Effects of molecular crowding environment on the acquisition of toxic properties of wild-type SOD1

Background: Mutants of Cu,Zn-superoxide dismutase (SOD1) exhibit cytotoxicity such as aggregation and pro-oxidation after denaturation, which is thought to be involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). In the present study, we investigated the possibility of the acquisition of toxic properties for wild-type SOD1 (WT) in the demetalated (apo) form after denaturation. Methods: Denaturation and subsequent pro-oxidant activity of SOD1 were confirmed by circular dichroism (CD) spectroscopy and fluorescence assay, respectively. The aggregation of SOD1 was investigated by native polyacrylamide gel electrophoresis (PAGE). Crowding environment was prepared by the addition of polyethylene glycol (PEG) into buffer solution. Results: The structural stability of SOD1 is reduced by demetallation. Nevertheless, high temperatures around 45 °C are required to induce denaturation of apo-WT. The generated denaturated apo-WT exhibits pro-oxidant activity after the rebinding of Cu²⁺. In molecular crowding environment mimicked by PEG, apo-WT is found to exhibit denaturation even at physiological temperature. The denatured WT in molecular crowding environment has both the activities of pro-oxidation and aggregation. The acquisition of the pro-oxidant activity is accelerated for H43R, which is an ALS-related mutant, in molecular crowding environment. Conclusions: Apo-WT acquires the toxic properties at physiological temperature when subjected to molecular crowding environment. Molecular crowding environment also accelerates the induction of the toxicity for H43R. General significance: Molecular crowding environment in living cells becomes an instability factor inducing denaturation and subsequent toxicity for SOD1. Apo-WT also has the toxic properties in molecular crowding environment, which can be related to the pathogenesis of ALS.