Study on the effects of various incubation conditions on aggregation of SOD1 variants: disulfide bond reduction and demetallation synergistically promote generation of non-amyloid amorphous aggregates from SOD1 mutants

Amyotrophic lateral sclerosis (ALS) is a progressive fetal neurodegenerative disease that affects neural cells in the brainstem and spinal cord, causing loss of muscle control, misfolding and aggregation of copper–zinc superoxide dismutase (SOD1). However, the particular mechanism by which mutant SOD1 causes neural death remains elusive. While metallated SOD1s were not aggregated in our previous study, the present study set out to determine whether dissociation/aggregation propensity of holo/apo SOD1 variants (WT, E100K and D125H), may increase under various incubation conditions/compositions or not. Therefore, the process of aggregation were analyzed by various techniques such as fluorescence, CD spectroscopy and XRD. Our results highlighted the importance of metal ions in misfolding/aggregation behavior of holo SOD1s and demonstrated that apo SOD1s had lower thermodynamic and/or kinetic stability compared to holo forms due to loss of metal ions in their structures. Also, the data indicated that incubation conditions/compositions were important in reducing of stability and increasing of aggregation propensity of apo/holo SOD1 variants. We demonstrated that any structural abnormalities such as loss of metal ions, reducing disulfide bonds and mutations that enhance the protein denaturation propensity, synergistically lead to decrease in thermodynamic stability of SOD1 variants and consequently facilitate unfolding transitions which cause formation of non-amyloid amorphous aggregates.