Farah Wazir

Introduction. Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease, is a neurodegenerative disease resulting in the loss of alpha motor neurons in the brain and spinal cord with no cure.^1,2 ALS results in difficulty controlling voluntary muscles and the eventual muscular paralysis.¹ ALS patients present with atrophy and spasticity and have a poor prognosis.¹ Familial ALS (fALS) arises primarily from a genetic origin involving major genes, including the superoxide dismutase 1 (Sod1) gene.³ Sod1 is a copper/zinc superoxide dismutase enzyme that converts superoxide radicals into oxygen and hydrogen peroxide and binds zinc and copper metal ions for proper maturation.¹ A copper chaperone (Ccs) promotes Sod1 protein maturation through facilitation of post-translational modifications.⁴ Sod1 gene mutations result in misfolded Sod1 protein in motor neurons and the accumulation of hydroxyl radicals leading to DNA damage.¹ Studies have found that Sod1 mutations hinder Ccs-mediated Sod1 maturation and affect zinc-binding affinities.⁵ Other studies have shown Ccs restores the maturation of Sod1 fALS mutants through the formation of stable Ccs-Zn complexes.⁶ These findings suggest a potential therapeutic target in Ccs mechanisms and metal ions in the proper folding of Sod1. Methods. The yeast form of Ccs and human form of Sod1 were utilized.⁵ Site-specific amino acid changes A4V, H80R, G85R, and G93A were done via quick-change mutagenesis to generate fALS-Sod1 mutants.⁵ Sod1 and Ccs proteins were expressed and purified.⁵ Purified ALS-Sod1 and Ccs protein samples were run on SDS-PAGE gels and visualized with Coomassie stain.⁵ Sod1 was fluorescently labeled and microplate binding assays were prepared.⁶ Equilibrium dialysis was used to measure Zn affinity values of isolated Sod1 mutants and mutant Sod1-Ccs complexes.⁵ Zinc binding affinities were quantified using dissociation constants (K_d).⁵ Results. The interaction of Ccs with fALS-Sod1 mutants was not significantly hindered.⁵ Ccs binding prevents oligomerization and insoluble aggregation in vitro of WT-like Sod1 mutants.⁵ Zinc dissociation constants for each fALS-Sod1 mutant differed at each stage of Sod1 maturation.⁵ Ccs was shown to have little effect on Zn affinity in G93A and G85R Sod1 mutants.⁵ A4V Sod1 mutant showed decreased Zn affinity with each stage of maturation.⁵ Conclusions. Studies show that fALS-Sod1 mutants have worse zinc-binding affinities than in WT Sod1 protein and provide a foundation for future studies in this field. These results indicate a significant role of copper-yeast chaperone in the proper acquisition of zinc in the maturation of Sod1 and a potential therapeutic target in fALS-Sod1 pathology.

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