Structural analysis of SAA-amyloid fibrils by cryo-EM and NMR

在所有蛋白质错折叠疾病中，AA淀粉样变性在研究致病淀粉样蛋白的结构、形成和传播方面具有独特的优势。有高度相关的体外培养、细胞培养和小鼠模型系统来复制淀粉样原纤维形成的关键步骤。淀粉样蛋白沉积相对较大，适合生化分析。小鼠SAA1是一种胞外蛋白，小到可以很好地进行核磁共振。这些特点使我们能够采取综合的结构生物学方法，其中我们使用冷冻电子显微镜(EM)和固态核磁共振光谱来分析纤维的结构。这些原纤维结构与疾病相关，因为它们会引起组织损伤，并导致疾病的传播和组织内蛋白质的错误折叠。该项目中使用的两种主要技术--冷冻-EM和固态核磁共振--是完全互补的。Cryo-EM可以确定总体电子密度、对称性和原丝亚结构，并服从于身体来源的纤维。固体核磁共振是表征聚集体内动态区域的理想工具。在这个项目中，我们希望通过检测小鼠脾、细胞培养来源和体外形成的纤维来补充应用这些技术来研究致病相关的AA淀粉样纤维的结构。

AA amyloidosis presents, among all protein misfolding diseases, unique advantages for studying the structure, formation and propagation of pathogenic amyloids. There are highly relevant in vitro, cell culture and mouse model systems to reproduce crucial steps of amyloid fibril formation. Amyloid deposits are comparatively large-sized and amenable for biochemical analysis. Murine SAA1 is an extracellular protein and small enough to be well accessible for NMR. These features enable us to take an integrative structural biology approach in which we use cryo-electron microscopy (EM) and solid-state NMR spectroscopy to analyze the structure of the fibrils. These fibril structures are relevant for disease because they give rise to tissue damage and underlie the spreading of disease and protein misfolding inside the tissue. The two major techniques employed in this project, cryo-EM and solid-state NMR, are perfectly complementary. Cryo-EM can determine the overall electron density, symmetry and protofilament substructure and is amenable to body-derived fibrils. Solid-state NMR is an ideal tool to characterize dynamic regions within the aggregate. In this project we want to complementarily apply these techniques to study the structure of pathogenically relevant AA amyloid fibrils by examining bona fide fibrils from murine spleen, cell culture derived and in vitro formed fibrils.