Neishaliz Cotto-Heredia, BS

Introduction: Over 100 missense mutations in SOD1 gene have been identified in a subset of familial Amyotrophic lateral sclerosis (fALS). This gene encodes for an antioxidant enzyme, superoxide dismutase 1 (SOD1), its role is protecting cells from oxidative stress catalyzing superoxide anion radical into hydrogen peroxide and molecular oxygen. Research suggests that mutant SOD1 proteins associated with ALS can impair mitophagy, leading to the accumulation of damaged mitochondria. The main goal is to determine the effects of SOD1(G92A) in mitophagy.

Methods: We made a report of conserved residues in all family members of the Sod1 protein. Human SOD1 was selected as the query sequence, and we found the members of the Cu-Zn SOD protein family using classification libraries. We searched for homologous proteins for each member of the protein family and performed a multiple sequence alignment (MSA). We investigated conservation of functionally relevant intramolecular interactions based on amino acid sequence alignments and analysis of spatially sidelong residue groups. To evaluate the mitochondria abundance in Sod1(G92A) yeast strains using Mitotracker Green dye. We optimized the concentration of dye to obtain a specific mitochondrial staining in the microscopy method.

Results: We found three fully conserved residues in all 88 sequences: Gly83, Asp84 and Asp125. Also, we identified that residue Gly94(Gly93), involved in ALS mutation G93A, is 80% conserved throughout the protein family. Gly93 has a role in the enzyme’s overall structure. Next, we will evaluate the structural motifs related to mutants SOD1 and docking analysis between protein-protein interactions. The results of the optimization was 15 nM of mitotracker green in 1×10⁷ cell/mL.

Conclusion: Next, we will also optimize the dye for the microplate method to evaluate the abundance of mitochondria in Sod1(G92A) strains. Investigating how the human SOD1G93A mutation affects mitophagy is essential for understanding the pathological mechanisms underlying ALS.

Acknowledgements: Research was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of Health under the grant number P20GM103475-19 and the Scientific Research Center at PUCPR.