Disulfide-Reduced ALS Variants of Cu, Zn Superoxide Dismutase Exhibit Increased Populations of Unfolded Species

JOURNAL OF MOLECULAR BIOLOGY

卷:398

Article

Kayatekin, Can¹  Zitzewitz, Jill A.¹  Matthews, C. Robert¹ 

[1] Univ Massachusetts, Sch Med, Dept Biochem & Mol Pharmacol, Worcester, MA 01605 USA

关键词: disulfide bond;    Zn binding;    protein folding;    aggregation;   

DOI  :  10.1016/j.jmb.2010.02.034

来源: Elsevier

【 摘 要 】

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 wildtype (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 and 37 degrees C, A4V, L38V, G93A, and L106V ranged from 50% to similar to 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. (C) 2010 Elsevier Ltd. All rights reserved.

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