LAUSR

Amyotrophic lateral sclerosis is a fatal, devastating, rapidly progressive, adult onset neurodegenerative disease, which is involved in the formation of proteinaceous virulent aggregates from superoxide dismutase 1 (SOD1) as a Cu/Zn metalloenzyme in motor neurons. However, the particular mechanism of mutant SOD1 toxicity has remained elusive. Here, we use complementary biochemical and biophysical methods to investigate thermodynamic parameters of wild-type (WT), E 100 K and D 125 H mutants in both metal-free (apo) and metalized (holo) forms of SOD1, to find out if there is any relationship between the structural/thermodynamic stability with aggregation propensity in apo and holo forms of WT/mutant SOD1 variants. Also, in this study, we compared the aggregation propensity of WT SOD1 and the mutants (E 100 K, D 125 H) under reducing conditions; the aggregation process and protein surface hydrophobicity were monitored by Thioflavin T (ThT) and ANS fluorescence, respectively. To determine molecular sizes, and intramolecular secondary structures, dynamic light scattering (DLS) and X-ray diffraction analyses were also applied, respectively. In addition to thermodynamic parameters measurements of both apo- and holo-proteins, melting temperature ( T m ) of apo proteins was assessed. Furthermore, for comparative evaluation of toxicity of SOD1 aggregates, LDH cytotoxicity as well as hemolysis assays were performed. The hydrodynamic diameters of all SOD1 variants were the same. ThT/ANS results, however, confirmed that DTT-mediated reduction in intramolecular S–S bonds induces gross conformational changes followed by the protein assembly. Thermodynamic studies showed that neither metal deficiency/mutation nor intramolecular S–S reduction, alone, can significantly reduce stability of SOD1 proteins, and demetallation (reduced stability) and intramolecular S–S reduction, together, are required for initiation of SOD1 misfolding/aggregation. CR binding, CD and XRD results, also together, confirmed the non-amyloid character of relatively toxic SOD1 aggregates. It appears that reduced thermodynamic stability as prerequisite for aggregation of SOD1 mutants must be accompanied by intramolecular disulfides reduction.