Publicação
The role of vacuolar membrane proteins in acetic acid-induced cell death
| Resumo: | Saccharomyces cerevisiae has been one of the most widely used model organism for understanding the molecular mechanisms underlying apoptosis. Apoptosis is a form of regulated cell death that can be triggered by a wide variety of external or internal stimuli, such as acetic acid (AA). This acid triggers, in the yeast, a cascade of intracellular apoptotic-like events, both at mitochondria and vacuole level. A similar process occurs during acetate-induced apoptosis in colorectal cancer (CRC) cells. Although not much is known about the role of vacuole/lysosome, their membrane permeabilization (VMP/LMP) appears to be crucial in the regulated cell death process triggered by AA/acetate. Herein, we aimed to assess the involvement of different vacuolar membrane proteins in AA-induced apoptosis, as well as to evaluate their putative role in VMP and translocation of the protease Pep4p from the vacuole to the cytosol. To this end, a functional genetic approach based on a set of mutants lacking the vacuolar membrane proteins Csc1p, Pep3p, Vma4p, Vma16p, Vtc4p and Zrt3p, together with biochemical and analytical techniques, were used. We found that absence of Zrt3p, Vtc4p, Csc1p and Vma4p render cells more resistant to AA. The resistant phenotype of csc1Δ and vtc4Δ mutants was associated with a delayed VMP and release of Pep4p to the cytosol, as previously shown for the zrt3Δ mutant. Altogether, these results indicate that these three proteins or their associated cellular functions, such as their contribution to the intracellular levels of zinc, calcium and polyphosphate, determine cell survival in response to AA. Particularly, and corroborating the involvement of zinc, we found that zinc availability influences survival of cells undergoing AA-induced cell death, reducing or enhancing cell survival under zinc limitation or supplementation, respectively. The levels of intracellular calcium also appear to play a role in AA-induced cell death, as suggested by Ca2+ lower levels in csc1Δ cells after AA treatment. While deletion of VMA16 does not affect cell survival in response to AA, PEP3 and VMA4 deletions do affect. However, further studies are required to characterize the phenotypes of these deletion mutants. In summary, this study allowed to unveil molecular components/cellular processes involved in AA-induced cell death, contributing to the elucidation of the underlying mechanisms and their modulation towards the improvement of yeast industrial strains and the design of a non-conventional therapy for CRC. |
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| Autores principais: | Teixeira, Marta Sofia Aguiar Carvalho de Oliveira |
| Assunto: | Acetic acid Apoptosis Pep4p Saccharomyces cerevisiae Vacuolar membrane permeabilization Vacuole Ácido acético Apoptose Permeabilização da membrana vacuolar Saccharomyces cerevisiae Vacuolo |
| Ano: | 2019 |
| 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: | Saccharomyces cerevisiae has been one of the most widely used model organism for understanding the molecular mechanisms underlying apoptosis. Apoptosis is a form of regulated cell death that can be triggered by a wide variety of external or internal stimuli, such as acetic acid (AA). This acid triggers, in the yeast, a cascade of intracellular apoptotic-like events, both at mitochondria and vacuole level. A similar process occurs during acetate-induced apoptosis in colorectal cancer (CRC) cells. Although not much is known about the role of vacuole/lysosome, their membrane permeabilization (VMP/LMP) appears to be crucial in the regulated cell death process triggered by AA/acetate. Herein, we aimed to assess the involvement of different vacuolar membrane proteins in AA-induced apoptosis, as well as to evaluate their putative role in VMP and translocation of the protease Pep4p from the vacuole to the cytosol. To this end, a functional genetic approach based on a set of mutants lacking the vacuolar membrane proteins Csc1p, Pep3p, Vma4p, Vma16p, Vtc4p and Zrt3p, together with biochemical and analytical techniques, were used. We found that absence of Zrt3p, Vtc4p, Csc1p and Vma4p render cells more resistant to AA. The resistant phenotype of csc1Δ and vtc4Δ mutants was associated with a delayed VMP and release of Pep4p to the cytosol, as previously shown for the zrt3Δ mutant. Altogether, these results indicate that these three proteins or their associated cellular functions, such as their contribution to the intracellular levels of zinc, calcium and polyphosphate, determine cell survival in response to AA. Particularly, and corroborating the involvement of zinc, we found that zinc availability influences survival of cells undergoing AA-induced cell death, reducing or enhancing cell survival under zinc limitation or supplementation, respectively. The levels of intracellular calcium also appear to play a role in AA-induced cell death, as suggested by Ca2+ lower levels in csc1Δ cells after AA treatment. While deletion of VMA16 does not affect cell survival in response to AA, PEP3 and VMA4 deletions do affect. However, further studies are required to characterize the phenotypes of these deletion mutants. In summary, this study allowed to unveil molecular components/cellular processes involved in AA-induced cell death, contributing to the elucidation of the underlying mechanisms and their modulation towards the improvement of yeast industrial strains and the design of a non-conventional therapy for CRC. |
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