IFM-Department of Physics, Chemistry and Biology, Linköping University, Linköping, Sweden; School of Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India.
IFM-Department of Physics, Chemistry and Biology, Linköping University, Linköping, Sweden.
Biochim Biophys Acta Proteins Proteom. 2019 Oct;1867(10):909-921. doi: 10.1016/j.bbapap.2019.03.010. Epub 2019 Mar 30.
Misfolding and aggregation of the human prion protein (PrP) cause neurodegenerative transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease. Mature native PrP is composed of 209 residues and is folded into a C-terminal globular domain (residues 125-209) comprising a small two-stranded β-sheet and three α-helices. The N-terminal domain (residues 23-124) is intrinsically disordered. Expression of truncated PrP (residues 90-231) is sufficient to cause prion disease and residues 90/100-231 is comprising the amyloid-like fibril core of misfolded infectious PrP. During PrP fibril formation under native conditions in vitro, the disordered N-terminal domain slows down fibril formation likely due to a mechanism of initial aggregation forming morphologically disordered aggregates. The morphological disordered aggregate is a transient phase. Nucleation of fibrils occurs from this initial aggregate. The aggregate phase is largely circumvented by seeding with preformed PrP fibrils. In vivo PrP is N-glycosylated at positions Asn181 and Asn197. Little is known about the importance of these positions and their glycans for PrP stability, aggregation and fibril formation. We have in this study taken a step towards that goal by mutating residues 181 and 197 for cysteines to study the positional impact on these processes. We have further by organic synthetic chemistry and chemical modification generated synthetic glycosylations in these positions. Our data shows that residue 181 when mutated to a cysteine is a key residue for self-chaperoning, rendering a trap in the initial aggregate preventing conformational changes towards amyloid fibril formation. Position 197 is less involved in the aggregate trapping and is more geared towards β-sheet structure conversion within amyloid fibrils. As expected, synthetic glycosylated 197 is less affected towards fibril formation compared to glycosylated 181. Our data are rather compatible with the parallel in-register intermolecular β-sheet model structure of the PrP90-231 fibril and sheds light on the misfolding transitions of PrP in vitro. We hypothesize that glycosylation of position 181 is a key site for prion strain differentiation in vivo.
朊病毒蛋白（PrP）的错误折叠和聚集导致神经退行性可传播海绵状脑病，如克雅氏病。成熟的天然 PrP 由 209 个残基组成，折叠成一个 C 端球状结构域（残基 125-209），包含一个小的双链β-折叠和三个α-螺旋。N 端结构域（残基 23-124）是无规卷曲的。表达截断的 PrP（残基 90-231）足以引起朊病毒病，并且残基 90/100-231 包含错误折叠的传染性 PrP 的淀粉样原纤维核心。在体外天然条件下 PrP 纤维形成过程中，无规卷曲的 N 端结构域会减缓纤维形成速度，可能是由于初始聚集形成形态无序聚集体的机制。形态无序聚集体是一个短暂的阶段。纤维的成核从这个初始聚集体开始。通过用预形成的 PrP 纤维进行接种，很大程度上避免了聚集体阶段。体内 PrP 在位置 Asn181 和 Asn197 处被 N-糖基化。对于这些位置及其聚糖对 PrP 稳定性、聚集和纤维形成的重要性，人们知之甚少。在这项研究中，我们通过将残基 181 和 197 突变为半胱氨酸来研究这些位置对这些过程的影响，朝着这个目标迈出了一步。我们还通过有机合成化学和化学修饰在这些位置生成了合成糖基化。我们的数据表明，当突变为半胱氨酸时，残基 181 是自我伴侣的关键残基，在初始聚集体中形成陷阱，阻止向淀粉样纤维形成的构象变化。位置 197 在聚集体捕获中的参与程度较低，更倾向于淀粉样纤维内的β-折叠结构转换。正如预期的那样，与糖基化 181 相比，合成糖基化 197 对纤维形成的影响较小。我们的数据与 PrP90-231 纤维的平行对位分子内β-折叠模型结构相当吻合，并阐明了 PrP 在体外的错误折叠转变。我们假设位置 181 的糖基化是体内朊病毒株分化的关键位点。
