Abstract
Cu, Zn superoxide dismutase (SOD1) is a representative antioxidant enzyme that catalyzes dismutation of reactive oxygen species in cells. However, (E,E)-SOD1 mutants in which both copper and zinc ions were deleted exhibit pro-oxidant activity, contrary to their antioxidant nature, at physiological temperatures, following denaturation and subsequent recombination of Cu2+. This oxidative property is likely related to the pathogenesis of amyotrophic lateral sclerosis (ALS); however, the mechanism by which Cu2+ re-binds to the denatured (E,E)-SOD1 has not been elucidated, since the concentration of free copper ions in cells is almost zero. In this study, we prepared the (Cu,E) form in which only a zinc ion was deleted using ALS-linked mutant H43R (His43→Arg) and found that (Cu,E)-H43R showed an increase in the pro-oxidant activity even at physiological temperature. The increase in the pro-oxidant activity of (Cu,E)-H43R was also observed in solution mimicking intracellular environment and at high temperature. These results suggest that the zinc-deficient (Cu,E) form can contribute to oxidative stress in cells, and that the formation of (E,E)-SOD1 together with the subsequent Cu2+ rebinding is not necessary for the acquisition of the pro-oxidant activity.
Highlights
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease in which the loss of motor neurons causes a decrease in, for example, muscular strength and respiratory function, eventually leading to death
More than 20 causative genes and proteins, including TAR DNA-binding protein 43 (TDP-43), fused in sarcoma (FUS), C9orf72, and ataxin-2, have been identified by recent genetic analysis of ALS patients and pathological examination of patient’s neurons, and these genes and proteins have been extensively investigated in terms of the pathology of ALS [4,5,6,7,8,9,10,11]
Fluorescent DCF is generated by the oxidation reaction between DCFH and reactive oxygen species (ROS) generated from H2 O2 catalyzed by SOD1
Summary
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease in which the loss of motor neurons causes a decrease in, for example, muscular strength and respiratory function, eventually leading to death. Neither the onset mechanism nor a fundamental treatment for ALS have been established [1,2,3]. Zn superoxide dismutase (SOD1) were first identified as causative proteins [12,13]. Native SOD1 is a metal-binding protein containing one Cu ion and one Zn ion in the metal-binding region, and its monomer has a molecular weight of 16 kDa (Figure 1). More than 180 SOD1 gene mutations have been identified in familial ALS patients, and SOD1-positive aggregates have been found, suggesting a relationship between SOD1 and the onset of ALS [14]. A proposed mechanism for Molecules 2020, 25, 3600; doi:10.3390/molecules25163600 www.mdpi.com/journal/molecules
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