Approximately 10% of all familial cases of amyotrophic lateral sclerosis (fALS) are linked to mutations in the SOD1 gene, which encodes the copper/zinc superoxide dismutase (CuZnSOD). Recently, wild-type CuZnSOD was shown to protect calcineurin, a calcium/calmodulin-regulated phosphoprotein phosphatase, from inactivation by reactive oxygen species. We asked whether the protective effect of CuZnSOD on calcineurin is affected by mutations associated with fALS. For this, we monitored calcineurin activity in the presence of mutant and wild-type SOD. We found that the degree of protection against inactivation of calcineurin by different SOD mutants correlates with the severity of the phenotype associated with the different mutations, suggesting a potential role for calcineurinŜ OD1 interaction in the etiology of fALS. ß
Mutations in the SOD1 gene are associated with familial amyotrophic lateral sclerosis (fALS). The mechanisms by which these mutations lead to anterior horn cell loss are unknown, however, increased binding of Hsps on the demetallated mutant SOD1 has been described which would make the HSPs unavailable for other purposes, and reduce the SOD1 concentration in mitochondria, thereby creating a proapoptotic situation finally leading to motor neuron death. Here we report the recombinant expression of four human copper/zinc superoxide dismutase (CuZnSOD) variants, including the wild-type enzyme and mutant proteins associated with familial ALS. The bacterial expression level of soluble mutated proteins was influenced by the mutations leading to drastically reduced levels of soluble CuZnSOD. Simultaneously, increasing levels of insoluble and probably aggregated mutated CuZnSOD were identified in bacterial cell pellets. In addition, altered reactivation kinetics of the purified mutant apoproteins after expression in bacterial culture was shown. Biophysical and biochemical analysis showed that zinc incorporation is severely reduced in the CuZnSOD proteins associated with the most severely forms of fALS (A4V, G93A). These data indicate that a reduced holoenzyme formation rate of mutant enzymes may be a critical factor in the etiopathology of fALS.
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