Abstract
Mutations in the gene coding for superoxide dismutase 1 (SOD1) are associated with familiar forms of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). These mutations are believed to result in a “gain of toxic function”, leading to neuronal degeneration. The exact mechanism is still unknown, but misfolding/aggregation events are generally acknowledged as important pathological events in this process. Recently, we observed that demetallated apoSOD1, with cysteine 6 and 111 substituted for alanine, is toxic to cultured neuroblastoma cells. This toxicity depended on an intact, high affinity Zn2+ site. It was therefor contradictory to discover that wild-type apoSOD1 was not toxic, despite of its high affinity for Zn2+. This inconsistency was hypothesized to originate from erroneous disulfide formation involving C6 and C111. Using high resolution non-reducing SDS-PAGE, we have in this study demonstrated that the inability of wild-type apoSOD1 to cause cell death stems from formation of non-native intra-molecular disulfides. Moreover, monomeric apoSOD1 variants capable of such disulfide scrambling aggregated into ThT positive oligomers under physiological conditions without agitation. The oligomers were stabilized by inter-molecular disulfides and morphologically resembled what has in other neurodegenerative diseases been termed protofibrils. Disulfide scrambling thus appears to be an important event for misfolding and aggregation of SOD1, but may also be significant for protein function involving cysteines, e.g. mitochondrial import and copper loading.
Highlights
Mutations in the superoxide dismutase 1 (SOD1) gene account for more than 20 % of the familial cases of amyotrophic lateral sclerosis (ALS), an aggressive neurodegenerative disorder characterized by degeneration of upper and lower motor neurons
The findings presented in Johansson et al are extended by data providing evidence for non-native intramolecular cysteine bond formation in wild-type apoSOD1 which renders the protein non-toxic, i.e. Zn2+ deficient
The level of cell toxicity of apoSOD1 C111A was virtually identical to the alkylated wild-type protein (Figure 1A), suggesting that primarily C111 is modified in the alkylation procedure
Summary
Mutations in the superoxide dismutase 1 (SOD1) gene account for more than 20 % of the familial cases of amyotrophic lateral sclerosis (ALS), an aggressive neurodegenerative disorder characterized by degeneration of upper and lower motor neurons. The lack of motor neuron disease in SOD1 knock-out mice [1] and the normal enzymatic activity conferred by some of the SOD1 mutants [2,3] suggest a cytotoxic gain of function similar to the disease causing proteins in other neurodegenerative disorders like Alzheimer’s disease and Parkinson’s disease. One hypothesis for the neuronal toxicity caused by mutant SOD1 is the formation of protein aggregates. In support of this idea, intracellular protein inclusions containing SOD1 have been identified in transgenic mice expressing human mutant SOD1 [4], as well as in familial ALS with SOD1 mutations [5] and in sporadic ALS [6]. Over-expression of mutant SOD1 in cultured cells in vitro results in aggregation and neurotoxicity linked to inter-molecular disulfide cross-linking of the free cysteines C6 and C111 [7,8]
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