Exploiting new fibril structures to understand the biophysical basis for oligomerization and toxicity of alpha-Synuclein

摘要 对帕金森病分子基础的研究最近发生了焦点的巨大转变 α-突触核蛋白 (aSyn) 的有毒早期寡聚物。了解这一有前景的新治疗靶点 偏离了对不溶性原纤维的研究，现在需要对 aSyn 错误折叠的生物物理学见解 单体以及随后组装这些有毒低聚物。这些低聚物种类的了解还少得多 比原纤维更难研究，这对生物物理学家提出了紧迫的挑战。 拟议工作的具体总体目标是确定内部和内部氨基酸相互作用的子集 在低聚物的组装和毒性中最重要的非同步单体之间的差异。多项新高 aSyn 原纤维的解析结构将作为一个令人兴奋的起点来展开详细的研究 组装早期阶段出现的结构图案。基于强有力的初步结果，我们 假设，尽管它们的结构相对混乱，但早期存在强大的、可靶向的结构基序 阶段低聚物在纤维化过程中持续存在。此外，这些主题的子集对于确定 毒性：一些促进有毒组装，而另一些则促进细胞保护性组装。高分辨率 早期低聚物的结构可能永远无法解决。如果没有结构，我们的数据将退而求其次 事情：它将指向稳定早期寡聚体的特定基序和残基，这应该是焦点 定向目标活动。 我们已经建立了一种高分辨率技术（时间和空间），即时间分辨 FRET， 使我们能够以极高的灵敏度研究细胞中 aSyn 聚集的早期阶段。我们将支持这些 通过严格的生物物理研究进行细胞观察，包括 19F NMR、双色 TIRF 显微镜和 计算建模。我们还将利用我们已建立的小分子发现技术进行创新 确定家族变体的寡聚体组装是否存在明显结构差异的方法 aSyn，以及这些组件在不同的神经元细胞系中是否有所不同。 总之，该提案将使该领域对生物物理基础有更深入的了解 aSyn 寡聚化的研究，并将得出关键氨基酸残基、折叠和毒性之间的新相关性。

Abstract Research into the molecular basis of Parkinson’s Disease has recently undergone a dramatic shift to focus on toxic, early stage oligomers of α-Synuclein (aSyn). Understanding this promising new therapeutic target, a departure from research on insoluble fibrils, now requires biophysical insight about the misfolding of aSyn monomers and subsequent assembly of these toxic oligomers. These oligomer species are far less understood than fibrils, and more difficult to study, presenting a pressing challenge to biophysicists. The specific overall goal of the proposed work is to identify a subset of amino acid interactions within and between aSyn monomers that are most important in the assembly and toxicity of oligomers. Several new high- resolution structures of aSyn fibrils will be used as an exciting starting point to launch detailed investigations into the structural motifs that are present in the early stages of assembly. Based on strong preliminary results, we hypothesize that, despite their relative structural disorder, there exist robust, targetable structural motifs in early stage oligomers that persist through fibrilization. Additionally, a subset of those motifs is essential in determining toxicity: some promote toxic assemblies while others promote cytoprotective assemblies. High-resolution structures of early-stage oligomers will likely never be solved. Absent structures, our data will do the next best thing: it will point to specific motifs and residues that stabilize early-stage oligomers and that should be the focus of directed targeting campaigns. We have established a highly resolved technology (both temporally and spatially), time-resolved FRET, that allows us to study with great sensitivity the early-stages of aSyn aggregation in the cell. We will support these cellular observations with rigorous biophysical studies including 19F NMR, two-color TIRF microscopy and computational modeling. We will also utilize our established small molecule discovery technology in an innovative way to establish whether there are clear structural differences in oligomeric assemblies of the familial variants of aSyn, and whether these assemblies vary in differing neuronal cell lines. In sum, the proposal will provide the field with a significantly deeper understanding of the biophysical basis of aSyn oligomerization and will draw new correlations between key amino-acid residues, folding and toxicity.