Amyotrophic lateral sclerosis (ALS) involves the abnormal posttranslational modifications and fibrillization of copper, zinc superoxide dismutase (SOD1) and TDP-43. However, how SOD1-catalyzed reaction product hydrogen peroxide affects amyloid formation of SOD1 and TDP-43 remains elusory. 90% of ALS cases are sporadic and the remaining cases are familial ALS. In this paper, 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, followed by the formation of sulfenic acid modified SOD1 oligomers. 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. Our Western blot and ELISA data demonstrate that sulfenic acid modified wild-type SOD1 level in cerebrospinal fluid of 15 sporadic ALS patients is significantly increased compared with 6 age-matched control patients. These findings can explain how H2O2 at pathologic concentrations regulates the misfolding and toxicity of SOD1 and TDP-43 associated with ALS, and suggest that sulfenic acid modification of wild-type SOD1 should play pivotal roles in the pathogenesis of sporadic ALS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease. Misfolded Cu, Zn-superoxide dismutase (SOD1) has been linked to both familial and sporadic ALS. SOD1 fibrils formed in vitro share toxic properties with ALS inclusions. Here we produced cytotoxic amyloid fibrils from full-length apo human SOD1 under reducing conditions and determined the atomic structure using cryo-EM. The SOD1 fibril consists of a single protofilament with a left-handed helix. The fibril core exhibits a serpentine fold comprising N-terminal segment (residues 3–55) and C-terminal segment (residues 86–153) with an intrinsic disordered segment. The two segments are zipped up by three salt bridge pairs. By comparison with the structure of apo SOD1 dimer, we propose that eight β-strands (to form a β-barrel) and one α-helix in the subunit of apo SOD1 convert into thirteen β-strands stabilized by five hydrophobic cavities in the SOD1 fibril. Our data provide insights into how SOD1 converts between structurally and functionally distinct states.

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.

Prion diseases and prion-like protein misfolding diseases involve the accumulation of abnormally aggregated forms of the normal host proteins, such as prion protein and Tau protein. These proteins are special because of their self-duplicating and transmissible characteristics. Such abnormally aggregated proteins mainly formed in neurons, cause the neurons dysfunction, and finally lead to invariably fatal neurodegenerative diseases. Prion diseases appear not only in animals, such as bovine spongiform encephalopathy in cattle and scrapie in sheep, but also in humans, such as Creutzfeldt -Jacob disease, and even the same prion or prion-like proteins can have many different phenotypes. A lot of biological evidence has suggested that the molecular basis for different strains of prions could be hidden in protein conformations, and the misfolded proteins with conformations different from the normal proteins have been proved to be the main cause for protein aggregation. Crowded physiological environments can be imitated in vitro to study how the misfolding of these proteins leads to the diseases in vivo. In this review, we provide an overview of the existing structural information for prion and prion-like proteins, and discuss the post-translational modifications of prion proteins and the difference between prion and other infectious pathogens. We also discuss what makes a misfolded protein become an infectious agent, and show some examples of prion-like protein aggregation, such as Tau protein aggregation and superoxide dismutase 1 aggregation, as well as some cases of prion-like protein aggregation in crowded physiological environments.

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal neurodegenerative disease characterized by the selective death of motor neurons. Misfolded Cu, Zn-superoxide dismutase (SOD1) has been linked to both familial ALS and sporadic ALS. SOD1 fibrils formed in vitro are able to incorporate into cells, transmit intercellularly, and share toxic properties with ALS inclusions. Here we produced amyloid fibrils in vitro from recombinant, full-length apo human SOD1 under semi-reducing conditions and determined the atomic structure using cryo-EM. The SOD1 fibril consists of a single protofibril with a left-handed helix. The fibril core exhibits a serpentine fold comprising N-terminal segment (residues 3 to 55) and C-terminal segment (residues 86 to 153) with a structural break. The two segments are zipped up by three salt bridge pairs. By comparison with the structure of apo SOD1 dimer, we propose that eight β-strands (to form a β-barrel) and one α-helix in the subunit of apo SOD1 convert into thirteen β-strands stabilized by five hydrophobic cavities in the SOD1 fibril. Our data provide insights into how SOD1 converts between structurally and functionally distinct states.