Phase separation of SOD1 is an intrinsic Zn cofactor regulated phenomenon

Motor neuron diseases are a group progressive neurodegenerative disorders that selectively targets motor neuron activity. ALS is one such motor neuron disease characterized by the loss of motor neurons in the brain and spinal cord. This results in loss of control over voluntary muscles with time, ultimately leading to respiratory muscle failure and death. One of the important pathological hallmarks of ALS is the aggregation of human Cu/Zn superoxide dismutase1 (SOD1). SOD1 is a metalloenzyme that scavenges reactive oxygen species from the cell, having two metal cofactors, Cu and Zn. Till date, more than 170 mutations of this protein have been linked to familial ALS. Our group had previously established a correlation between the stability, aggregation, and disease severity for a subset of these mutants. In the present study, we hypothesized that SOD1 undergoes liquid-liquid phase separation (LLPS) since it has been reported to accumulate within stress granules. Stress granules are membraneless organelles, believed to originate via a process called LLPS. Our results have revealed that SOD1 indeed undergoes LLPS when Zn is removed from it by chelation or when it is unable to bind the protein due to disease mutation. We found that conformational disorder in the protein drives its LLPS and Zn coordination prevents LLPS by reducing disorderedness. Prolonged LLPS inducing conditions cause the protein to undergo liquid to solid transition culminating in aggregation. SOD1 LLPS is reversible by addition of Zn but its aggregation is an irreversible process. Furthermore, we have shown that the phase separated condensates of SOD1 induce higher cytotoxicity than its aggregates.