DNA-Triggered Aggregation of Copper, Zinc Superoxide Dismutase in the Presence of Ascorbate

Abstract

The oxidative damage hypothesis proposed for the function gain of copper, zinc superoxide dismutase (SOD1) maintains that both mutant and wild-type (WT) SOD1 catalyze reactions with abnormal substrates that damage cellular components critical for viability of the affected cells. However, whether the oxidative damage of SOD1 is involved in the formation of aggregates rich in SOD1 or not remains elusive. Here, we sought to explore the oxidative aggregation of WT SOD1 exposed to environments containing both ascorbate (Asc) and DNA under neutral conditions. The results showed that the WT SOD1 protein was oxidized in the presence of Asc. The oxidation results in the higher affinity of the modified protein for DNA than that of the unmodified protein. The oxidized SOD1 was observed to be more prone to aggregation than the WT SOD1, and the addition of DNA can significantly accelerate the oxidative aggregation. Moreover, a reasonable relationship can be found between the oxidation, increased hydrophobicity, and aggregation of SOD1 in the presence of DNA. The crucial step in aggregation is neutralization of the positive charges on some SOD1 surfaces by DNA binding. This study might be crucial for understanding molecular forces driving the protein aggregation.

Molecular targets of oxidative stress

“…Furthermore, increased levels of carbonylated aggregates are observed in patients with age-related disorders such as Parkinson's disease, Alzheimer's disease and cancer. Even wild-type Sod1p is prone to oxidative destabilization and aggregation in vitro [59,100], consistent with this protein's susceptibility to carbonylation (see the section entitled 'Protein oxidation'). Even wild-type Sod1p is prone to oxidative destabilization and aggregation in vitro [59,100], consistent with this protein's susceptibility to carbonylation (see the section entitled 'Protein oxidation').…”

Polyanion binding accelerates the formation of stable and low‐toxic aggregates of ALS‐linked SOD1 mutant A4V

“…The report that the proteinaceous inclusions as a hallmark of ALS do not have the classic fibrillar appearance of amyloid prompted us to examine morphology of the A4V SOD1 aggregates formed with polyanions under TEM. The aggregates of both wtSOD1 and its oxidized form in the presence of DNA were observed to have the heterogeneous profiles similar to those identified in the diseased neurons and tissues under acidic and neutral conditions …”

“…Although an increase in the extrinsic thioflavin T (ThT) fluorescence is a well-used method to characterize the formation of fibrillar protein amyloids, the increased ThT signal caused by the formation of amyloid fibrils cannot be used to monitor the formation of protein aggregates with DNA, not only because of the formation of non-fibrillar protein aggregates in the presence of DNA, but also because the ThT signal increased by interactions with DNA can mask the ThT signal enhanced by protein aggregation. [43][44][45] To prove the availability of Trp fluorescence to follow the aggregation with polyanions, first, the time course of A4V aggregation in the presence of LMWHep was simultaneously monitored with three methods at pH 4.0, because the presence of LMWHep does not affect ThT fluorescence. The data showed that following a lag phase of 20 s, Trp fluorescence was quenched with incubation time, whereas both ThT fluorescence and LS value were enhanced [ Fig.…”

“…However, we have observed in solutions that the DNA binding can accelerate the conversion of wtSOD1 and its oxidized form into aggregates under conditions tested. This accelerating role has been called as a template effect in the SOD1 aggregation . Indeed, the template effect of heparin and its analogs had been observed in the formation of gelsolin and α‐synuclein fibrils .…”

“…This accelerating role has been called as a template effect in the SOD1 aggregation. [43][44][45][46] Indeed, the template effect of heparin and its analogs had been observed in the formation of gelsolin and a-synuclein fibrils. 47,48 In general, the template polyanions were found to remain within the resulted protein aggregates.…”