Strategies for stabilizing superoxide dismutase (SOD1), the protein destabilized in the most common form of familial amyotrophic lateral sclerosis

Abstract

Amyotrophic lateral sclerosis (ALS) is a disorder characterized by the death of both upper and lower motor neurons and by 3- to 5-yr median survival postdiagnosis. The only US Food and Drug Administration-approved drug for the treatment of ALS, Riluzole, has at best, moderate effect on patient survival and quality of life; therefore innovative approaches are needed to combat neurodegenerative disease. Some familial forms of ALS (fALS) have been linked to mutations in the Cu/Zn superoxide dismutase (SOD1). The dominant inheritance of mutant SOD1 and lack of symptoms in knockout mice suggest a "gain of toxic function" as opposed to a loss of function. A prevailing hypothesis for the mechanism of the toxicity of fALS-SOD1 variants, or the gain of toxic function, involves dimer destabilization and dissociation as an early step in SOD1 aggregation. Therefore, stabilizing the SOD1 dimer, thus preventing aggregation, is a potential therapeutic strategy. Here, we report a strategy in which we chemically cross-link the SOD1 dimer using two adjacent cysteine residues on each respective monomer (Cys111). Stabilization, measured as an increase in melting temperature, of ∼20 °C and ∼45 °C was observed for two mutants, G93A and G85R, respectively. This stabilization is the largest for SOD1, and to the best of our knowledge, for any disease-related protein. In addition, chemical cross-linking conferred activity upon G85R, an otherwise inactive mutant. These results demonstrate that targeting these cysteine residues is an important new strategy for development of ALS therapies.

Fig. 1.

Model of SOD1 after cross-linking DTME with adjacent C111 residues at the dimer interface. (Top, A) Free (unreacted) DTME. (B) Maleimide-chemistry mediated, “cross-linked” reaction product of SOD1 and DTME, with DTME bridging individual SOD1 monomers. SOD1 Cys111 constituents are shown in red, and the two SOD1 monomer/subunits are labeled “suba” and “subb.” Cysteine111 rotomers are oriented such that the cysteinyl sulfur spacing is 13 Å (the minor rotomer observed in crystal structures). (C) Thiol-disulfide exchange- and maleimide-chemistry mediated cross-linked reaction product of SOD1 and DTME. Cysteine111 rotomers are oriented such that the cysteinyl sulfur spacing is 9 Å (the major rotomer observed in crystal structures). Note that thiomaleimidoethane (TME) is comprised of half of the DTME structure. TME results from both binding a maleimide moiety to one subunit (Left), and the reaction of the Cysteine111 thiol of the second subunit with the disulfide of DTME (Right). Note that the Cys111 thiol (red) exchanges with a disulfide sulfur of DTME (green) to form a new disulfide bond. (Bottom) Cysteine 111 sulfur (PDB ID code 2C9V) (44) is denoted by yellow balls. TME, which results from thiol-disulfide exchange (Fig. S3) and maleimide cross-linking as seen in the bottom panel, is shown in cyan. This model, including site of cross-linking, was confirmed via LC-FTMS analysis (Fig. S4).

Fig. 2.

Cross-linker dependent dimerization of SOD1. (A) Extracted ion chromatogram for the 15 H+ charge state of the SOD1 monomer (red) and (B) 23 H+ charge state of the DTME-cross-linked dimer (blue) demonstrate the stoichiometric conversation of monomeric as-isolated SOD1 to dimeric cross-linked SOD1. (C) Mass spectrum of as-isolated (non-cross-linked) G93A SOD1 and (D) DTME cross-linked G93A SOD1 confirm cross-linker dependent dimerization. (E) Chemical cross-linking of WT SOD1 with a reductively labile molecule. One of the maleimide cross-linkers tested, DTME, has a disulfide bond in its spacer arm that can be cleaved by reducing agents such as DTT. WT SOD1 was cross-linked with a 1∶1 molar ratio of DTME and analyzed by SDS-PAGE gel in both the presence and in the absence of reducing agent, DTT. The SOD1 cross-linked dimer became a monomer in the presence of reducing agent (Fig. 2B, lane 2), suggesting that the dimer being formed is specific to the cross-linkers being used, and was not the result of cross-linker catalyzed dimer formation.

Fig. 3.

Stabilization of fALS-associated SOD1 variants by chemical cross-linking. Stability of mutant SOD1 measured by thermofluorescence assay (see Fig. S5). Ten micromolar G93A (A) and G85R SOD1 (C) were incubated with concentrations of BMOE ranging from 2.5–20 μM. Ten micromolar G93A (B) and G85R (D) were incubated with 20 μM copper/zinc and concentrations of BMOE ranging from 2.5–20 μM. The cross-linkers used here were resuspended in DMSO; therefore, SOD1 in 4% DMSO controls were used. WT SOD1 was also investigated; however, due to its near-boiling point melting temperature, the assay used here is not capable of detecting whether or not stabilization occurred. G85R and G93A were stabilized in a cross-linker concentration-dependent manner by approximately 45 °C and 20 °C, respectively. The addition of copper and zinc had little effect on the wild-type like (metallated) G93A variant; however, the addition of copper and zinc to G85R, a metal-deficient variant, along with cross-linkers stabilizes the protein to almost wild-type levels. Fig. 4 illustrates that this highly stable form of G85R has regained wild-type-like levels of SOD1 activity. The above graphs represent the average of three replicates of each respective concentration; these experiments were repeated in triplicate.

Fig. 4.

Rescue of fALS-variant G85R SOD1 activity by chemical cross-linking (see Fig. S6). G85R is a metal-deficient and inactive variant. In the presence of excess copper, zinc, or BMOE G85R activity increases. Most notably, G85R in the presence of copper, zinc, and DTME confers WT-like levels of activity. Thus, the increase in stability of G85R also corresponds to an increase in activity (Fig. S6). As expected WT SOD1 and G93A SOD1, a fully active mutant, appear to be unaffected.