Mechanisms Of The Folding And Misfolding Of Superoxide Dismutase

The structural integrity of the ubiquitous enzyme copper, zinc superoxide dismutase (SOD1) depends critically on the correct coordination of zinc and copper. We investigate here the roles of the stoichiometric zinc and copper ions in modulating the oxidative refolding of reduced, denatured bovine erythrocyte SOD1 at physiological pH and room temperature. Fluorescence experiments show that the oxidative refolding of the demetalated SOD1 (apo-SOD1) is biphasic, and the addition of stoichiometric Zn2+ ions into the refolding buffer remarkably accelerates both the fast phase and the slow phase of the oxidative refolding, compared with no Zn2+. Aggregation of apo-SOD1 in the presence of stoichiometric Zn2+ ions is remarkably slower than that in the absence of Zn2+. In contrast, the effects of stoichiometric Cu2+ on both the rates of the oxidative refolding and the aggregation of apo-SOD1 are not remarkable. Experiments of resistance to proteinase K show that apo-SOD1 forms a conformation with low-level proteinase K resistance during refolding and stoichiometric Cu2+ has no obvious effect on the resistance to proteinase K. In contrast, when the refolding buffer contains stoichiometric zinc, SOD1 forms a compact conformation with high-level proteinase K resistance during refolding. Our data here demonstrate that stoichiometric zinc plays an important role in the oxidative refolding of low micromolar bovine SOD1 by accelerating the oxidative refolding, suppressing the aggregation during refolding and helping the protein to form a compact conformation with high protease resistance activity.Misfolding and aggregation of mutant human copper, zinc superoxide dismutase (SOD1) is associated with the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS). Recent studies have shown that the co-expression of mutant and wild-type SOD1 (WT SOD1) in mice accelerates ALS of mice and thus provides evidence that WT SOD1 may be an important factor in mutant SOD 1-mediated ALS. However, the mechanism of why the relative stable WT SOD1 aggregates in vivo is not well understood. We report here an enhancing effect of human SOD1 mutant A4V (A4V SOD1) on the aggregation of WT SOD1. The results obtained from thioflavin T binding and turbidity experiments show that the addition of A4V SOD1 at 0.05-0.2 mg/ml dramatically accelerates aggregate formation of WT SOD1 on the first events of misfolding. The results from transmission electron microscopy and ANS binding suggest that the aggregates, produced from WT SOD1 induced by A4V SOD1, resemble aggregates found in vivo and may be also toxic to the cells and responsible for the onset of neurodegenerative disease. Taken together, these results indicate that A4V SOD1 strongly induces the aggregation of WT SOD1, providing a good explanation why co-expression of mutant and WT SOD1 in mice accelerates ALS of mice. The increased propensity of WT SOD1 to aggregate described here implies that SOD1 could gain toxic function via other ways, such as inducement, rather than mutation.