Department of Biochemistry, Institute of Chemistry, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
Faculty of Pharmacy, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
FASEB J. 2020 Jan;34(1):365-385. doi: 10.1096/fj.201901897R. Epub 2019 Nov 22.
Structural conversion of cellular prion protein (PrP) into scrapie PrP (PrP) and subsequent aggregation are key events associated with the onset of transmissible spongiform encephalopathies (TSEs). Experimental evidence supports the role of nucleic acids (NAs) in assisting this conversion. Here, we asked whether PrP undergoes liquid-liquid phase separation (LLPS) and if this process is modulated by NAs. To this end, two 25-mer DNA aptamers, A1 and A2, were selected against the globular domain of recombinant murine PrP (rPrP) using SELEX methodology. Multiparametric structural analysis of these aptamers revealed that A1 adopts a hairpin conformation. Aptamer binding caused partial unfolding of rPrP and modulated its ability to undergo LLPS and fibrillate. In fact, although free rPrP phase separated into large droplets, aptamer binding increased the number of droplets but noticeably reduced their size. Strikingly, a modified A1 aptamer that does not adopt a hairpin structure induced formation of amyloid fibrils on the surface of the droplets. We show here that PrP undergoes LLPS, and that the PrP interaction with NAs modulates phase separation and promotes PrP fibrillation in a NA structure and concentration-dependent manner. These results shed new light on the roles of NAs in PrP misfolding and TSEs.
细胞朊病毒蛋白（PrP）的结构转换为瘙痒 PrP（PrP），随后发生聚集，是与传染性海绵状脑病（TSE）发作相关的关键事件。实验证据支持核酸（NAs）在协助这种转换中的作用。在这里，我们询问 PrP 是否经历液-液相分离（LLPS），以及这一过程是否被 NAs 调节。为此，我们使用 SELEX 方法针对重组鼠 PrP（rPrP）的球形结构域选择了两个 25 个碱基对的 DNA 适体 A1 和 A2。对这些适体的多参数结构分析表明，A1 采用发夹构象。适体结合导致 rPrP 部分展开，并调节其进行 LLPS 和原纤维形成的能力。事实上，尽管游离 rPrP 相分离成大液滴，但适体结合增加了液滴的数量，但明显减小了其尺寸。引人注目的是，不采用发夹结构的修饰 A1 适体诱导在液滴表面形成淀粉样纤维。我们在这里表明 PrP 经历 LLPS，并且 PrP 与 NAs 的相互作用以 NA 结构和浓度依赖的方式调节相分离并促进 PrP 原纤维形成。这些结果为 NAs 在 PrP 错误折叠和 TSE 中的作用提供了新的视角。
