Phase separation and protein aggregation

Phase separation of biomolecules has recently been recognised as an important cellular process governing homogeneous organisation which is a driving force for cellular self-assembly. Multivalent interactions between biomolecules give rise to condensed phases with a spectrum of material properties from liquids to solids. The liquid-like properties (wetting, fusion, and dynamic exchange of internal components) of membraneless organelles such as P granules, stress granules, and the nucleolus have been demonstrated to be based on Liquid–liquid phase separation. The process of protein aggregation - disordered proteins which pathologically self-assemble into insoluble fibres that further aggregate into the plaques, tangles, or inclusions has long been believed to be related to a common, systemic origin and mechanism of toxicity. The potential for disordered proteins to assemble via phase separation into soft condensed material states raises questions about the relationship between phase separation and neurodegeneration. The evidence in support of a relationship between droplet properties, protein aggregation, and neurodegenerative pathology is rapidly growing.

Protein liquid–liquid phase separation (LLPS) is studied in cellular subcompartments like membraneless organelles or in vitro. Protocols for protein LLPS in vitro depend on the studied proteins since different proteins undergo LLPS under different conditions according to their properties. Generally, LLPS can be achieved by decreasing the ionic strength of the buffer, addition of precipitators, co-addition of interacting partners, and removal of the recombinant tag by protease. Several assays and standards are performed to determine whether the proteins undergo LLPS, including assessing the turbidity of the solution, microscopic examination of liquid droplets, monitoring the fusion and fission of liquid droplets, assessing their wetting properties, and subjecting the mixture to sedimentation to analyze the partitioning of proteins between the different phases.

Researchers from Max Planck Institute of Biochemistry in Martinsried, Germany have recently published an article (Frottin et al. 2019) in the renowned scientific journal Science where they study protein aggregation in the nucleolus, which is the largest non–membrane bound subcompartment in the nucleus. The nucleolus consists of liquid-like phases that do not intermix, giving rise to distinct zones. They found that, during stress, misfolded proteins enter the liquid-like GC phase of the nucleolus, where irreversible co-aggregation of different misfolded protein species is prevented, allowing Hsp70-mediated extraction and refolding (or degradation) upon recovery from stress. In contrast, disruption of the GC phase causes the formation of stable protein aggregates which they detected using Amytracker 680. Prolonged stress resulted in a transition of the nucleolar matrix from liquid-like to solid and prevents nucleolar quality control.

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