Liquid–liquid phase separation technologies of α-synuclein governed by its C-terminus via electrostatic interactions: unveiling potential therapeutic targets for Parkinson’s disease

JOURNAL/atin/04.03/02274269-202503000-00001/figure1/v/2025-03-08T073941Z/r/image-tiff Liquid‒liquid phase separation of α-synuclein, a key pathogenic protein in Parkinson’s disease, is increasingly recognized for its role in the aggregation process. However, the underlying mechanisms remain elusive. This study aimed to elucidate the liquid‒liquid phase separation mechanism of α-synuclein, focusing on the critical role of its C-terminus and electrostatic interactions. Through turbidity and microscopic imaging assays, we identified an optimal liquid‒liquid phase separation system for α-synuclein under specific buffer conditions. We discovered that the C-terminus is a critical domain for α-synuclein liquid‒liquid phase separation, as evidenced by the liquid droplets formed by the truncation peptides. Furthermore, phase diagrams of NaCl and 1,6-hexanediol assays highlighted the significant role of electrostatic forces in driving α-synuclein liquid‒liquid phase separation. Notably, mutations in four glycine residues at positions 31, 68, 84, and 86 substantially altered liquid‒liquid phase separation behavior. In conclusion, this work clarifies the liquid‒liquid phase separation mechanism of α-synuclein, highlighting the importance of the C-terminus and electrostatic interactions, and suggests potential avenues for therapeutic intervention in Parkinson’s disease. The clinical significance lies in the potential to develop targeted therapies that modulate α-synuclein liquid‒liquid phase separation, potentially arresting disease progression and improving patient outcomes.