Aberrant structural formations of Cu/Zn superoxide dismutase enzyme (SOD1) are the probable mechanism by which circumscribed mutations in the SOD1 gene cause familial amyotrophic lateral sclerosis (ALS1). SOD1 forms aberrant structures which can proceed by nucleation to insoluble aggregates. Here, the SOD1 aggregation reaction was investigated predominantly by time-course studies on ALS1 variants G85R, G37R, D101G, and D101N in human embryonic kidney cells (HEK293FT), with analysis by detergent ultracentrifugation extractions and high-resolution PAGE methodologies. Nucleation was found to be pseudo-zeroth order and dependent on time and concentration at constant 37.0 °C and pH 7.4. The predominant subsets of the total SOD1 expression set which comprised the nucleation phase were both soluble and insoluble inactive monomers, trimers, and hexamers with reduced intra-disulfide bonds. Superoxide exposure via paraquat initiated the formation of SOD1 trimers in untransfected SH-SY5Y cells and increased the aggregation propensity of G85R in HEK293FT. These data show the kinetic formation of aberrant SOD1 subsets implicated in ALS1 and indicate that superoxide substrate may initiate its radical polymerization. In an instance of the utility of methodological reductionism in molecular theory: though many ALS1 variants retain their global enzymatic activity, the SOD1 subsets most implicated in causing ALS1 do not retain their specific activity.
Keywords: Familial amyotrophic lateral sclerosis (ALS); High-resolution colorless native PAGE (hrcN-PAGE); Polyacrylamide gel electrophoresis (PAGE); Superoxide dismutase 1 (SOD1).