Centre for Protein Science, Design and Engineering, Indian Institute of Science Education and Research Mohali, Mohali, Punjab 140306, India.
Department of Chemical Sciences, Indian Institute of Science Education and Research Mohali, Mohali, Punjab 140306, India.
Proc Natl Acad Sci U S A. 2023 Jan 10;120(2):e2216338120. doi: 10.1073/pnas.2216338120. Epub 2023 Jan 3.
Biomolecular condensates formed via phase separation of proteins and nucleic acids are thought to perform a wide range of critical cellular functions by maintaining spatiotemporal regulation and organizing intracellular biochemistry. However, aberrant phase transitions are implicated in a multitude of human diseases. Here, we demonstrate that two neuronal proteins, namely tau and prion, undergo complex coacervation driven by domain-specific electrostatic interactions to yield highly dynamic, mesoscopic liquid-like droplets. The acidic N-terminal segment of tau interacts electrostatically with the polybasic N-terminal intrinsically disordered segment of the prion protein (PrP). We employed a unique combination of time-resolved tools that encompass several orders of magnitude of timescales ranging from nanoseconds to seconds. These studies unveil an intriguing symphony of molecular events associated with the formation of heterotypic condensates comprising ephemeral, domain-specific, short-range electrostatic nanoclusters. Our results reveal that these heterotypic condensates can be tuned by RNA in a stoichiometry-dependent manner resulting in reversible, multiphasic, immiscible, and ternary condensates of different morphologies ranging from core-shell to nested droplets. This ternary system exhibits a typical three-regime phase behavior reminiscent of other membraneless organelles including nucleolar condensates. We also show that upon aging, tau:PrP droplets gradually convert into solid-like co-assemblies by sequestration of persistent intermolecular interactions. Our vibrational Raman results in conjunction with atomic force microscopy and multi-color fluorescence imaging reveal the presence of amorphous and amyloid-like co-aggregates upon maturation. Our findings provide mechanistic underpinnings of overlapping neuropathology involving tau and PrP and highlight a broader biological role of complex phase transitions in physiology and disease.
生物分子凝聚体通过蛋白质和核酸的相分离形成，被认为通过维持时空调节和组织细胞内生物化学来执行广泛的关键细胞功能。然而，异常的相转变与多种人类疾病有关。在这里，我们证明了两种神经元蛋白，即 tau 和朊病毒，通过特定于结构域的静电相互作用经历复杂的凝聚作用，产生高度动态的、介观的液态样液滴。tau 的酸性 N 端片段与朊病毒蛋白（PrP）的多碱性 N 端无规卷曲结构域静电相互作用。我们采用了一种独特的组合，包括跨越从纳秒到秒的几个数量级的时间尺度的时间分辨工具。这些研究揭示了与形成包含短暂的、特定于结构域的、短程静电纳米簇的异质凝聚体相关的一系列有趣的分子事件。我们的结果表明，这些异质凝聚体可以通过 RNA 以依赖于化学计量的方式进行调节，从而导致不同形态的可逆的、多相的、不混溶的和三相凝聚体，从核仁凝聚体到嵌套液滴。这种三相系统表现出典型的三区域相行为，类似于其他无膜细胞器，包括核仁凝聚体。我们还表明，tau:PrP 液滴在老化过程中通过持久的分子间相互作用的隔离逐渐转化为固态共组装体。我们的振动拉曼结果与原子力显微镜和多色荧光成像相结合，揭示了在成熟过程中存在无定形和类淀粉样共聚集物。我们的发现为涉及 tau 和 PrP 的重叠神经病理学提供了机制基础，并强调了复杂相变在生理和疾病中的更广泛的生物学作用。
