Progress towards structural understanding of infectious sheep PrP-amyloid

The still elusive structural difference of non-infectious and infectious amyloid of the mammalian prion protein (PrP) is a major pending milestone in understanding protein-mediated infectivity in neurodegenerative diseases. Preparations of PrP-amyloid proven to be infectious have never been investigated with a high-resolution technique. All available models to date have been based on low-resolution data. Here, we establish protocols for the preparation of infectious samples of full-length recombinant (rec) PrP-amyloid in NMR-sufficient amounts by spontaneous fibrillation and seeded fibril growth from brain extract. We link biological and structural data of infectious recPrP-amyloid, derived from bioassays, atomic force microscopy, and solid-state NMR spectroscopy. Our data indicate a semi-mobile N‑terminus, some residues with secondary chemical shifts typical of α‑helical secondary structure in the middle part between ∼115 to ∼155, and a distinct β‑sheet core C‑terminal of residue ∼155. These findings are not in agreement with all current models for PrP-amyloid. We also provide evidence that samples seeded from brain extract may not differ in the overall arrangement of secondary structure elements, but rather in the flexibility of protein segments outside the β-core region. Taken together, our protocols provide an essential basis for the high-resolution characterization of non-infectious and infectious PrP-amyloid in the near future.

哺乳动物朊蛋白（PrP）的非感染性及感染性淀粉样蛋白的结构差异至今仍是一个难以捉摸的重要里程碑，对于理解蛋白质介导的神经退行性疾病中的感染性具有重要意义。至今为止，对于已被证实具有感染性的PrP-淀粉样蛋白的制备从未使用过高分辨率技术进行过研究。迄今为止的所有模型均基于低分辨率数据。在本研究中，我们建立了通过自发性纤维化和从脑提取物中进行的种子纤维生长，以制备全长重组（rec）PrP-淀粉样蛋白感染性样本的方案，并且这些样本在核磁共振（NMR）条件下可以充分制备。我们通过生物试验、原子力显微镜和固态核磁共振光谱学，将感染性重组PrP-淀粉样蛋白的生物和结构数据联系起来。我们的数据表明，N-端具有半流动性，中间部分（约115至155之间）存在一些具有α-螺旋二级结构典型次级化学位移的残基，以及一个从约155开始的明确的β-折叠核心C端。这些发现与目前所有关于PrP-淀粉样蛋白的模型并不一致。我们还提供了证据，表明从脑提取物中种子培养的样本在二级结构元素的总体排列上可能并无差异，但在β核心区域外的蛋白质片段的灵活性上可能存在差异。综上所述，我们的方案为未来对非感染性和感染性PrP-淀粉样蛋白进行高分辨率表征提供了不可或缺的基础。