The yeast Saccharomyces cerevisiae expresses one member of metacaspase Cys protease family,encoded by YCA1 gene. Combination of proteomics and metabolomics data showed YCA1deletion down-regulated glycolysis, TCA cycle and alcoholic fermentation as compared with WTcells. ?yca1 cells also showed a down-regulation of the pentose phosphate pathway and anaccumulation of pyruvate, correlated with higher levels of certain amino acids found in thesecells. Accordingly, there is a decrease in protein biosynthesis, and up-regulation of specific stressresponse protein like Ahp1p, which possibly provides these cells with a better protection againststress. Moreover, in agreement with the down-regulation of protein biosynthesis machinery in?yca1 cells, we have found that regulation of transcription, co-translational protein folding andprotein targeting to different subcellular locations were also down-regulated.Metabolomics analysis of the nucleotide content showed a significant reduction in ?yca1 cells incomparison with the WT, except for GTP content which remained unchanged. Thus, ourcombined proteome/metabolome approach added a new dimension to the non-apoptotic functionof yeast metacaspase, which can specifically affect cell metabolism through as yet unknownmechanisms and possibly stress-response pathways, like HOG and cell wall integrity pathways.Certainly, YCA1 deletion may induce compensatory changes in stress response proteins offering abetter protection against apoptosis to ?yca1 cells rather than a loss in a pro-apoptotic YCA1-associated activity.

Positive-strand RNA [(+)RNA] viruses, the largest class of viruses, include many important pathogens of humans, animals and plants, sharing common replication mechanisms. A highly conserved feature of (+)RNA virus replication is the association of the viral replication complex with specific intracellular membranes, which are induced to proliferate and are extensively rearranged to form vesicles (or spherules). These partially closed vesicular enclaves constitute the confined environment in which virus and host factors concentrate to allow for a productive viral RNA synthesis, under conditions protected from host defense reactions. Virus-encoded proteins are responsible for the intracellular localization of the replication complex and for the formation of spherules. The association of viral replicase proteins with the outer membrane of mitochondria has been studied in details with Carnation Italian ringspot virus (CIRV, genus Tombusvirus, family Tombusviridae), a virus with a (+)RNA genome 4.8 kb in size, containing five ORFs. In infected plants, CIRV replication takes place in membranous structures originating from vesiculation of the mitochondrial outer membrane. The signals targeting and anchoring CIRV replication complex to the mitochondrial membrane are contained in the 36-kDa product of ORF1 (p36). Most traits of CIRV replication can be reconstituted in Saccharomyces cerevisiae cells, thus representing a good model for virus-host interaction studies. Heterologous expression of p36 protein fused or not to GFP localizes to mitochondria in yeast cells and causes organelle and membrane proliferation. To gain insights into the interaction between p36 and mitochondria, the effects were studied of p36 heterologous expression on yeast cell viability as well as on programmed cell death induced by acetic acid. It was shown that p36 affects cell viability and seems to exert an inhibitory effect on the nature of acetic acid-induced cell death. Due to the conservation of replication mechanisms between (+)RNA viruses, data obtained with simple model viruses, like CIRV, in a simple eukaryotic host, could be extended to pathogens of higher eukaryotes.

To gain further insight into yeast acetic acid-induced programmed cell death (AA-PCD) we analyzed the effects of the antioxidant N-acetyl-L-cysteine (NAC) on cell viability, hydrogen peroxide (H2O2)production, DNA fragmentation, cytochrome c (cyt c) release and caspase-like activation in wild type (wt) and metacaspase and/or cyt c-lacking cells. We found that NAC prevents AA-PCD in wt cells, by scavenging H2O2 and by inhibiting both cyt c release and caspase-like activation. This shows the occurrence of a reactive oxygen species (ROS)-dependent AA-PCD. Contrarily no NAC dependentchange in AA-PCD of mutant cells was detectable, showing that a ROS-independent AA-PCD can also occur.

Fino a 15 anni fa si pensava che la morte cellulare programmata, la cui forma più nota è l'apoptosi, fosse una peculiarità degli organismi pluricellulari per garantire il corretto sviluppo embrionale, il differenziamento cellulare e la regolazione della risposta immunitaria. La scoperta che anche gli esseri unicellulari come il lievito vanno incontro a morte cellulare programmata ha smentito questa convinzione, destando stupore nella comunità scientifica: a che scopo un organismo unicellulare dovrebbe attivare un programma di morte geneticamente controllato?

Mitochondrial dysfunction has been associated with cancer development and progression. Recent evidences suggest that pathogenic mutations or depletion of the mitochondrial genome can contribute to development of chemoresistance in malignant tumors. In this review we will describe the current knowledge on the role of mitochondrial dysfunction in the development of chemoresistance in cancer. We will also discuss the significance of this research topic in the context of development of more effective, targeted therapeutic modalities and diagnostic strategies for cancer patients, with a particular focus on the potential use of PARP inhibitors in cancer patients displaying mitochondrial DNA mutations. We will discuss recent studies highlighting the importance of the cross-talk between the tumor microenvironment and mitochondrial functionality in determining selective response to certain chemotherapeutic drugs. Finally, owing to the similarities between cancer and yeast cell metabolism, we will point out the use of yeast as a model system to study cancer-related genes and for anti-cancer drugs screening. © 2014 Elsevier Inc. All rights reserved.

Beyond its classical biotechnological applications such as food and beverage production or as a cell factory, the yeast Saccharomyces cerevisiae is a valuable model organism to study fundamental mechanisms of cell response to stressful environmental changes. Acetic acid is a physiological product of yeast fermentation and it is a well-known food preservative due to its antimicrobial action. Acetic acid has recently been shown to cause yeast cell death and aging. Here we shall focus on the molecular mechanisms of S. cerevisiae stress adaptation and programmed cell death in response to acetic acid. We shall elaborate on the intracellular signaling pathways involved in the cross-talk of pro-survival and pro-death pathways underlying the importance of understanding fundamental aspects of yeast cell homeostasis to improve the performance of a given yeast strain in biotechnological applications.

Caspase proteases are responsible for the regulated disassembly of the cell into apoptotic bodies during mammalian apoptosis. Structural homologues of the caspase family (called metacaspases) are involved in programmed cell death in single-cell eukaryotes, yet the molecular mechanisms that contribute to death are currently undefined. Recent evidence revealed that a programmed cell death process is induced by acetic acid (AA-PCD) in Saccharomyces cerevisiae both in the presence and absence of metacaspase encoding gene YCA1. Here, we report an unexpected role for the yeast metacaspase in protein quality and metabolite control. By using an "omics" approach, we focused our attention on proteins and metabolites differentially modulated en route to AA-PCD either in wild type or YCA1-lacking cells. Quantitative proteomic and metabolomic analyses of wild type and ?yca1 cells identified significant alterations in carbohydrate catabolism, lipid metabolism, proteolysis and stress-response, highlighting the main roles of metacaspase in AA-PCD. Finally, deletion of YCA1 led to AA-PCD pathway through the activation of ceramides, whereas in the presence of the gene yeast cells underwent an AA-PCD pathway characterized by the shift of the main glycolytic pathway to the pentose phosphate pathway and a proteolytic mechanism to cope with oxidative stress. Significance: The yeast metacaspase regulates both proteolytic activities through the ubiquitin-proteasome system and ceramide metabolism as revealed by proteome and metabolome profiling of YCA1-knock-out cells during acetic-acid induced programmed cell death.

Silencing of the tumor suppressor protein BRCA2 and its detection by conventional biochemical analyses represent a great technical challenge owing to the large size of the human BRCA2 protein (approximately 390 kDa). We report modifications of standard siRNA transfection and immunoblotting protocols to silence human BRCA2 and detect endogenous BRCA2 protein, respectively, in human epithelial cell lines. Key steps include a high siRNA to transfection reagent ratio and two subsequent rounds of siRNA transfection within the same experiment. Using these and other modifications to the standard protocol we consistently achieve more than 70% silencing of the human BRCA2 gene as judged by immunoblotting analysis with anti-BRCA2 antibodies. In addition, denaturation of the cell lysates at 55 °C instead of the conventional 70-100 °C and other technical optimizations of the immunoblotting procedure allow detection of intact BRCA2 protein even when very low amounts of starting material are available or when BRCA2 protein expression levels are very low. Efficient silencing of BRCA2 in human cells offers a valuable strategy to disrupt BRCA2 function in cells with intact BRCA2, including tumor cells, to examine new molecular pathways and cellular functions that may be affected by pathogenic BRCA2 mutations in tumors. Adaptation of this protocol for efficient silencing and analysis of other 'large' proteins like BRCA2 should be readily achievable.

Adhesion of normal epithelial cells to the extracellular matrix (ECM) is essential for survival. Cell detachment from ECM induces a specific form of programmed cell death (PCD) termed anoikis. BRCA2, a tumor suppressor gene whose mutations confer predisposition to cancer, has been implicated in the regulation of DNA repair, transcription, cell proliferation, and apoptosis. However, the potential role of BRCA2 in the regulation of anoikis has not been investigated. Here, we found that suppression of BRCA2 expression by short hairpin RNA promoted resistance to anoikis in prostate, breast and thyroid normal epithelial cells, which was accompanied by reduced caspases 3/7 levels and activity. Using yeast as a model, we assessed that expression of human BRCA2 does not induce cell death by itself but it can promote acetic acid-induced PCD (AA-PCD). Induction of BRCA2 expression decreased cell survival and increased the number of cells positive to different apoptotic markers, including DNA fragmentation and phosphatidylserine externalization en route to AA-PCD. A higher increase in ROS levels occurred in the early phase of AA-PCD in BRCA2-expressing yeast cells compared with non-expressing cells. Accordingly, a delay in the initial burst of ROS levels was observed in BRCA2-knockdown anoikis-resistant human cells. Treatment with the antioxidants N-acetylcysteine or ascorbic acid reduced sensitivity to anoikis in human cells and inhibited AA-PCD in yeast cells expressing BRCA2. Taken together, these results show a new function of BRCA2 protein as modulator of anoikis sensitivity through an evolutionarily-conserved molecular mechanism involving regulation of ROS production and/or detoxification by BRCA2 during PCD processes.

Mitochondrial diseases are a plethora of inherited neuromuscular disorders sharing defects in mitochondrial respiration, but largely different from one another for genetic basis and pathogenic mechanism. Whole exome sequencing was performed in a familiar trio (trio-WES) with a child affected by severe epileptic encephalopathy associated with respiratory complex I deficiency and mitochondrial DNA depletion in skeletal muscle. By trio-WES we identified biallelic mutations in SLC25A10, a nuclear gene encoding a member of the mitochondrial carrier family. Genetic and functional analyses conducted on patient fibroblasts showed that SLC25A10 mutations are associated with reduction in RNA quantity and aberrant RNA splicing, and to absence of SLC25A10 protein and its transporting function. The yeast SLC25A10 ortholog knockout strain showed defects in mitochondrial respiration and mitochondrial DNA content, similarly to what observed in the patient skeletal muscle, and growth susceptibility to oxidative stress. Albeit patient fibroblasts were depleted in the main antioxidant molecules NADPH and glutathione, transport assays demonstrated that SLC25A10 is unable to transport glutathione. Here, we report the first recessive mutations of SLC25A10 associated to an inherited severe mitochondrial neurodegenerative disorder. We propose that SLC25A10 loss-of-function causes pathological disarrangements in respiratory-demanding conditions and oxidative stress vulnerability.

When the glucose supply is high, despite the presence of oxygen Saccharomyces cerevisiae uses fermentation as its main metabolic pathway and switches to oxidative metabolism only when this carbon source is limited. There are similarities between glucose-induced repression of oxidative metabolism of yeast and metabolic reprogramming of tumor cells. The glucose-induced repression of oxidative metabolism is regulated by oncogene homologues in yeast, such as Ras and Sch9p, the yeast homologue of Akt. Yeast also undergoes an apoptosis-like programmed cell death process sharing several features with mammalian apoptosis, including oxidative stress and a major role played by mitochondria. Evasion of apoptosis and sustained proliferative signalling are hallmarks of cancer. This, together with the possibility of heterologous expression of human genes in yeast, has allowed new insights to be obtained into the function of mammalian oncogenes/oncosuppressors. Here we elaborate on the similarities between tumor and yeast cells underpinning the use of this model organism in cancer research. We also review the achievements obtained through heterologous expression in yeast of p53, BRCA1 and BRCA2 which are among the best known cancer susceptibility genes, with the aim of understanding their role in tumorigenesis. Yeast cell-based functional assays for cancer genetic testing will also be dealt with. This article is protected by copyright. All rights reserved.

The replication of positive-strand RNA [(+)RNA] viruses is always associated with specific cell membranes. The association of viral replicase proteins with mitochondria has been studied with Carnation Italian ringspot virus (CIRV), a (+)RNA plant virus replicating in membranous structures originating from vesiculation of the mitochondrial outer membrane. The replication-associated protein p36 is responsible for targeting and anchoring CIRV replication complex to the mitochondrial membrane. Yet, the mechanisms by which (+)RNA viruses control host cell fate are still unknown.Programmed cell death (PCD) is a primordial response of virus-infected cells, whose mechanism is conserved from yeast to mammals.Taking advantage of the successful CIRV replication in the yeast Saccharomyces cerevisiae,we investigated whether and how the heterologous expression of the mitochondria-targeted CIRV p36 alone or in combination with the replicase protein p95 and DI RNA could affect yeast cell viability or stress-induced PCD.S. cerevisiae YPH499 cells were transformed with plasmids containing the CIRV p36 sequence, fused or not to GFP, cloned under the control of the inducible GAL1 promoter. Acetic acid-induced PCD (AA-PCD) was triggered and cell viability measured as in Giannattasio et al. (2005).The type of yeast cell death was analyzed by measuring plasma membrane integrity and PS externalization by propidium iodide and FITC-annexin V staining, respectively, and fluorescence microscopy.Heterologous expression of p36/p95 did not affect yeast cell viability up to 72 h whereas p36 alone slowed down cell growth after 24 h expression compared to control cells, with virtually no loss in plasma membrane integrity. Interestingly,after AA-PCD induction, p36-expressing cells lost viability at the same rate as the controls but showed a change in the type of cell death judged by apoptotic and necrotic marker analysis.S. cerevisiae is a powerful model organism for virus-host interaction studies. Due to the conservation of replication mechanisms among (+)RNA viruses, the data obtained with plain model viruses in this simple eukaryotic host can potentially be transferred to pathogens of higher eukaryotes. In addition, this system can be extended to investigate cellular pathways such as PCD.

Programmed cell death can occur through two separate pathways caused by treatment of Saccharomyces cerevisiaewith acetic acid (AA-PCD), which differ from one another essentially with respect to their sensitivity to Nacetylcysteine(NAC) and to the role played by cytochrome c and metacaspase YCA1. Moreover, yeast can also undergomacroautophagy which occurs in NAC-insensitive manner. In order to gain some insight into the relationship betweenAA-PCD and macroautophagy use was made of WT and knock-out cells lacking YCA1 and/or cytochrome c. We showthat i. macroautophagy is modulated by YCA1 and by cytochrome c in a negative and positive manner, respectively, ii.the NAC-insensitive AA-PCD and macroautophagy differ from one another and iii. NAC-insensitive AA-PCD pathwaytakes place essentially without macroautophagy, even if the shift of extracellular pH to acidic values required for AA-PCDto occur leads itself to increased or decreased macroautophagy in YCA1 or cytochrome c-lacking cells.

Mammalian apoptosis and yeast programmed cell death (PCD) share a variety of features including reactive oxygen species production, protease activity and a major role played by mitochondria. In view of this, and of the distinctive characteristics differentiating yeast and multicellular organism PCD, the mitochondrial contribution to cell death in the genetically tractable yeast Saccharomyces cerevisiae has been intensively investigated. In this mini-review we report whether and how yeast mitochondrial function and proteins belonging to oxidative phosphorylation, protein trafficking into and out of mitochondria, and mitochondrial dynamics, play a role in PCD. Since in PCD many processes take place over time, emphasis will be placed on an experimental model based on acetic acid-induced PCD (AA-PCD) which has the unique feature of having been investigated as a function of time. As will be described there are at least two AA-PCD pathways each with a multifaceted role played by mitochondrial components, in particular by cytochrome c.

Cell homeostasis results from the balance between cell capability to adapt or succumb to environmental stress. Mitochondria, inaddition to supplying cellular energy, are involved in a range of processes deciding about cellular life or death. The crucial role ofmitochondria in cell death is well recognized.Mitochondrial dysfunction has been associated with the death process and the onsetof numerous diseases. Yet, mitochondrial involvement in cellular adaptation to stress is still largely unexplored. Strong interestexists in pharmacologicalmanipulation ofmitochondrialmetabolism and signaling. The yeast Saccharomyces cerevisiae has provena valuablemodel organism in which several intracellular processes have been characterized in great detail, including the retrograderesponse to mitochondrial dysfunction and, more recently, programmed cell death. In this paper we review experimental evidencesof mitochondrial involvement in cytoprotection and propose yeast as a model system to investigate the role of mitochondria in thecross-talk between prosurvival and prodeath pathways.

To investigate the role of cytochrome c (cyt c) release in yeast acetic acid-induced programmed celldeath (AA-PCD), wild type (wt) and cells lacking metacaspase (Dyca1), cytochrome c (Dcyc1,7) andboth (Dcyc1,7Dyca1) were compared for AA-PCD occurrence, hydrogen peroxide (H2O2) productionand caspase activity. AA-PCD occurs in Dcyc1,7 and Dcyc1,7Dyca1 cells slower than in wt, but similarto that in Dyca1 cells, in which no cytochrome c release occurs. Both H2O2 production and caspaseactivation occur in these cells with early and extra-activation in Dcyc1,7 cells. We conclude thatalternative death pathways can be activated in yeast AA-PCD, one dependent on cyt c release, whichrequires YCA1, and the other(s) independent on it.

Mitochondrial retrograde signaling is a mitochondria-to-nucleus communication pathway, conserved from yeast to humans, by which dysfunctional mitochondria relay signals that lead to cell stress adaptation in physiopathological conditions by changes in nuclear gene expression. The best comprehension of components and regulation of retrograde signaling have been obtained in Saccharomyces cerevisiae, where retrograde target gene expression is regulated by RTG genes. In this chapter, we describe the methods to measure mitochondrial retrograde pathway activation in yeast cells by monitoring the mRNA levels of RTG target genes, such as those encoding for peroxisomal citrate synthase, aconitase, and NAD(+)-specific isocitrate dehydrogenase subunit 1, as well as the phosphorylation status of Rtg1/3p transcriptional factor which controls RTG target gene transcription.

Remembering the ancient latin saying omnes viae Romam ducunt, the yeast cell death community came to the eternal city to attend the 9th International Meeting on Yeast Apoptosis (IMYA), from 17-20 September 2012. More than one hundred investigators from around the world presented and discussed their researches on programmed cell death (PCD) and its role in stress responses, aging and development employing yeast as model organism.

In order to investigate whether and how a modification of mitochondrialmetabolism can affect yeast sensitivityto programmed cell death (PCD) induced by acetic acid (AA-PCD), yeast cells were grown on raffinose, as a sole carbon source, which, differently from glucose, favours mitochondrial respiration. We found that, differently from glucose-grown cells, raffinose-grown cells were mostly resistant to AA-PCD and that this was due to the activation of mitochondrial retrograde (RTG) response, which increased with time, as revealed by the upregulationof the peroxisomal isoform of citrate synthase and isocitrate dehydrogenase isoform 1, RTG pathwaytarget genes. Accordingly, the deletion of RTG2 and RTG3, a positive regulator and a transcription factor of the RTG pathway, resulted in AA-PCD, as shown by TUNEL assay. Neither deletion in raffinose-grown cells of HAP4,encoding the positive regulatory subunit of the Hap2,3,4,5 complex nor constitutive activation of the RTG pathway in glucose-grown cells due to deletion of MKS1, a negative regulator of RTG pathway, had effect on yeast AA-PCD. The RTG pathway was found to be activated in yeast cells containing mitochondria, in which membrane potential wasmeasured, capable to consume oxygen in amanner stimulated by the uncoupler CCCP and inhibited by the respiratory chain inhibitor antimycin A. AA-PCD resistance in raffinose-grown cells occurs with a decrease in both ROS production and cytochrome c release as compared to glucose-grown cells en route to AA-PCD.