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.

Figure 1. Observations of Asc-mediated WT SOD1 oxidation by one- and two-dimensional gel electrophoresis.

A) Non-reducing SDS-PAGE gels for observation of Asc dose-dependent SOD1 oxidation. 10 µM SOD1 was incubated at 37°C for 2 h with 0–8 mM Asc. B) 2-DE gels for observation of pI and molecular mass of the SOD1 protein treated with Asc. 10 µM SOD1 was incubated with 4 mM Asc at 37°C for 2 h.

Figure 2. Comparison of ANS binding property of SOD1 in the presence and absence of both Asc and DNA in 20 mM Tris-HCl buffer (pH 7.4).

A) Fluorescence spectra of 20 µM ANS added into the mixtures containing 4 µM SOD1, 4 µM SOD1 and 7.5 µM ctDNA, or 4 µM SOD1, 7.5 µM ctDNA and 2 mM Asc. B) Change in the emission of 20 µM ANS added into the mixtures containing 4 µM SOD1 and 7.5 µM ctDNA with Asc dose. C) Change in the emission of 20 µM ANS added into the mixtures containing 4 µM SOD1, 2 mM Asc, and 7.5 µM ctDNA with incubation time. Reactions were first incubated at 37°C for 2 h (A and B) or 0–72 h (C), and then re-incubated for 10 min at 37°C after addition of 20 µM ANS prior to measurement.

Figure 3. ITC analysis of oxidized SOD1 proteins binding to ssDNA.

A) The upper panel shows the raw calorimetric data of the titration of ssDNA (40 µM) into WT SOD1 (5 µM) at 25°C in the 20 mM Tris-HCl buffer (pH 7.4) containing 4 mM Asc, and the lower panel shows the corresponding integrated injection heats, corrected for the heat of dilution. B) ITC data of the titration of ssDNA (40 µM) into SOD1 (5 µM) at 25°C in the 20 mM Tris-HCl buffer (pH 7.4) without Asc. All samples were incubated for 2 h at 37°C prior to titration under the identical conditions. The curve in the lower figure represents the best least-squares fits to the one-site binding model.

Figure 4. Dependences of DNA-triggered aggregation of oxidized SOD1 proteins on reaction conditions in 20 mM Tris-HCl buffer (pH 7.4).

A) Reactions without stirring were incubated at 37°C for 0–120 min after addition of 7.5 µM ctDNA into the mixtures containing 4 µM SOD1 and 2 mM Asc. B) For DNA dose dependence, 0–75 µM ctDNA were added into the mixtures containing 4 µM SOD1 and 2 mM Asc were incubated at 37°C for 2 h. C) SOD1 dose dependence was observed by incubating reactions consisted of 0–16 µM SOD1, 2 mM Asc, and 7.5 µM ctDNA at 37°C for 2 h.

Figure 5. Inhibition of DNA-triggered aggregation of oxidized SOD1 proteins in 20 mM Tris-HCl buffer (pH 7.4).

A) Effect of ionic strength was observed by incubating reactions containing 4 µM SOD1, 2 mM Asc, and 7.5 µM ctDNA at 37°C for 2 h in the presence of 0–800 mM NaCl. Here, the inhibition degree of aggregation is expressed by [(RALS)₀– (RALS)_NaCl]/(RALS)₀×100%, (RALS)₀ and (RALS)_NaCl represent RALS values in the absence and in the presence of NaCl at each concentration, respectively. B) The inhibitory effect of GdmCl was monitored by RALS measurements. Reactions containing 4 µM SOD1, 2 mM Asc, and 7.5 µM ctDNA were incubated at 37°C for 2 h in the presence of 0–6 M GdmCl. The disaggregation degree of aggregation is expressed as (A). C) The disaggregation and inhibition of aggregates was monitored by agarose gel electrophoresis. The aggregates were produced by incubating reactions containing 4 µM SOD1, 2 mM Asc, and 15 µM pBR322 DNA for 24 h at 37°C, and re-incubated for 1 min with 0–6 M GdmCl before loading onto gels. D) The inhibition of aggregation caused by EDTA was monitored by agarose gel electrophoresis. 4 µM SOD1 and 2 mM ascorbate were incubated for 2 h at 37°C with 15 µM pBR322 DNA in the presence of 0–100 mM EDTA before loading onto gels.

Figure 6. Visualization of aggregate and control samples under TEM.

The controls were 7.5 µM λDNA (A) and 4 µM SOD1 (B) and incubated for 2 h at 37°C in the buffer (pH 7.4) containing 2 mM Asc prior to observation. C) Aggregate monomers were produced by incubating reactions containing 4 µM SOD1, 2 mM Asc and 7.5 µM λDNA for 2 h at 37°C.