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Ligand binding and aggregation of pathogenic SOD1.

Wright GS, Antonyuk SV, Kershaw NM, Strange RW, Samar Hasnain S - Nat Commun (2013)

Bottom Line: Superoxide dismutase-1 mutations decrease protein stability and promote aggregation.We find both compounds interact with superoxide dismutase-1 at a key region identified at the core of the superoxide dismutase-1 fibrillar aggregates, β-barrel loop II-strand 3, rather than the proposed dimer interface site.This illustrates the need for direct structural observations when developing compounds for protein-targeted therapeutics.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biophysics Group, Institute of Integrative Biology, Faculty of Health and Life Sciences, University of Liverpool, Liverpool L69 7ZB, UK.

ABSTRACT
Mutations in the gene encoding Cu/Zn superoxide dismutase-1 cause amyotrophic lateral sclerosis. Superoxide dismutase-1 mutations decrease protein stability and promote aggregation. The mutant monomer is thought to be an intermediate in the pathway from the superoxide dismutase-1 dimer to aggregate. Here we find that the monomeric copper-apo, zinc-holo protein is structurally perturbed and the apo-protein aggregates without reattainment of the monomer-dimer equilibrium. Intervention to stabilize the superoxide dismutase-1 dimer and inhibit aggregation is regarded as a potential therapeutic strategy. We describe protein-ligand interactions for two compounds, Isoproterenol and 5-fluorouridine, highlighted as superoxide dismutase-1 stabilizers. We find both compounds interact with superoxide dismutase-1 at a key region identified at the core of the superoxide dismutase-1 fibrillar aggregates, β-barrel loop II-strand 3, rather than the proposed dimer interface site. This illustrates the need for direct structural observations when developing compounds for protein-targeted therapeutics.

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Aggregation of apo-A4V and apo-I113T SOD1 in the presence of Isoproterenol and 5-FUrd.Size-exclusion chromatograms of apo-SOD1 with Isoproterenol and 5-FUrd after 48 h incubation at 37 °C compared with similarly treated protein without ligand and the fresh protein used as starting material for aggregation experiments measured at 280 nm. (a) 100 μM dimeric A4V SOD1 with 300 μM and 3 mM Isoproterenol. (b) 100 μM monomeric A4V SOD1 with 300 μM and 3 mM Isoproterenol. (c) 100 μM dimeric I113T SOD1 with 300 μM and 3 mM Isoproterenol. (d) 25 μM dimeric A4V SOD1 with 75 and 750 μM Isoproterenol. (e) 25 μM monomeric A4V SOD1 with 75 and 750 μM Isoproterenol. (f) 25 μM monomeric I113T SOD1 with 75 and 750 μM Isoproterenol. (g) 100 μM dimeric A4V SOD1 with 300 μM and 3 mM 5-FUrd. (h) 100 μM monomeric A4V SOD1 with 300 μM and 3 mM 5-FUrd. (i) 100 μM dimeric I113T SOD1 with 300 μM and 3 mM 5-FUrd. (j) 25 μM dimeric A4V SOD1 with 75 and 750 μM 5-FUrd. (k) 25 μM monomeric A4V SOD1 with 75 and 750 μM 5-FUrd. (l) 25 μM monomeric I113T SOD1 with 75 and 750 μM 5-FUrd. Each line is representative of a single chromatography run; however, figures showing similar chromatograms resulting from the average of triplicate separations of I113T with both 5-FUrd and Isoproterenol can be found in Supplementary Figs S1–S4.
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f3: Aggregation of apo-A4V and apo-I113T SOD1 in the presence of Isoproterenol and 5-FUrd.Size-exclusion chromatograms of apo-SOD1 with Isoproterenol and 5-FUrd after 48 h incubation at 37 °C compared with similarly treated protein without ligand and the fresh protein used as starting material for aggregation experiments measured at 280 nm. (a) 100 μM dimeric A4V SOD1 with 300 μM and 3 mM Isoproterenol. (b) 100 μM monomeric A4V SOD1 with 300 μM and 3 mM Isoproterenol. (c) 100 μM dimeric I113T SOD1 with 300 μM and 3 mM Isoproterenol. (d) 25 μM dimeric A4V SOD1 with 75 and 750 μM Isoproterenol. (e) 25 μM monomeric A4V SOD1 with 75 and 750 μM Isoproterenol. (f) 25 μM monomeric I113T SOD1 with 75 and 750 μM Isoproterenol. (g) 100 μM dimeric A4V SOD1 with 300 μM and 3 mM 5-FUrd. (h) 100 μM monomeric A4V SOD1 with 300 μM and 3 mM 5-FUrd. (i) 100 μM dimeric I113T SOD1 with 300 μM and 3 mM 5-FUrd. (j) 25 μM dimeric A4V SOD1 with 75 and 750 μM 5-FUrd. (k) 25 μM monomeric A4V SOD1 with 75 and 750 μM 5-FUrd. (l) 25 μM monomeric I113T SOD1 with 75 and 750 μM 5-FUrd. Each line is representative of a single chromatography run; however, figures showing similar chromatograms resulting from the average of triplicate separations of I113T with both 5-FUrd and Isoproterenol can be found in Supplementary Figs S1–S4.

Mentions: Using an in silico screening approach, Lansbury and co-workers2122 identified a diverse array of chemical structures, which inhibited aggregation of A4V SOD1 in vitro. These compounds were postulated to bind a hydrophobic pocket created by opposing residues Val7, Gly147 and Val148 at the dimer interface. This was thought to stabilize the SOD1 dimer, and inhibit monomerization and slow aggregation. 5-FUrd, Isoproterenol and norepinephrine were found to inhibit SOD1 aggregation to such an extent that ~90% of the SOD1 dimer remained after a 48-h incubation in comparison with 40% if the compounds were not present22. To investigate the effect these compounds have on the aggregation of SOD1, the aggregation assay of Nowak et al.22 was replicated using 25 and 100 μM monomeric and dimeric A4V SOD1. Two compound concentrations, a 1:3 and 1:30 molar excess of ligand, were used to ensure consistency and attempt to provide a sufficiency of bound ligand, respectively. Identical experiments were also performed with I113T SOD1, because aggregation has been well characterized for this mutant and has been proven inhibitable in vitro by licensed drug compounds3031. Figure 3 shows the size exclusion chromatograms for aggregated SOD1 in the presence of Isoproterenol and 5-FUrd. At 25 μM dimeric A4V and 75 μM compound (Fig. 3d), there is no inhibition of either dimer loss or accumulation of higher molecular weight species when compared with the control. This result is directly contradictory to the work of Nowak et al.22 and is also seen at higher protein concentrations (Fig. 3a). Indeed, the chromatograms of SOD1 with 5-FUrd are almost indistinguishable from the controls at any of the concentrations assayed (Fig. 3g–l and Supplementary Figs. S1–2) and are highly repeatable (Supplementary Figs S1 and S2). Isoproterenol concentrations of 750 μM or 3 mM cause an increase in 280-nm absorbance over a region corresponding to high molecular weight species (7–13 ml; Fig. 3a–f and Supplementary Figs S3 and S4). Chromatography of 3 mM Isoproterenol after incubation without SOD1 indicates this compound spontaneously forms high molecular weight species (Supplementary Fig. S5). This may relate to oxidation of the catechol alcohols and formation of melanin-like macromolecules, but these products do not elute concurrently with monomeric, dimeric or aggregated SOD1. In the case of A4V SOD1, there is a slight reduction in prevalence of the dimeric or monomeric starting material when compared with the control and low Isoproterenol concentration (75 and 300 μM) (Fig. 3a, b, d and e). These observations suggest high Isoproterenol concentrations may promote SOD1 oligomerization or the ligand is becoming incorporated into growing SOD1 aggregates causing an increase in 280 nm absorption.


Ligand binding and aggregation of pathogenic SOD1.

Wright GS, Antonyuk SV, Kershaw NM, Strange RW, Samar Hasnain S - Nat Commun (2013)

Aggregation of apo-A4V and apo-I113T SOD1 in the presence of Isoproterenol and 5-FUrd.Size-exclusion chromatograms of apo-SOD1 with Isoproterenol and 5-FUrd after 48 h incubation at 37 °C compared with similarly treated protein without ligand and the fresh protein used as starting material for aggregation experiments measured at 280 nm. (a) 100 μM dimeric A4V SOD1 with 300 μM and 3 mM Isoproterenol. (b) 100 μM monomeric A4V SOD1 with 300 μM and 3 mM Isoproterenol. (c) 100 μM dimeric I113T SOD1 with 300 μM and 3 mM Isoproterenol. (d) 25 μM dimeric A4V SOD1 with 75 and 750 μM Isoproterenol. (e) 25 μM monomeric A4V SOD1 with 75 and 750 μM Isoproterenol. (f) 25 μM monomeric I113T SOD1 with 75 and 750 μM Isoproterenol. (g) 100 μM dimeric A4V SOD1 with 300 μM and 3 mM 5-FUrd. (h) 100 μM monomeric A4V SOD1 with 300 μM and 3 mM 5-FUrd. (i) 100 μM dimeric I113T SOD1 with 300 μM and 3 mM 5-FUrd. (j) 25 μM dimeric A4V SOD1 with 75 and 750 μM 5-FUrd. (k) 25 μM monomeric A4V SOD1 with 75 and 750 μM 5-FUrd. (l) 25 μM monomeric I113T SOD1 with 75 and 750 μM 5-FUrd. Each line is representative of a single chromatography run; however, figures showing similar chromatograms resulting from the average of triplicate separations of I113T with both 5-FUrd and Isoproterenol can be found in Supplementary Figs S1–S4.
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f3: Aggregation of apo-A4V and apo-I113T SOD1 in the presence of Isoproterenol and 5-FUrd.Size-exclusion chromatograms of apo-SOD1 with Isoproterenol and 5-FUrd after 48 h incubation at 37 °C compared with similarly treated protein without ligand and the fresh protein used as starting material for aggregation experiments measured at 280 nm. (a) 100 μM dimeric A4V SOD1 with 300 μM and 3 mM Isoproterenol. (b) 100 μM monomeric A4V SOD1 with 300 μM and 3 mM Isoproterenol. (c) 100 μM dimeric I113T SOD1 with 300 μM and 3 mM Isoproterenol. (d) 25 μM dimeric A4V SOD1 with 75 and 750 μM Isoproterenol. (e) 25 μM monomeric A4V SOD1 with 75 and 750 μM Isoproterenol. (f) 25 μM monomeric I113T SOD1 with 75 and 750 μM Isoproterenol. (g) 100 μM dimeric A4V SOD1 with 300 μM and 3 mM 5-FUrd. (h) 100 μM monomeric A4V SOD1 with 300 μM and 3 mM 5-FUrd. (i) 100 μM dimeric I113T SOD1 with 300 μM and 3 mM 5-FUrd. (j) 25 μM dimeric A4V SOD1 with 75 and 750 μM 5-FUrd. (k) 25 μM monomeric A4V SOD1 with 75 and 750 μM 5-FUrd. (l) 25 μM monomeric I113T SOD1 with 75 and 750 μM 5-FUrd. Each line is representative of a single chromatography run; however, figures showing similar chromatograms resulting from the average of triplicate separations of I113T with both 5-FUrd and Isoproterenol can be found in Supplementary Figs S1–S4.
Mentions: Using an in silico screening approach, Lansbury and co-workers2122 identified a diverse array of chemical structures, which inhibited aggregation of A4V SOD1 in vitro. These compounds were postulated to bind a hydrophobic pocket created by opposing residues Val7, Gly147 and Val148 at the dimer interface. This was thought to stabilize the SOD1 dimer, and inhibit monomerization and slow aggregation. 5-FUrd, Isoproterenol and norepinephrine were found to inhibit SOD1 aggregation to such an extent that ~90% of the SOD1 dimer remained after a 48-h incubation in comparison with 40% if the compounds were not present22. To investigate the effect these compounds have on the aggregation of SOD1, the aggregation assay of Nowak et al.22 was replicated using 25 and 100 μM monomeric and dimeric A4V SOD1. Two compound concentrations, a 1:3 and 1:30 molar excess of ligand, were used to ensure consistency and attempt to provide a sufficiency of bound ligand, respectively. Identical experiments were also performed with I113T SOD1, because aggregation has been well characterized for this mutant and has been proven inhibitable in vitro by licensed drug compounds3031. Figure 3 shows the size exclusion chromatograms for aggregated SOD1 in the presence of Isoproterenol and 5-FUrd. At 25 μM dimeric A4V and 75 μM compound (Fig. 3d), there is no inhibition of either dimer loss or accumulation of higher molecular weight species when compared with the control. This result is directly contradictory to the work of Nowak et al.22 and is also seen at higher protein concentrations (Fig. 3a). Indeed, the chromatograms of SOD1 with 5-FUrd are almost indistinguishable from the controls at any of the concentrations assayed (Fig. 3g–l and Supplementary Figs. S1–2) and are highly repeatable (Supplementary Figs S1 and S2). Isoproterenol concentrations of 750 μM or 3 mM cause an increase in 280-nm absorbance over a region corresponding to high molecular weight species (7–13 ml; Fig. 3a–f and Supplementary Figs S3 and S4). Chromatography of 3 mM Isoproterenol after incubation without SOD1 indicates this compound spontaneously forms high molecular weight species (Supplementary Fig. S5). This may relate to oxidation of the catechol alcohols and formation of melanin-like macromolecules, but these products do not elute concurrently with monomeric, dimeric or aggregated SOD1. In the case of A4V SOD1, there is a slight reduction in prevalence of the dimeric or monomeric starting material when compared with the control and low Isoproterenol concentration (75 and 300 μM) (Fig. 3a, b, d and e). These observations suggest high Isoproterenol concentrations may promote SOD1 oligomerization or the ligand is becoming incorporated into growing SOD1 aggregates causing an increase in 280 nm absorption.

Bottom Line: Superoxide dismutase-1 mutations decrease protein stability and promote aggregation.We find both compounds interact with superoxide dismutase-1 at a key region identified at the core of the superoxide dismutase-1 fibrillar aggregates, β-barrel loop II-strand 3, rather than the proposed dimer interface site.This illustrates the need for direct structural observations when developing compounds for protein-targeted therapeutics.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biophysics Group, Institute of Integrative Biology, Faculty of Health and Life Sciences, University of Liverpool, Liverpool L69 7ZB, UK.

ABSTRACT
Mutations in the gene encoding Cu/Zn superoxide dismutase-1 cause amyotrophic lateral sclerosis. Superoxide dismutase-1 mutations decrease protein stability and promote aggregation. The mutant monomer is thought to be an intermediate in the pathway from the superoxide dismutase-1 dimer to aggregate. Here we find that the monomeric copper-apo, zinc-holo protein is structurally perturbed and the apo-protein aggregates without reattainment of the monomer-dimer equilibrium. Intervention to stabilize the superoxide dismutase-1 dimer and inhibit aggregation is regarded as a potential therapeutic strategy. We describe protein-ligand interactions for two compounds, Isoproterenol and 5-fluorouridine, highlighted as superoxide dismutase-1 stabilizers. We find both compounds interact with superoxide dismutase-1 at a key region identified at the core of the superoxide dismutase-1 fibrillar aggregates, β-barrel loop II-strand 3, rather than the proposed dimer interface site. This illustrates the need for direct structural observations when developing compounds for protein-targeted therapeutics.

Show MeSH
Related in: MedlinePlus