Publicação
Elucidation of molecular pathways involved in Saccharomyces cerevisiae apoptotic cell death
| Resumo: | The yeast Saccharomyces cerevisiae undergoes apoptosis upon either external or physiological stimuli, through the intervention of several orthologues of mammalian apoptotic regulators. Evidence indicated that like mammalian cells, yeast present distinct pathways that lead to an apoptotic cell death. However, integrative studies allowing the definition of key pathways in yeast apoptosis were still scarce at the moment this work began. Such studies are crucial for increasing the knowledge on the intrinsic yeast apoptotic mechanisms, allowing a better understanding of the roots of metazoan apoptosis. Using proteomic, biochemistry and functional analysis, data was obtained contributing to a further understanding of the apoptotic pathways induced by three well described apoptotic inducers, acetic acid, hydrogen peroxide (H2O2) and chronological aging. We revealed that contrarily to H2O2-induced apoptosis, acetic acid promotes intracellular amino acids starvation, and induces alterations in the levels of proteins directly or indirectly linked with the target of rapamycin (TOR) pathway, implicating this pathway in the progress of apoptosis. Further analysis demonstrated that the role of TOR pathway in acetic acid-induced apoptosis requires the downstream phosphatases Pph21p, Pph22p, but not Sit4p. We also demonstrated that general amino-acid control (GAAC) system, through its main players Gcn2p and Gcn4p, cooperates with TOR in the triggering of yeast apoptosis. The translation factor eEF1A appeared as a key factor on acetic acid-induced apoptotic mechanism, suggesting a role on the promotion of actin cytoskeleton alterations and mitochondrial dysfunction that culminate in cell death. Analysis of yeast H2O2-induced apoptotic process revealed the induction of a totally distinct mechanism. Upon H2O2 treatment, we demonstrated that yeast cells are able to synthesize the signaling molecule nitric oxide (NO), which in turn acts in two different ways: it boosts the intracellular levels of reactive oxygen species (ROS), and promotes, as in mammalian cells, S-nitrosation of the yeast glycolytic enzyme glyceraldehyde-3- phosphate dehydrogenase (GAPDH). Additionally, we demonstrated that these molecular events also occur during the physiological triggering of apoptosis during chronological aging. By further analyzing the role of GAPDH in the commitment of yeast cells to apoptosis, we revealed that this protein is, at the present date, the only described yeast metacaspase substrate, being cleaved upon NO signaling. NO control of metacaspase-mediated GAPDH cleavage might rely on its capacity to activate yeast metacaspase or in the promotion of GAPDH S-nitrosation as a signal for cleavage. Preliminary results indicate that GAPDH is cleaved by metacaspase in order to impair its role in survival through participation in autophagy. Since yeast cells display an endogenous apoptotic machinery, besides contributing for the elucidation of metazoan apoptosis, yeast also offers the opportunity for screening compounds with antifungal properties. In fact, the discovery of antifungal compounds able to promote cell death on antifungal-resistant fungi could be one of the major contributions of yeast for biomedical research. Thus, ciclopirox olamine (CPO), a fungicidal agent widely used in clinical practice but which mechanism of action was still elusive, was shown to induce in yeast cells a programmed cell death (PCD) process, characterized by nuclear morphology alterations, chromatin condensation associated with the appearance of a population in the sub-G0/G1 cell cycle phase and an arrest in the G2/M phases. Subsequent analysis on CPO-mediated cell death indicated that the PCD process induced by this antifungal agent is atypical, as it was neither associated with ROS signaling nor with a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive phenotype, or with the yeast homologue of apoptosis-inducing factor (AIF) and metacaspase. On the contrary, CPO effects seem to be dependent on unknown aspartic protease activity, indicating that CPO could also be employed for the further uncovering of yeast endogenous cell death machinery. In summary, the results presented in the scope of this thesis contributed to the knowledge on the yeast apoptotic processes, reinforcing yeast as an extremely valuable model for the study of metazoan apoptosis and for the screening of effective antifungal compounds able to modulate its endogenous apoptotic machinery. |
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| Autores principais: | Almeida, Bruno |
| Ano: | 2009 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | português |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | The yeast Saccharomyces cerevisiae undergoes apoptosis upon either external or physiological stimuli, through the intervention of several orthologues of mammalian apoptotic regulators. Evidence indicated that like mammalian cells, yeast present distinct pathways that lead to an apoptotic cell death. However, integrative studies allowing the definition of key pathways in yeast apoptosis were still scarce at the moment this work began. Such studies are crucial for increasing the knowledge on the intrinsic yeast apoptotic mechanisms, allowing a better understanding of the roots of metazoan apoptosis. Using proteomic, biochemistry and functional analysis, data was obtained contributing to a further understanding of the apoptotic pathways induced by three well described apoptotic inducers, acetic acid, hydrogen peroxide (H2O2) and chronological aging. We revealed that contrarily to H2O2-induced apoptosis, acetic acid promotes intracellular amino acids starvation, and induces alterations in the levels of proteins directly or indirectly linked with the target of rapamycin (TOR) pathway, implicating this pathway in the progress of apoptosis. Further analysis demonstrated that the role of TOR pathway in acetic acid-induced apoptosis requires the downstream phosphatases Pph21p, Pph22p, but not Sit4p. We also demonstrated that general amino-acid control (GAAC) system, through its main players Gcn2p and Gcn4p, cooperates with TOR in the triggering of yeast apoptosis. The translation factor eEF1A appeared as a key factor on acetic acid-induced apoptotic mechanism, suggesting a role on the promotion of actin cytoskeleton alterations and mitochondrial dysfunction that culminate in cell death. Analysis of yeast H2O2-induced apoptotic process revealed the induction of a totally distinct mechanism. Upon H2O2 treatment, we demonstrated that yeast cells are able to synthesize the signaling molecule nitric oxide (NO), which in turn acts in two different ways: it boosts the intracellular levels of reactive oxygen species (ROS), and promotes, as in mammalian cells, S-nitrosation of the yeast glycolytic enzyme glyceraldehyde-3- phosphate dehydrogenase (GAPDH). Additionally, we demonstrated that these molecular events also occur during the physiological triggering of apoptosis during chronological aging. By further analyzing the role of GAPDH in the commitment of yeast cells to apoptosis, we revealed that this protein is, at the present date, the only described yeast metacaspase substrate, being cleaved upon NO signaling. NO control of metacaspase-mediated GAPDH cleavage might rely on its capacity to activate yeast metacaspase or in the promotion of GAPDH S-nitrosation as a signal for cleavage. Preliminary results indicate that GAPDH is cleaved by metacaspase in order to impair its role in survival through participation in autophagy. Since yeast cells display an endogenous apoptotic machinery, besides contributing for the elucidation of metazoan apoptosis, yeast also offers the opportunity for screening compounds with antifungal properties. In fact, the discovery of antifungal compounds able to promote cell death on antifungal-resistant fungi could be one of the major contributions of yeast for biomedical research. Thus, ciclopirox olamine (CPO), a fungicidal agent widely used in clinical practice but which mechanism of action was still elusive, was shown to induce in yeast cells a programmed cell death (PCD) process, characterized by nuclear morphology alterations, chromatin condensation associated with the appearance of a population in the sub-G0/G1 cell cycle phase and an arrest in the G2/M phases. Subsequent analysis on CPO-mediated cell death indicated that the PCD process induced by this antifungal agent is atypical, as it was neither associated with ROS signaling nor with a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive phenotype, or with the yeast homologue of apoptosis-inducing factor (AIF) and metacaspase. On the contrary, CPO effects seem to be dependent on unknown aspartic protease activity, indicating that CPO could also be employed for the further uncovering of yeast endogenous cell death machinery. In summary, the results presented in the scope of this thesis contributed to the knowledge on the yeast apoptotic processes, reinforcing yeast as an extremely valuable model for the study of metazoan apoptosis and for the screening of effective antifungal compounds able to modulate its endogenous apoptotic machinery. |
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