PhD Student Universidade de São Paulo São Paulo, Sao Paulo, Brazil
Caroline Goncalves de Góes (Universidade de São Paulo)| Victor Faria (Universidade de São Paulo)| Fernando Gomes (Universidade de São Paulo)| Luis Netto (Universidade de São Paulo)
We are interested in characterization of the mechanisms by which cells decompose peroxides during conditions of peroxisome biogenesis, when H2O2 is generated during beta-oxidation of fatty acids. Catalase is a well-known protective system in peroxisome, but other systems cooperate to protect cells from oxidative insults.
Saccharomyces cerevisiae is a model organism that contains eight Cys-based peroxidases. Catalase A (CTA1) is a peroxisomal enzyme involved in H2O2 detoxification in yeast. However, knockout of yeast for catalase (Δcta1) displays similar resistance did not render cells more sensitive to H2O2 than wild-type cells, suggesting the existence of other peroxide removing systems.
Indeed, glutathione peroxidase 1 (Gpx1) is a peroxisomal enzyme, whose expression is induced in medium containing oleate. Alkylhydroperoxide reductase 1 (Ahp1) is an atypical 2-Cys peroxiredoxin (Prx), belonging to the Prx5 sub-family that is located in the cytosol, when yeast cells are grown on glucose. However, Ahp1 might also be located in the peroxisome, when cells are grown in oleate-containing medium, as its amino acid sequence displays a C-terminal AHL motif, compatible with the peroxisomal targeting signal 1 (PTS1).
Here, we aim to gain insights on the roles of Ahp1 in yeast cells in distinct physiological conditions, using a Δahp1 (cells with the Ahp1 gene deleted) strain. Notably, yeast cells treated with 1mM tert.-butyl hydroperoxyde (tBHP) underwent a growth arrest, in medium containing glucose. The lag time to recover the cell growth in glucose was longer for ΔAhp1 (about 72 hours) than wild type cells (WT), ΔGpx1 and ΔCta1 strains (about 48 hours). In ethanol medium, the growth of all strains showed lag time about 72 hours and all mutant cells displayed a lag time of about 24 hours when treated with 0.5 mM tBHP. On the other hand, all strains when grown in medium containing oleate as the sole carbon source showed no lag time.
We also measured the amounts of peroxides remaining in medium, using the ferrous oxidation-xylenol orange (FOX) assay. The observed lag times were correlated the amounts of tBHP remaining in culture medium. As tBHP levels dropped to certain threshold levels, yeast growth was restored for all strains. Ahp1 expression increased in Δtsa1 (cells with the Tsa1 gene deleted) strain grown in oleate as carbon source, indicating compensatory effects. In contrast, Ahp1 levels were not altered in the Δtsa1 strain grown in a glucose-containing medium with the 0.5 mM tBHP treatment. Together, our results suggest that Ahp1 is involved in the response of yeast to tBHP insult.