Yeast as a tool to study mitochondrial retrograde pathway en route to cell stress response

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

Yeast acetic-acid induced programmed cell death resistance in raffinose is controlled by co-operation of ADR1 and CAT8 with the mitochondrial retrograde regulator RTG2

Nutrient availability and the differentiation state determine cell fate in multicellular organisms. The yeast Saccharomyces cerevisiae is a valuable model organism to study how nutrient availability determines cell fate in different growth conditions. In an attempt to understand how metabolism controls cell death and survival in actively dividing and proliferating cells, we have shown that exponential growing yeast cells in the presence of the preferred carbon source glucose (GLU-WT), which inhibits respiration through carbon catabolite repression (CR) pathway, undergo programmed cell death (PCD) in response to acetic acid (AA) treatment. On the contrary, yeast cells grown in raffinose (RAF-WT) are resistant to AA-PCD in a manner dependent on both the activation of mitochondrial retrograde (RTG) pathway, which senses mitochondrial dysfunction, and glucose de-repression of mitochondrial respiration. To study the relationships between CR and RTG pathway in yeast cell death and survival in response to AA, we compared WT cells and a set of yeast mutants lacking negative or positive regulators of either RTG or CR pathway as for their viability after AA treatment. We found that glucose-grown cells lacking MIG1 and HXK2, which repress transcription of mitochondrial respiratory genes in the presence of glucose, undergo AA-PCD as GLU-WT cells, as judged by decrease in viability and increase in DNA fragmentation. On the other hand, differently from RAF-WT cells, raffinose-grown cells lacking transcription factors ADR1 or CAT8, which are activated by alternative carbon sources, or RTG2, a positive regulator of RTG pathway, underwent AA-PCD. Cells lacking HAP4, the regulatory subunit of HAP complex active in raffinose, remain fully resistant to AA-PCD. Interestingly, double knock-out ∆adr1∆rtg2 and ∆cat8∆rtg2 cells were found nearly as much resistant to AA-PCD as RAF-WT cells. RTG pathway activation was studied in raffinose-grown WT and knock-out cells after AA treatment by analyzing the prototypical RTG-target gene CIT2 mRNA level. Results showed that ADR1 and CAT8 are positive regulators of RTG2-dependent transcription and suggest that RTG and CR pathways co-operate in the control of cell fate through interaction between RTG2 and CAT8 or ADR1

Differential proteome-metabolome profiling of YCA1-knock-out and wild type cells reveals novel metabolic pathways and cellular processes dependent on the yeast metacaspase

The yeast Saccharomyces cerevisiae expresses one member of the metacaspase Cys protease family, encoded by the YCA1 gene. Combination of proteomics and metabolomics data showed that YCA1 deletion down-regulated glycolysis, the TCA cycle and alcoholic fermentation as compared with WT cells. Δyca1 cells also showed a down-regulation of the pentose phosphate pathway and accumulation of pyruvate, correlated with higher levels of certain amino acids found in these cells. Accordingly, there is a decrease in protein biosynthesis, and up-regulation of specific stress response proteins like Ahp1p, which possibly provides these cells with a better protection against stress. Moreover, in agreement with the down-regulation of protein biosynthesis machinery in Δyca1 cells, we have found that regulation of transcription, co-translational protein folding and protein targeting to different subcellular locations were also down-regulated. Metabolomics analysis of the nucleotide content showed a significant reduction in Δyca1 cells in comparison with the WT, except for GTP content which remained unchanged. Thus, our combined proteome-metabolome approach added a new dimension to the non-apoptotic function of yeast metacaspase, which can specifically affect cell metabolism through as yet unknown mechanisms and possibly stress-response pathways, like HOG and cell wall integrity pathways. Certainly, YCA1 deletion may induce compensatory changes in stress response proteins offering a better protection against apoptosis to Δyca1 cells rather than a loss in pro-apoptotic YCA1-associated activity

The transcription factors ADR1 or CAT8 are required for RTG pathway activation and evasion from yeast acetic acid-induced programmed cell death in raffinose

Yeast Saccharomyces cerevisiae grown on glucose undergoes programmed cell death (PCD) induced by acetic acid (AA-PCD), but evades PCD when grown in raffinose. This is due to concomitant relief of carbon catabolite repression (CCR) and activation of mitochondrial retrograde signaling, a mitochondria-to-nucleus communication pathway causing up-regulation of various nuclear target genes, such as CIT2, encoding peroxisomal citrate synthase, dependent on the positive regulator RTG2 in response to mitochondrial dysfunction. CCR down-regulates genes mainly involved in mitochondrial respiratory metabolism. In this work, we investigated the relationships between the RTG and CCR pathways in the modulation of AA-PCD sensitivity under glucose repression or de-repression conditions. Yeast single and double mutants lacking RTG2 and/or certain factors regulating carbon source utilization, including MIG1, HXK2, ADR1, CAT8, and HAP4, have been analyzed for their survival and CIT2 expression after acetic acid treatment. ADR1 and CAT8 were identified as positive regulators of RTG-dependent gene transcription. ADR1 and CAT8 interact with RTG2 and with each other in inducing cell resistance to AA-PCD in raffinose and controlling the nature of cell death. In the absence of ADR1 and CAT8, AA-PCD evasion is acquired through activation of an alternative factor/pathway repressed by RTG2, suggesting that RTG2 may play a function in promoting necrotic cell death in repressing conditions when RTG pathway is inactive. Moreover, our data show that simultaneous mitochondrial retrograde pathway activation and SNF1-dependent relief of CCR have a key role in central carbon metabolism reprogramming which modulates the yeast acetic acid-stress response

Proteome and metabolome profiling of wild-type and YCA1-knock-out yeast cells during acetic acid-induced programmed cell death

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 Δyca1 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

Metabolic Plasiticy in Cancers—Distinct Role of Glycolytic Enzymes GPI, LDHs or Membrane Transporters MCTs

Research on cancer metabolism has recently re-surfaced as a major focal point in cancer field with a reprogrammed metabolism no longer being considered as a mere consequence of oncogenic transformation, but as a hallmark of cancer. Reprogramming metabolic pathways and nutrient sensing is an elaborate way by which cancer cells respond to high bioenergetic and anabolic demands during tumorigenesis. Thus, inhibiting specific metabolic pathways at defined steps should provide potent ways of arresting tumor growth. However, both animal models and clinical observations have revealed that this approach is seriously limited by an extraordinary cellular metabolic plasticity. The classical example of cancer metabolic reprogramming is the preference for aerobic glycolysis, or Warburg effect, where cancers increase their glycolytic flux and produce lactate regardless of the presence of the oxygen. This allows cancer cells to meet the metabolic requirements for high rates of proliferation. Here, we discuss the benefits and limitations of disrupting fermentative glycolysis for impeding tumor growth at three levels of the pathway: (i) an upstream block at the level of the glucose-6-phosphate isomerase (GPI), (ii) a downstream block at the level of lactate dehydrogenases (LDH, isoforms A and B), and (iii) the endpoint block preventing lactic acid export (MCT1/4). Using these examples of genetic disruption targeting glycolysis studied in our lab, we will discuss the responses of different cancer cell lines in terms of metabolic rewiring, growth arrest, and tumor escape and compare it with the broader literature