Mechanisms Of Misfolding And Toxicity Of Human Superoxide Dismutase Induced By Hydrogen Peroxide

Amyotrophic lateral sclerosis(ALS)involves the abnormal posttranslational modifications and fibrillization of copper,zinc superoxide dismutase(SOD1)and TDP-43.However,how SOD 1-catalyzed reaction product hydrogen peroxide affects amyloid formation of SOD1 and TDP-43 remains elusory.Here we investigate the effects of hydrogen peroxide on the misfolding and cytotoxicity of wild-type human SOD1 and TDP-43.We demonstrate that H2O2 at pathological concentrations triggers the fibrillization of wild-type SOD1 both in vitro and in SH-SY5Y cells.Using an anti-dimedone antibody that detects sulfenic acid modification of proteins,we found that Cys-111 in wild-type SOD1 is oxidized to C-SOH by pathological concentration of H2O2 both in vitro and in cells,followed by the formation of sulfenic acid modified SOD1 oligomers,that sulfenic acid modified SOD1 level in cerebrospinal fluid of some patients with sporadic ALS is significantly increased compared with age-matched non-ALS control patients.Furthermore,we show that such SOD1 oligomers propagate in a prion-like manner,and not only drive wild-type SOD1 to form fibrils in the cytoplasm but also induce cytoplasm mislocalization and the subsequent fibrillization of wild-type TDP-43,thereby inducing apoptosis of living cells.Thus,we propose that H2O2 at pathological concentrations triggers the fibrillization of wild-type SOD1 and subsequently induces SOD1 toxicity and TDP-43 toxicity in neuronal cells via sulfenic acid modification of Cys-111 in SOD1.These findings can explain how H2O2 at pathologic concentrations regulates the misfolding and toxicity of SOD1 and TDP-43 associated with ALS and suggest a role of sulfenic acid modified SOD1 as a diagnostic biomarker in sporadic ALS.SOD1 is a homodimeric metalloenzyme containing one Cu and one Zn ion in each subunit.The catalytic copper ion is coordinate with four histidines(His-46,His-48,His-63 and His-120)and the structural zinc ion is bound by three histidines(His-63,His--71 and His80)and one aspartate(Asp-83).Here we study the effect of Cys-111 on the metal binding to SOD1 and fibrillization of SOD1 induced by hydrogen peroxide(H2O2).As evidenced by thioflavin T binding assays,Cys-111 mutation or Zn2+/Cu2+at the molar ratio of 2 or 4 could inhibit the fibrillization of SOD1 in the presence of H2O2.As revealed by isothermal titration calorimetry,substitution of Cys-111 with Ala or Ser changed the binding modes remarkably and dramatically decreased the number of binding sites to Zn2+ and Cu2+ with SOD1.The results from the analysis of SOD1’s activity show that Cys-111 affects the metal binding of SOD1 but did increase the activity of SOD1.Our findings demonstrate that Cys-111 is very important for the structure and function of SOD1.