Screening Of Small Molecule Inhibitors Of TIA1 Protein Aggregation And Its Mechanism

Liquid-liquid phase separation(LLPS)of proteins is a physicochemical event that is thought to be a key mechanism for forming large molecules such as proteins and nucleic acids into membrane-free organelles such as stress granules in the cytoplasm.When the liquid-liquid phase separation of proteins is abnormal,it is transformed into irreversible protein aggregates,known as amyloid fibrils.Neurodegenerative diseases are manifested by irreversible progressive loss.A key feature of neurodegenerative diseases is the aggregation of proteins in the brain of patients and in populations of selective neurons,which makes effective treatment relatively difficult.Therefore,the inhibition of amyloid aggregation may be a viable strategy for therapeutic intervention in neurodegenerative diseases.In recent decades,several small molecule compounds have been extensively studied and they are considered as an important therapeutic and preventive strategy to effectively inhibit the amyloid aggregation process and the associated cytotoxicity.T cell restricted intracellular antigen 1(TIA1)protein has been shown to be associated with pathological processes in a variety of neurodegenerative diseases.In this paper,five commonly used small molecule compounds were co-incubated with TIA1,and the effects of aggregation and phase separation were investigated by thioflavin T experiments,confocal microscopy imaging,turbidity experiments,and fluorescent bleaching recovery experiments.It was shown that baicalein would maintain TIA1 in a phase-separated droplet state without aggregation.Then six structural analogues of baicalein were co-incubated with TIA1 protein to study the aggregation and phase separation of TIA1.It was determined that when phenolic hydroxyl groups were present at the fifth,sixth and seventh positions in the parent ring structure,which can effect on TIA1 particularly.Next,two fragments of TIA1 protein(TIA1-LCD and TIA1-ΔLCD)were expressed and purified separately,and the study determined that baicalein mainly acts on TIA1-LCD.Then,the potential mechanism of baicalein’s effect on TIA1 aggregation and phase separation was further investigated by molecular simulation and molecular docking methods,which showed that baicalein mainly interacts with TIA1 through hydrogen bonding.In summary,the results of this paper revealed that baicalein inhibits the aggregation of TIA1 by promoting the phase separation of TIA1 and maintaining TIA1 in the phase separated droplet phase.Further studies revealed that the phenolic hydroxyl groups at the fifth,sixth and seventh positions of the parent ring structure of the flavonoid are particularly important for this role,and further studies revealed that the potential mechanism of baicalein action on TIA1 protein through hydrogen bonding.This finding provides a new idea for the protein aggregation inhibition pathway and also provides a possible mechanism for subsequent studies.