Cupric Ions Induce the Oxidation and Trigger the Aggregation of Human Superoxide Dismutase 1

Cheng Li,Fu-Quan Yang,Yi Liang,Kai Pan,Dai-Wen Pang,Jiao Hu,Jie Chen,Wen-Chang Xu,Zhen-Sheng Xie,Jose M Sanchez-Ruiz

doi:10.1371/journal.pone.0065287

Abstract

BackgroundAmyotrophic lateral sclerosis (ALS), partly caused by the mutations and aggregation of human copper, zinc superoxide dismutase (SOD1), is a fatal degenerative disease of motor neurons. Because SOD1 is a major copper-binding protein present at relatively high concentration in motor neurons and copper can be a harmful pro-oxidant, we want to know whether aberrant copper biochemistry could underlie ALS pathogenesis. In this study, we have investigated and compared the effects of cupric ions on the aggregation of ALS-associated SOD1 mutant A4V and oxidized wild-type SOD1.Methodology/Principal FindingsAs revealed by 90° light scattering, dynamic light scattering, SDS-PAGE, and atomic force microscopy, free cupric ions in solution not only induce the oxidation of either apo A4V or Zn2-A4V and trigger the oligomerization and aggregation of oxidized A4V under copper-mediated oxidative conditions, but also trigger the aggregation of non-oxidized form of such a pathogenic mutant. As evidenced by mass spectrometry and SDS-PAGE, Cys-111 is a primary target for oxidative modification of pathological human SOD1 mutant A4V by either excess Cu2+ or hydrogen peroxide. The results from isothermal titration calorimetry show that A4V possesses two sets of independent binding sites for Cu2+: a moderate-affinity site (106 M-1) and a high-affinity site (108 M-1). Furthermore, Cu2+ binds to wild-type SOD1 oxidized by hydrogen peroxide in a way similar to A4V, triggering the aggregation of such an oxidized form.Conclusions/SignificanceWe demonstrate that excess cupric ions induce the oxidation and trigger the aggregation of A4V SOD1, and suggest that Cu2+ plays a key role in the mechanism of aggregation of both A4V and oxidized wild-type SOD1. A plausible model for how pathological SOD1 mutants aggregate in ALS-affected motor neurons with the disruption of copper homeostasis has been provided.

Highlights

Amyotrophic lateral sclerosis (ALS), partly caused by the mutations and aggregation of human copper, zinc superoxide dismutase (SOD1, EC 1.15.1.1), is a fatal degenerative disease of motor neurons [1]
By using several biophysical methods, such as 90u light scattering, dynamic light scattering, atomic force microscopy (AFM), mass spectrometry (MS), size-exclusion chromatography, and isothermal titration calorimetry (ITC), we investigated and compared the effects of cupric ions on the aggregation of ALS-associated SOD1 mutant A4V and wild-type SOD1 oxidized by hydrogen peroxide
It has been reported that the loss of zinc from SOD1 results in the remaining copper in SOD1 to become extremely toxic to motor neurons in ALS [57]

Summary

Introduction

Amyotrophic lateral sclerosis (ALS), partly caused by the mutations and aggregation of human copper, zinc superoxide dismutase (SOD1, EC 1.15.1.1), is a fatal degenerative disease of motor neurons [1]. Copper is too redox active to exist in an unbound form in the cell without causing oxidative damage [12,13,14], and disruption of copper homeostasis is implicated in a number of neurodegenerative diseases, including Alzheimer disease and ALS [10,11,12,13]. Amyotrophic lateral sclerosis (ALS), partly caused by the mutations and aggregation of human copper, zinc superoxide dismutase (SOD1), is a fatal degenerative disease of motor neurons. Because SOD1 is a major copper-binding protein present at relatively high concentration in motor neurons and copper can be a harmful pro-oxidant, we want to know whether aberrant copper biochemistry could underlie ALS pathogenesis. We have investigated and compared the effects of cupric ions on the aggregation of ALS-associated SOD1 mutant A4V and oxidized wild-type SOD1

Methods

Results

Conclusion