Department of Biotechnology, National Institute of Pharmaceutical Education and Research, Sector 67, S.A.S. Nagar, Sahibzada Ajit Singh Nagar, Punjab, 160062, India.
Eur Biophys J. 2021 Jan;50(1):59-67. doi: 10.1007/s00249-020-01486-1. Epub 2021 Jan 2.
Intracellular macromolecular crowding can lead to increased aggregation of proteins, especially those that lack a natively folded conformation. Crowding may also be mimicked by the addition of polymers like polyethylene glycol (PEG) in vitro. α-Synuclein is an intrinsically disordered protein that exhibits increased aggregation and amyloid fibril formation in a crowded environment. Two hypotheses have been proposed to explain this observation. One is the excluded volume effect positing that reduced water activity in a crowded environment leads to increased effective protein concentration, promoting aggregation. An alternate explanation is that increased crowding facilitates conversion to a non-native form increasing the rate of aggregation. In this work, we have segregated these two hypotheses to investigate which one is operating. We show that mere increase in concentration of α-synuclein is not enough to induce aggregation and consequent fibrillation. In vitro, we find a complex relationship between PEG concentrations and aggregation, in which smaller PEGs delay fibrillation; while, larger ones promote fibril nucleation. In turn, while PEG600 did not increase the rate of aggregation, PEG1000 did and PEG4000 and PEG12000 slowed it but led to a higher overall fibril burden in the latter to cases. In cells, PEG4000 reduces the aggregation of α-synuclein but in a way specific to the cellular environment/due to cellular factors. The aggregation of the similarly sized, globular lysozyme does not increase in vitro when at the same concentrations with either PEG8000 or PEG12000. Thus, natively disordered α-synuclein undergoes a conformational transition in specific types of crowded environment, forming an aggregation-prone conformer.
细胞内大分子拥挤会导致蛋白质聚集增加，尤其是那些缺乏天然折叠构象的蛋白质。在体外，通过添加聚乙二醇（PEG）等聚合物也可以模拟拥挤。α-突触核蛋白是一种无序蛋白，在拥挤的环境中会增加聚集和淀粉样纤维形成。有两种假说可以解释这一观察结果。一种是排除体积效应，假设拥挤环境中的水活度降低会导致有效蛋白质浓度增加，从而促进聚集。另一种解释是，增加拥挤度有助于转化为非天然形式，从而增加聚集的速率。在这项工作中，我们将这两种假说分开来研究哪种假说在起作用。我们表明，仅仅增加α-突触核蛋白的浓度不足以诱导聚集和随后的纤维化。在体外，我们发现 PEG 浓度与聚集之间存在复杂的关系，其中较小的 PEG 延迟纤维化；而较大的则促进纤维核的形成。相反，虽然 PEG600 不会增加聚集的速率，但 PEG1000 会，而 PEG4000 和 PEG12000 则会减缓它，但会导致后两种情况下纤维负担更高。在细胞中，PEG4000 减少了 α-突触核蛋白的聚集，但方式是特定于细胞环境/由于细胞因素。同样大小的球状溶菌酶在相同浓度下与 PEG8000 或 PEG12000 一起在体外时不会增加聚集。因此，天然无序的 α-突触核蛋白在特定类型的拥挤环境中发生构象转变，形成易于聚集的构象。
