One of the recognized motor neuron degenerative disorders is amyotrophic lateral sclerosis (ALS). By now, several mutations have been reported and linked to ALS patients, some of which are induced by mutations in the human superoxide dismutase (hSOD1) gene. The ALS-provoking mutations are located throughout the structure of hSOD1 and promote the propensity to aggregate. Despite numerous investigations, the underlying mechanism related to the toxicity of mutant hSOD1 through the gain of a toxic function is still vague. We surveyed two mutant forms of hSOD1 by removing and adding cysteine at positions 146 and 72, respectively, to investigate the biochemical characterization and amyloid formation. Our findings predicted the harmful and destabilizing impact of two SOD1 mutants using multiple programs. The specific activity of the wild-type form was about 1.42- and 1.92-fold higher than that of C146R and G72C mutants, respectively. Comparative structural studies using CD spectropolarimetry, and intrinsic and ANS fluorescence showed alterations in secondary structure content, exposure of hydrophobic patches, and structural compactness of WT-hSOD1 vs. mutants. We demonstrated that two mutants were able to promote amyloid-like aggregates under amyloid induction circumstances (50-mM Tris–HCl pH 7.4, 0.2-M KSCN, 50-mM DTT, 37 °C, 190 rpm). Monitoring aggregates were done using an enhancement in thioflavin T fluorescence and alterations in Congo red absorption. The mutants accelerated fibrillation with subsequently greater fluorescence amplitude and a shorter lag time compared to WT-SOD1. These findings support the aggregation of ALS-associated SOD1 mutants as an integral part of ALS pathology.
