FUNCTIONAL ANALYSIS OF HUMAN SOD2 VARIANTS
M. D. C. DE CARVALHO1, J. F. DE MESQUITA2 AND E. C. A. ELEUTHERIO1
1 Programa de Pós-graduação de Bioquímica, Instituto de Química, Universidade Federal do Rio de Janeiro
2 Programa de Pós-graduação em Genética e Biologia Molecular, Instituto Biomédico, Universidade Federal do Estado do Rio de Janeiro,
Superoxide dismutase 2 (Mn-Sod or Sod2) protects against oxidative stress, a main factor influencing cellular longevity. Polymorphisms in Sod2 have been associated with the development of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s disease, as well as psychiatric disorders, such as schizophrenia, depression and bipolar disorder. In this work, we performed in silico analysis of all of the described natural variants (S10I, A16V, E66V, G76R, I82T and R156W) using eight different algorithms: SNPeffect, PolyPhen-2, PhD-SNP, PMUT, SIFT, SNAP, SNPs&GO and nsSNPAnalyzer. Structural theoretical models were created for variants through comparative modelling performed using the MHOLline server (which includes MODELLER and PROCHECK) and ab initio modelling, using the I-Tasser server. The predicted models were evaluated using TM-align, and the results showed that the models were constructed with high accuracy. The RMSD values of the modelled mutants indicated likely pathogenicity for all missense mutations. Structural phylogenetic analysis using ConSurf revealed that human Sod2 is highly conserved. To investigate the effect of those amino acid substitutions in vivo, Saccharomyces cerevisiae was used as experimental model. Contrary to human, yeast can tolerate Sod2 absence. However, as expected, Sod2 deficiency considerably impaired respiratory metabolism (growth on glycerol) and reduced tolerance to oxidative stress. Currently, we are testing the effect of I65T variant of Saccharomyces cerevisiae (equivalent to I82T human variant) on Sod2 activity and oxidative stress tolerance. According to our results, I82T seems to harmfully affect the protein according to four algorithms. Furthermore, conservation analysis revealed that the I82 amino acid of Sod2 has a high degree of conservation across species, suggesting that isoleucine at position 82 is yet an important amino acid for Sod2.
Financial Support: CAPES, FAPERJ ; Area: Biochemistry
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