Abstract
BackgroundAcetic acid is mostly known as a toxic by-product of alcoholic fermentation carried out by Saccharomyces cerevisiae, which it frequently impairs. The more recent finding that acetic acid triggers apoptotic programmed cell death (PCD) in yeast sparked an interest to develop strategies to modulate this process, to improve several biotechnological applications, but also for biomedical research. Indeed, acetate can trigger apoptosis in cancer cells, suggesting its exploitation as an anticancer compound. Therefore, we aimed to identify genes involved in the positive and negative regulation of acetic acid-induced PCD by optimizing a functional analysis of a yeast Euroscarf knock-out mutant collection.ResultsThe screen consisted of exposing the mutant strains to acetic acid in YPD medium, pH 3.0, in 96-well plates, and subsequently evaluating the presence of culturable cells at different time points. Several functional categories emerged as greatly relevant for modulation of acetic acid-induced PCD (e.g.: mitochondrial function, transcription of glucose-repressed genes, protein synthesis and modifications, and vesicular traffic for protection, or amino acid transport and biosynthesis, oxidative stress response, cell growth and differentiation, protein phosphorylation and histone deacetylation for its execution). Known pro-apoptotic and anti-apoptotic genes were found, validating the approach developed. Metabolism stood out as a main regulator of this process, since impairment of major carbohydrate metabolic pathways conferred resistance to acetic acid-induced PCD. Among these, lipid catabolism arose as one of the most significant new functions identified. The results also showed that many of the cellular and metabolic features that constitute hallmarks of tumour cells (such as higher glycolytic energetic dependence, lower mitochondrial functionality, increased cell division and metabolite synthesis) confer sensitivity to acetic acid-induced PCD, potentially explaining why tumour cells are more susceptible to acetate than untransformed cells and reinforcing the interest in exploiting this acid in cancer therapy. Furthermore, our results clearly establish a connection between cell proliferation and cell death regulation, evidencing a conserved developmental role of programmed cell death in unicellular eukaryotes.ConclusionsThis work advanced the characterization of acetic acid-induced PCD, providing a wealth of new information on putative molecular targets for its control with impact both in biotechnology and biomedicine.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-14-838) contains supplementary material, which is available to authorized users.
Highlights
Acetic acid is mostly known as a toxic by-product of alcoholic fermentation carried out by Saccharomyces cerevisiae, which it frequently impairs
We found that metabolism is a major regulator of cell death, since impairment of major carbohydrate and amino acid metabolic pathways resulted in increased resistance to acetic acid-induced apoptosis
Cellular processes involved in positive regulation of acetic acid-induced programmed cell death (PCD) Amino acid biosynthesis Of the genes whose deletion caused resistance to acetic acid-induced PCD, and are involved in mediation of this process, “Amino acid transport” was the most significantly enriched term, with “Cellular amino acid biosynthetic process” significantly enriched (Table 2). Grouped under these terms there were genes encoding proteins involved in assimilation of ammonia, metabolism of urea cycle, creatine and polyamines (e.g. ALD3, SPE1, SPE3), metabolism of glutamate (GDH1 and GDH2), metabolism of amino acids of the aspartate family, threonine (e.g. HOM3, THR1), arginine (e.g. ARG1-3, ARG80-82, CPA1) and methionine (e.g. MET1, MET2, SAM4), metabolism of amino acids of the pyruvate family, alanine (e.g. AGX1), isoleucine (e.g. BAT2), leucine (e,g, LEU1, LEU4, LEU9) and valine (e.g. LPD1), metabolism of tryptophan (e.g. BNA2, BNA3, BNA4), histidine (e.g. HIS2-4, HIS6, HIS7), glycine (e.g. AGX1, GCV1, LPD1, SHM2), serine (e.g. SER2, SER3, SER33), cysteine (e.g. STR2) and phenylalanine (e.g. AAD3-4, ARO1, ARO8-10). These results show that tolerance to acetic acid-induced PCD is connected with the incapacity of the cell to promote the biosynthesis of amino acids, contrary to what was observed in a study assessing determinants of growth in the presence of acetic acid [16]
Summary
Acetic acid is mostly known as a toxic by-product of alcoholic fermentation carried out by Saccharomyces cerevisiae, which it frequently impairs. Acetic acid can affect cell viability and lead to a programmed cell death (PCD) process with features similar to mammalian apoptosis, such as chromatin condensation along the nuclear envelope, DNA fragmentation, ROS accumulation, hyperpolarization followed by depolarization of the mitochondrial membrane, exposure of phosphatidylserine on the outer leaflet of the cytoplasmic membrane and release of cytochrome c (cyt c) from mitochondria [7,8]. This PCD process can proceed via pathways dependent or independent of the yeast metacaspase Yac1p [9,10,11]. The discovery that acetate triggers apoptotic cell death in cancer cells [12,13] reinforced the importance of elucidating the mechanisms underlying this process for the biomedical field
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