title: Disulfide-reduced ALS variants of Cu, Zn superoxide dismutase exhibit increased populations of unfolded species.
abstract: Cu,Zn superoxide dismutase (SOD1) is a dimeric metal-binding enzyme responsible for the dismutation of toxic superoxide to hydrogen peroxide and oxygen in cells. Mutations at dozens of sites in SOD1 induce amyotrophic lateral sclerosis (ALS), a fatal gain-of-function neurodegenerative disease whose molecular basis is unknown. To obtain insights into effects of the mutations on the folded and unfolded populations of immature monomeric forms whose aggregation or self-association may be responsible for ALS, the thermodynamic and kinetic folding properties of a set of disulfide-reduced and disulfide-oxidized Zn-free and Zn-bound stable monomeric SOD1 variants were compared to properties of the wild-type (WT) protein. The most striking effect of the mutations on the monomer stability was observed for the disulfide-reduced metal-free variants. Whereas the WT and S134N monomers are >95% folded at neutral pH and 37 degrees C, A4V, L38V, G93A, and L106V ranged from 50% to approximately 90% unfolded. The reduction of the disulfide bond was also found to reduce the apparent Zn affinity of the WT monomer by 750-fold, into the nanomolar range, where it may be unable to compete for free Zn in the cell. With the exception of the S134N metal-binding variant, the Zn affinity of disulfide-oxidized SOD1 monomers showed little sensitivity to amino acid replacements. These results suggest a model for SOD1 aggregation where the constant synthesis of ALS variants of SOD1 on ribosomes provides a pool of species in which the increased population of the unfolded state may favor aggregation over productive folding to the native dimeric state.
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors.forename: Can
authors.initials: C
authors.lastname: Kayatekin
authors.forename: Jill A
authors.initials: JA
authors.lastname: Zitzewitz
authors.forename: C Robert
authors.initials: CR
authors.lastname: Matthews
mesh_terms: D000690:Amyotrophic Lateral Sclerosis / Q000201:enzymology* / Q000235:genetics; D018360:Crystallography, X-Ray; D004220:Disulfides / Q000737:chemistry*; D004795:Enzyme Stability; D006801:Humans; D009154:Mutation; D010084:Oxidation-Reduction; D017510:Protein Folding; D055503:Protein Multimerization; D012270:Ribosomes / Q000378:metabolism; D013482:Superoxide Dismutase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D000072105:Superoxide Dismutase-1; D015032:Zinc / Q000737:chemistry*
chemical_list: D004220:Disulfides; C000606290:SOD1 protein, human; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; D015032:Zinc