Publicação
Role of apoptotic regulators in the protective effect of acetic acid against ethanol - induced cell death in Saccharomyces cerevisiae
| Resumo: | Ethanol is a well-know end product of alcoholic fermentation carried by Saccharomyces cerevisiae. At relatively low concentrations it is an inhibitor of yeast growth, while, high ethanol concentrations reduces cell viability (Birch and Walker, 2000). During fermentation, acetic acid produced by yeast metabolism may accumulate in growth medium and enhance ethanol toxicity (Gibson et al., 2007). By contrast, studies conducted in our laboratory have shown that S. cerevisiae cells exposed simultaneously to toxic concentrations of ethanol and low concentrations of acetic acid displayed a higher survival than cells treated only with ethanol (Vieira et al., unpublished results). Later, Trindade showed that Hog1p and Fps1p, involved in acetic acid resistance, do not have a role in the protective effect of acetic acid seen by Vieira (Trindade, 2009). Subsequently, it was also shown that the osmolyte trehalose, the heat shock protein Hsp12p, the MAP kinase Slt2/MPK1 and functional mitochondria play a role in the protection by the undissociated form of acetic acid against ethanol induced cytotoxicity (Afonso, 2011). With the present master thesis we intended to further understand how acetic acid protects against ethanol-induced cytotoxicity, determining the role of known apoptotic regulators in this protection. We also intended to characterize the death of cells exposed to toxic concentrations of ethanol since this knowledge is important to dissect the role of acetic acid in its inhibition. This study showed that the deletion in CYC3 or ATP2, ATP10 and NUC1 abolish and decrease, respectively, the protection of acetic acid against loss of cell viability, loss of plasma membrane integrity and the accumulation of superoxide anion, induced by ethanol. Death induced by ethanol was associated with typical markers of apoptosis but also of necrosis. In particular caspase activation and exposure of phosphatidylserine was detected mainly in cells with compromised plasma membrane, but also HMGB1/Nhp6A translocates from the nucleus to the cytosol in response to ethanol. Moreover, the absence of known apoptotic regulatory proteins abrogate (atp2Δ, atp10Δ, cyc3Δ, por1Δ, cpr3Δ and yca1Δ mutants), exacerbate (aif1Δ, nuc1Δ, and cyc1Δcyc7Δ mutants) or have no effect (pep4Δ mutant) in ethanol induced cell death. Altogether the results obtained with the mutants phenotypes and the characterization of cell death markers indicate that ethanol triggers a regulated cell death process that shares features typical of both apoptosis and necrosis. Whether the necrotic features reflect a programmed necrosis or a secondary necrosis of apoptotic cells committed to death in response to ethanol, requires further studies. |
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| Autores principais: | Rocha, Natália Cristina Alves |
| Ano: | 2013 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Ethanol is a well-know end product of alcoholic fermentation carried by Saccharomyces cerevisiae. At relatively low concentrations it is an inhibitor of yeast growth, while, high ethanol concentrations reduces cell viability (Birch and Walker, 2000). During fermentation, acetic acid produced by yeast metabolism may accumulate in growth medium and enhance ethanol toxicity (Gibson et al., 2007). By contrast, studies conducted in our laboratory have shown that S. cerevisiae cells exposed simultaneously to toxic concentrations of ethanol and low concentrations of acetic acid displayed a higher survival than cells treated only with ethanol (Vieira et al., unpublished results). Later, Trindade showed that Hog1p and Fps1p, involved in acetic acid resistance, do not have a role in the protective effect of acetic acid seen by Vieira (Trindade, 2009). Subsequently, it was also shown that the osmolyte trehalose, the heat shock protein Hsp12p, the MAP kinase Slt2/MPK1 and functional mitochondria play a role in the protection by the undissociated form of acetic acid against ethanol induced cytotoxicity (Afonso, 2011). With the present master thesis we intended to further understand how acetic acid protects against ethanol-induced cytotoxicity, determining the role of known apoptotic regulators in this protection. We also intended to characterize the death of cells exposed to toxic concentrations of ethanol since this knowledge is important to dissect the role of acetic acid in its inhibition. This study showed that the deletion in CYC3 or ATP2, ATP10 and NUC1 abolish and decrease, respectively, the protection of acetic acid against loss of cell viability, loss of plasma membrane integrity and the accumulation of superoxide anion, induced by ethanol. Death induced by ethanol was associated with typical markers of apoptosis but also of necrosis. In particular caspase activation and exposure of phosphatidylserine was detected mainly in cells with compromised plasma membrane, but also HMGB1/Nhp6A translocates from the nucleus to the cytosol in response to ethanol. Moreover, the absence of known apoptotic regulatory proteins abrogate (atp2Δ, atp10Δ, cyc3Δ, por1Δ, cpr3Δ and yca1Δ mutants), exacerbate (aif1Δ, nuc1Δ, and cyc1Δcyc7Δ mutants) or have no effect (pep4Δ mutant) in ethanol induced cell death. Altogether the results obtained with the mutants phenotypes and the characterization of cell death markers indicate that ethanol triggers a regulated cell death process that shares features typical of both apoptosis and necrosis. Whether the necrotic features reflect a programmed necrosis or a secondary necrosis of apoptotic cells committed to death in response to ethanol, requires further studies. |
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