SERF Protein Promotes A-Synuclein Protein Aggregation Through Liquid-liquid Phase Separation

Amyloid aggregation is closely associated with the development of neurodegenerative diseases.α-Synuclein(α-Syn)aggregation is a major contributor to the pathogenesis of Parkinson’s disease(PD).It has been found that α-Syn protein forms multimers through liquid-liquid phase separation(LLPS),which oligomers exhibit significantly higher toxicity than fibrils,and deposition in vivo severely interferes with normal cellular physiological functions.Therefore,modulation of oligomers may be a key way to reduce the toxicity of aggregated α-Syn.A small disordered protein,SERF(small EDRK-rich factor),which blocks aggregation by specifically accelerating primary nucleation of α-Syn,however,the exact molecular mechanism remains unclear.The present work reveals a novel mechanism that SERF accelerates the rapid conversion of α-Syn from highly toxic oligomers to less toxic fibrils via LLPS,reducing the cytotoxicity caused by α-Syn aggregationFirstly,we constructed p ET28a-EGFP-SERF and p GEX-6P-1-SERF recombinant plasmids by molecular cloning technique and expressed high purity SERF recombinant protein with green fluorescent tag and tag-free SERF protein.According to the in vitro experiments,which combined with SDS-PAGE,confocal microscopy experiments,fluorescent bleaching recovery,turbidity experiments and other experimental methods,we have successfully demonstrated that SERF proteins are able to undergo LLPS driven by positive charge-dominated electrostatic interactions,low salt conditions and polyanions have a regulatory effect on droplet formation.In addition,this force also drives the onset of SERF/α-Syn protein co-phase separation,with the SERF protein remaining in a highly disordered structural state in the droplet during the pre-binding phase and then detaching from the droplet,while the α-Syn protein gradually solidifies in the droplet.This binding significantly accelerates the conversion of α-Syn protein from oligomers to fibrils.Furthermore,cell experiments,immunofluorescence techniques and cell transfection,showed that stress conditions could trigger the co-phase separation of the two proteins at cellular level,and the accelerated aggregation of α-Syn protein by SERF protein significantly reduced the toxicity of its polymorphs to cells.In conclusion,our results reveal a molecular mechanism,which SERF proteins can reduce cytotoxicity by regulating α-Syn protein aggregation through LLPS.This finding opens a new way of investigating amyloid aggregation and its cytotoxicity.