A Study On The Phase Separation And Aggregation Mechanism Of Amyotrophic Lateral Sclerosis-related Protein SOD1

Copper/Zn superoxide dismutase 1(SOD1)has a strong propensity to misfold and form abnormal aggregates when subjected to oxidative stress or when carrying mutations associated with amyotrophic lateral sclerosis(ALS).However,how the transition from functionally soluble SOD1 to aggregated SOD1 occurs is not fully understood.Currently,it is shown that many aggregation-prone proteins involved in neurodegenerative diseases tend to undergo liquid-liquid phase separation(LLPS)before forming stable aggregated species.The intracellular condensates formed by LLPS are usually complex,adopt a heterogeneous multilayer structure,and have partially solid-like characteristics.Solid-like phases can arise from sub-stable liquid condensates and further form intracellular aggregates,which are relevant for biological functions and many diseases.In this study,it is proposed for the first time that LLPS represents a biophysical process that converts soluble SOD1 into aggregated SOD1.Firstly,in this study,the SOD1 recombinant protein and its mutant were successfully constructed and expressed in vitro,while the SOD1 protein carrying a green-fluorescent tag was successfully expressed intracellularly by cell transfection.Secondly,through confocal microscopy,turbidity assay,fluorescent bleaching recovery,in vitro protein aggregation kinetics,and polyacrylamide gel electrophoresis,this study demonstrated that SOD1 can undergo LLPS in vitro and in cells under oxidative stress.aberrant oxidation of SOD1 induces the maturation of droplets formed by LLPS,which eventually leads to protein aggregation and fibrosis,which is associated with residues Cys111 and Trp32 are closely related.Also,pathological mutations in SOD1 associated with ALS alter the morphology and physical state of the droplets and promote the transformation of SOD1 to solid-like amyloid oligomers.In addition,amyloid aggregates formed by both pathways have significant toxic effects on neuronal cells.These combined results suggest that LLPS may play a major role in pathological SOD1 aggregation,which provides a new way to explore the pathogenesis of SOD1-related ALS.