elucidated using our 3 complementary structural analysis tools.