Background: Mutations in a number of proteins (SOD1, FUS, optineurin, TDP43 etc) are associated with familial amyotrophic lateral sclerosis (fALS). Superoxide dismutase 1 (SOD1) is the most well understood of these proteins in relation to fALS, yet a causal realtionship with the fatal neurodegenerative disease is yet to be established.

Problem: Aggregation of SOD1 is associated with fALS pathology. It is accepted that aggregation is dependent upon dissociation of the native homodimer, to monomers, and that mutation can increase dimer dissociation propensity. However, mutation alone is not always enough to dissociate the homodimer. Recently, it was suggested that the naturally occurring modification 'glutathionylation' can enhance the dissociation of SOD1 dimers.

Methodology: To determine the effect of glutathionylation on SOD1 dimer dissociation, a broad range of  recombinant SOD1 variants were purified and subject to mass spectrometry (MS). A dilution series ranging from 20 µM to 0.6 µM was analysed to determine the dissociation constants of the SOD1 dimer.

Results:  MS analysis revealed that all SOD1 variants were glutathionylated. Using MS it was possible to determine the dissociation constants of both non-glutathionylated and glutathionylated SOD1 in a heterogeneous mixture. Glutathionylation increases dissociation of all SOD1 variants analysed, with the greatest increase occurring for SOD1^G93A (12 nM to 160 nM) compared to SOD1^WT (9 nM to 34nM).

Conclusion: Previous reports show that SOD1 mutants have a higher aggregation potential than SOD1^WT, yet we report multiple SOD1 mutants having dissociation constants similar to SOD1^WT. We propose that glutathionylation plays a substantial role in the pathway that leads to SOD1 associated fALS pathogenesis, via dissociation of the native homodimer. Dimer dissociation leads to the formation of monomer that may undertake a toxic conformation or aggregate into a toxic species.