Molecular Mechanisms For The Misfolding Of Superoxide Dismutase1(SOD1) Induced By Copper And The Role Of Wild-type SOD1in ALS

1. Amyotrophic lateral sclerosis (ALS), partly caused by the mutations and aggregation of human copper, zinc superoxide dismutase (SOD1), is a fatal degenerative disease of motor neurons. Because SOD1is a major copper-binding protein present at relatively high concentration in motor neurons and copper can be harmfully pro-oxidant, we want to know whether aberrant copper biochemistry could underlie ALS pathogenesis. In this study, we have investigated and compared the effects of copper on the aggregation of ALS-associated SOD1mutant A4V and oxidized wild-type SOD1. Our data demonstrated for the first time that Cys-111is a primary target for oxidative modification of pathological human SOD1mutant A4V by Cu2+. A4V possesses two sets of independent binding sites for Cu2+:a moderate-affinity site (186nM) and a high-affinity site (1.83nM). High micromolar copper not only induces the oxidation of A4V and triggers the oligomerization and aggregation of oxidized A4V easier than wild-type SOD1, but also triggers the aggregation of non-oxidized form of such a pathogenic mutant. Furthermore, Cu2+binds to oxidized wild-type SOD1in a way similar to A4V, triggering the aggregation of such an oxidized form. We thus suggest that Cu2+plays a key role in both steps of nucleation and elongation of aggregation of both A4V and oxidized wild-type SOD1. Our data provide a plausible model to explain why and how pathological SOD1mutants aggregate in ALS-affected motor neurons, and will be helpful to the understanding of the role of aberrant copper biochemistry in the pathogenesis of ALS.2. 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 SOD1may be an important factor in mutant SOD1-mediated ALS. However, the mechanism of why the relative stable WT SOD1aggregates in vivo is not well understood. The results obtained from thioflavin T binding, turbidity and SDS-PAGE experiments show that shows that human SOD1mutant A4V (A4V SOD1) enhance the aggregation of WT SOD1in the TFE condition or the neutral reducing environment. It shows WT SOD1can promote the lag time of A4V SOD1aggregates through the physiological condition at pH7.4PBS buffer. The mutant SOD1lacking111free sulfhydryl group (C111S) decrease the lag time of the enhancing process compared with WT SOD1which demonstrate111free sulfhydryl group are important factors for the WT SOD1enhancing effect. Taken together, these results indicate that WT SOD1participate in the aggregation of A4V SOD1, providing a good explanation why co-expression of mutant and WT SOD1in mice accelerates ALS of mice. The increased propensity of WT SOD1to aggregate described here implies that SOD1could gain toxic function via other ways, such as inducement, rather than mutation.3. Converging evidence indicates that the fibrillization of human copper, zinc superoxide dismutase (SOD1) could play an important role in the etiology of both familial amyotrophic lateral sclerosis (ALS) and sporadic ALS. The mechanism by which this highly stable enzyme misfolds to form amyloid fibrils is currently poorly understood, as are the stresses that initiate misfolding. The oxidative damage hypothesis proposes that its normal free radical scavenger role puts SOD1at risk of oxidative damage and that it is this damage that triggers the misfolding of the protein. Here, by using SDS-PAGE and mass spectroscopy methods, we demonstrate that with the treatment of20-200μM hydrogen peroxide, human SODl forms amyloid fibrils and generates an upper shift band in reducing SDS-PAGE, which turns out to be the results of oxidative modification of cysteine111. As revealed by circular dichroism and transmission electron microscopy, such an oxidative modification could be crucial to the fibrillization of human SOD1accompanied by the major structural changes. In brief, hydrogen peroxide triggers the fibrillization of human SOD1via oxidative modification of Cys-111.