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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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Guanidine-induced unfolding of apo-I113T and apo-A4V SOD1 in the presence of Isoproterenol and 5-FUrd.(a) Apo-dimeric I113T unfolding in GdnHCl at 0.2 M concentration intervals with Isoproterenol or 5-FUrd monitored by ellipticity at 220 nm. The mid-point of the unfolding transition is found at 0.95, 0.87 and 0.81 M GdnHCl. (b) Apo-dimeric A4V unfolding in GdnHCl at 0.2-M concentration intervals with Isoproterenol or 5-FUrd monitored by ellipticity at 220 nm with unfolding transitions at 0.57, 0.81 and 0.73 M GdnHCl. In each case, unfolding is monitored by a single circular dichroism measurement.
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f4: Guanidine-induced unfolding of apo-I113T and apo-A4V SOD1 in the presence of Isoproterenol and 5-FUrd.(a) Apo-dimeric I113T unfolding in GdnHCl at 0.2 M concentration intervals with Isoproterenol or 5-FUrd monitored by ellipticity at 220 nm. The mid-point of the unfolding transition is found at 0.95, 0.87 and 0.81 M GdnHCl. (b) Apo-dimeric A4V unfolding in GdnHCl at 0.2-M concentration intervals with Isoproterenol or 5-FUrd monitored by ellipticity at 220 nm with unfolding transitions at 0.57, 0.81 and 0.73 M GdnHCl. In each case, unfolding is monitored by a single circular dichroism measurement.

Mentions: The instability of mutant SOD1 in comparison with the wild-type enzyme is well documented141532, and stabilization of the protein is regarded as a viable strategy for drug development18. Isoproterenol and 5-FUrd were not assayed for a protective effect against chemically induced SOD1 denaturation by Nowak et al.22; however, Lansbury and co-workers have used this method extensively to complement in silico docking and in vitro aggregation experiments2122. Figure 4 shows the guanidine hydrochloride (GdnHCl)-induced unfolding of I113T and dimeric A4V SOD1 in the presence of these two compounds. The unfolding transition shown is monitored using circular dichroism spectroscopy at 220 nm, which is typical for β-sheet-rich proteins33; however, measurements at 230, 218, 215, 212, 210 and 208 nm indicated identical transitions. At low guanidine concentration (<2 M) apo-SOD1 is known to unfold34, and this is replicated here for both apo-I113T and apo-A4V with unfolding transitions at 0.81 and 0.57 M GdnHCl, respectively. This transition is not significantly shifted by the presence of a fourfold molar excess of Isoproterenol or 5-FUrd.


Ligand binding and aggregation of pathogenic SOD1.

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

Guanidine-induced unfolding of apo-I113T and apo-A4V SOD1 in the presence of Isoproterenol and 5-FUrd.(a) Apo-dimeric I113T unfolding in GdnHCl at 0.2 M concentration intervals with Isoproterenol or 5-FUrd monitored by ellipticity at 220 nm. The mid-point of the unfolding transition is found at 0.95, 0.87 and 0.81 M GdnHCl. (b) Apo-dimeric A4V unfolding in GdnHCl at 0.2-M concentration intervals with Isoproterenol or 5-FUrd monitored by ellipticity at 220 nm with unfolding transitions at 0.57, 0.81 and 0.73 M GdnHCl. In each case, unfolding is monitored by a single circular dichroism measurement.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3644087&req=5

f4: Guanidine-induced unfolding of apo-I113T and apo-A4V SOD1 in the presence of Isoproterenol and 5-FUrd.(a) Apo-dimeric I113T unfolding in GdnHCl at 0.2 M concentration intervals with Isoproterenol or 5-FUrd monitored by ellipticity at 220 nm. The mid-point of the unfolding transition is found at 0.95, 0.87 and 0.81 M GdnHCl. (b) Apo-dimeric A4V unfolding in GdnHCl at 0.2-M concentration intervals with Isoproterenol or 5-FUrd monitored by ellipticity at 220 nm with unfolding transitions at 0.57, 0.81 and 0.73 M GdnHCl. In each case, unfolding is monitored by a single circular dichroism measurement.
Mentions: The instability of mutant SOD1 in comparison with the wild-type enzyme is well documented141532, and stabilization of the protein is regarded as a viable strategy for drug development18. Isoproterenol and 5-FUrd were not assayed for a protective effect against chemically induced SOD1 denaturation by Nowak et al.22; however, Lansbury and co-workers have used this method extensively to complement in silico docking and in vitro aggregation experiments2122. Figure 4 shows the guanidine hydrochloride (GdnHCl)-induced unfolding of I113T and dimeric A4V SOD1 in the presence of these two compounds. The unfolding transition shown is monitored using circular dichroism spectroscopy at 220 nm, which is typical for β-sheet-rich proteins33; however, measurements at 230, 218, 215, 212, 210 and 208 nm indicated identical transitions. At low guanidine concentration (<2 M) apo-SOD1 is known to unfold34, and this is replicated here for both apo-I113T and apo-A4V with unfolding transitions at 0.81 and 0.57 M GdnHCl, respectively. This transition is not significantly shifted by the presence of a fourfold molar excess of Isoproterenol or 5-FUrd.

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