Centre for Protein Science, Design and Engineering, Indian Institute of Science Education and Research Mohali, Punjab 140306, India.
Department of Biological Sciences, Indian Institute of Science Education and Research Mohali 140306 Punjab, India.
Proc Natl Acad Sci U S A. 2021 Nov 9;118(45). doi: 10.1073/pnas.2100968118.
Biomolecular condensation via liquid-liquid phase separation of intrinsically disordered proteins/regions (IDPs/IDRs) along with other biomolecules is proposed to control critical cellular functions, whereas aberrant phase transitions are associated with a range of neurodegenerative diseases. Here, we show that a disease-associated stop codon mutation of the prion protein (PrP) at tyrosine 145 (Y145Stop), resulting in a truncated, highly disordered, N-terminal IDR, spontaneously phase-separates into dynamic liquid-like droplets. Phase separation of this highly positively charged N-terminal segment is promoted by the electrostatic screening and a multitude of weak, transient, multivalent, intermolecular interactions. Single-droplet Raman measurements, in conjunction with an array of bioinformatic, spectroscopic, microscopic, and mutagenesis studies, revealed a highly mobile internal organization within the liquid-like condensates. The phase behavior of Y145Stop is modulated by RNA. Lower RNA:protein ratios promote condensation at a low micromolar protein concentration under physiological conditions. At higher concentrations of RNA, phase separation is abolished. Upon aging, these highly dynamic liquid-like droplets gradually transform into ordered, β-rich, amyloid-like aggregates. These aggregates formed via phase transitions display an autocatalytic self-templating characteristic involving the recruitment and binding-induced conformational conversion of monomeric Y145Stop into amyloid fibrils. In contrast to this intrinsically disordered truncated variant, the wild-type full-length PrP exhibits a much lower propensity for both condensation and maturation into amyloids, hinting at a possible protective role of the C-terminal domain. Such an interplay of molecular factors in modulating the protein phase behavior might have much broader implications in cell physiology and disease.
生物分子通过无序蛋白质/区域（IDPs/IDRs）与其他生物分子的液-液相分离进行凝聚被认为可以控制关键的细胞功能，而异常的相转变与一系列神经退行性疾病有关。在这里，我们表明，朊病毒蛋白（PrP）在酪氨酸 145 处的疾病相关终止密码子突变（Y145Stop）导致截短的、高度无序的 N 端 IDR 自发地相分离成动态的液态样液滴。这种带正电荷的 N 端片段的相分离是通过静电屏蔽和大量弱、瞬时、多价、分子间相互作用来促进的。单液滴拉曼测量，结合一系列生物信息学、光谱学、显微镜学和诱变研究，揭示了液态样凝聚物内高度可移动的内部组织。Y145Stop 的相行为受 RNA 调节。在生理条件下，较低的 RNA:蛋白质比在低微摩尔蛋白质浓度下促进凝聚。在较高浓度的 RNA 下，相分离被取消。随着老化，这些高度动态的液态样液滴逐渐转化为有序的、富含β的、类淀粉样聚集物。这些通过相转变形成的聚集物表现出自催化的自我模板特征，涉及单体 Y145Stop 的募集和结合诱导构象转换为淀粉样纤维。与这种内在无序的截断变体相反，野生型全长 PrP 对凝聚和成熟为淀粉样的倾向要低得多，这暗示 C 端结构域可能具有保护作用。这种调节蛋白质相行为的分子因素的相互作用可能在细胞生理学和疾病中有更广泛的意义。
