Paula Ludovico

Saccharomyces cerevisiae commits to a programmed cell death process in response to acetic acid

Recent evidence has revealed the occurrence of an apoptotic phenotype in Saccharomyces cerevisiae that is inducible with oxidative stress. Here, exposure of S. cerevisiae to 20-200 mM acetic acid for 200 min at pH 3.0 resulted in cell death. Yeast mortality induced by 120-200 mM acid was not inhibited by cycloheximide and was accompanied by ultrastructural alterations typical of necrosis. In contrast, alterations associated with cell death induced by 20-80 mM acetic acid included: (i) cycloheximide-inhibitable chromatin condensation along the nuclear envelope; (ii) exposure of phosphatidylserine on the surface of the cytoplasmic membrane, revealed by the FITC-annexin V reaction; and (iii) the occurrence of DNA strand breaks, demonstrated by the TUNEL assay. These results show that a programmed cell death process sharing common features with an apoptotic phenotype can be induced by acetic acid in S. cerevisiae. This observation raises the possibility of this mode of cell death being more generalized in yeasts than previously considered and extended to cell death induced by other stress agents.

An AIF orthologue regulates apoptosis in yeast

Apoptosis-inducing factor (AIF), a key regulator of cell death, is essential for normal mammalian development and participates in pathological apoptosis. The proapoptotic nature of AIF and its mode of action are controversial. Here, we show that the yeast AIF homologue Ynr074cp controls yeast apoptosis. Similar to mammalian AIF, Ynr074cp is located in mitochondria and translocates to the nucleus of yeast cells in response to apoptotic stimuli. Purified Ynr074cp degrades yeast nuclei and plasmid DNA. YNR074C disruption rescues yeast cells from oxygen stress and delays age-induced apoptosis. Conversely, overexpression of Ynr074cp strongly stimulates apoptotic cell death induced by hydrogen peroxide and this effect is attenuated by disruption of cyclophilin A or the yeast caspase YCA1. We conclude that Ynr074cp is a cell death effector in yeast and rename it AIF-1 (Aif1p, gene AIF1).

Caloric restriction or catalase inactivation extends yeast chronological lifespan by inducing H2O2 and superoxide dismutase activity

The free radical theory of aging posits oxidative damage to macromolecules as a primary determinant of lifespan. Recent studies challenge this theory by demonstrating that in some cases, longevity is enhanced by inactivation of oxidative stress defenses or is correlated with increased, rather than decreased reactive oxygen species and oxidative damage. Here we show that, in Saccharomyces cerevisiae, caloric restriction or inactivation of catalases extends chronological lifespan by inducing elevated levels of the reactive oxygen species hydrogen peroxide, which activate superoxide dismutases that inhibit the accumulation of superoxide anions. Increased hydrogen peroxide in catalase-deficient cells extends chronological lifespan despite parallel increases in oxidative damage. These findings establish a role for hormesis effects of hydrogen peroxide in promoting longevity that have broad implications for understanding aging and age-related diseases.

Hyperosmotic stress induces metacaspase - and mitochondria - dependent apoptosis in Saccharomyces cerevisiae

During the last years, several reports described an apoptosis‐like programmed cell death process in yeast in response to different environmental aggressions. Here, evidence is presented that hyperosmotic stress caused by high glucose or sorbitol concentrations in culture medium induces in Saccharomyces cerevisiae a cell death process accompanied by morphological and biochemical indicators of apoptotic programmed cell death, namely chromatin condensation along the nuclear envelope, mitochondrial swelling and reduction of cristae number, production of reactive oxygen species and DNA strand breaks, with maintenance of plasma membrane integrity. Disruption of AIF1 had no effect on cell survival, but lack of Yca1p drastically reduced metacaspase activation and decreased cell death indicating that this death process was associated to activation of this protease. Supporting the involvement of mitochondria and cytochrome c in caspase activation, the mutant strains cyc1Δcyc7Δ and cyc3Δ, both lacking mature cytochrome c, displayed a decrease in caspase activation associated to increased cell survival when exposed to hyperosmotic stress. These findings indicate that hyperosmotic stress triggers S. cerevisiae into an apoptosis‐like programmed cell death that is mediated by a caspase‐dependent mitochondrial pathway partially dependent on cytochrome c.

NO-mediated apoptosis in yeast

Nitric oxide (NO) is a small molecule with distinct roles in diverse physiological functions in biological systems, among them the control of the apoptotic signalling cascade. By combining proteomic, genetic and biochemical approaches we demonstrate that NO and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are crucial mediators of yeast apoptosis. Using indirect methodologies and a NO-selective electrode, we present results showing that H2O2-induced apoptotic cells synthesize NO that is associated to a nitric oxide synthase (NOS)-like activity as demonstrated by the use of a classical NOS kit assay. Additionally, our results show that yeast GAPDH is a target of extensive proteolysis upon H2O2-induced apoptosis and undergoes S-nitrosation. Blockage of NO synthesis with Nω-nitro-L-arginine methyl ester leads to a decrease of GAPDH S-nitrosation and of intracellular reactive oxygen species (ROS) accumulation, increasing survival. These results indicate that NO signalling and GAPDH S-nitrosation are linked with H2O2-induced apoptotic cell death. Evidence is presented showing that NO and GAPDH S-nitrosation also mediate cell death during chronological life span pointing to a physiological role of NO in yeast apoptosis.

SNCA (α-synuclein)-induced toxicity in yeast cells is dependent on sirtuin 2 (Sir2)-mediated mitophagy

SNCA (α-synuclein) misfolding and aggregation is strongly associated with both idiopathic and familial forms of Parkinson disease (PD). Evidence suggests that SNCA has an impact on cell clearance routes and protein quality control systems such as the ubiquitin-proteasome system (UPS) and autophagy. Recent advances in the key role of the autosomal recessive PARK2/PARKIN and PINK1 genes in mitophagy, highlighted this process as a prominent new pathogenic mechanism. Nevertheless, the role of autophagy/mitophagy in the pathogenesis of sporadic and autosomal dominant familial forms of PD is still enigmatic. The yeast Saccharomyces cerevisiae is a powerful “empty room” model that has been exploited to clarify different molecular aspects associated with SNCA toxicity, which combines the advantage of being an established system for aging research. The contribution of autophagy/mitophagy for the toxicity induced by the heterologous expression of the human wild-type SNCA gene and the clinical A53T mutant during yeast chronological life span (CLS) was explored. A reduced CLS together with an increase of autophagy and mitophagy activities were observed in cells expressing both forms of SNCA. Impairment of mitophagy by deletion of ATG11 or ATG32 resulted in a CLS extension, further implicating mitophagy in the SNCA toxicity. Deletion of SIR2, essential for SNCA toxicity, abolished autophagy and mitophagy, thereby rescuing cells. These data show that Sir2 functions as a regulator of autophagy, like its mammalian homolog, SIRT1, but also of mitophagy. Our work highlights that increased mitophagy activity, mediated by the regulation of ATG32 by Sir2, is an important phenomenon linked to SNCA-induced toxicity during aging.

Drug-induced apoptosis in yeast

In order to alter the impact of diseases on human society, drug development has been one of the most invested research fields. Nowadays, cancer and infectious diseases are leading targets for the design of effective drugs, in which the primary mechanism of action relies on the modulation of programmed cell death (PCD). Due to the high degree of conservation of basic cellular processes between yeast and higher eukaryotes, and to the existence of an ancestral PCD machinery in yeast, yeasts are an attractive tool for the study of affected pathways that give insights into the mode of action of both antitumour and antifungal drugs. Therefore, we covered some of the leading reports on drug-induced apoptosis in yeast, revealing that in common with mammalian cells, antitumour drugs induce apoptosis through reactive oxygen species (ROS) generation and altered mitochondrial functions. The evidence presented suggests that yeasts may be a powerful model for the screening/development of PCD-directed drugs, overcoming the problem of cellular specificity in the design of antitumour drugs, but also enabling the design of efficient antifungal drugs, targeted to fungal-specific apoptotic regulators that do not have major consequences for human cells.

Yeast protein expression profile during acetic acid‐induced apoptosis indicates causal involvement of the TOR pathway

Although acetic acid has been shown to induce apoptosis in yeast, the exact apoptotic mechanisms remain unknown. Here, we studied the effects of acetic acid treatment on yeast cells by 2‐DE, revealing alterations in the levels of proteins directly or indirectly linked with the target of rapamycin (TOR) pathway: amino‐acid biosynthesis, transcription/translation machinery, carbohydrate metabolism, nucleotide biosynthesis, stress response, protein turnover and cell cycle. The increased levels of proteins involved in amino‐acid biosynthesis presented a counteracting response to a severe intracellular amino‐acid starvation induced by acetic acid. Deletion of GCN4 and GCN2 encoding key players of general amino‐acid control (GAAC) system caused a higher resistance to acetic acid indicating an involvement of Gcn4p/Gcn2p in the apoptotic signaling. Involvement of the TOR pathway in acetic acid‐induced apoptosis was also reflected by the higher survival rates associated to a terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling (TUNEL)‐negative phenotype and lower reactive oxygen species levels of Δtor1 cells. In addition, deletion mutants for several downstream mediators of the TOR pathway revealed that apoptotic signaling involves the phosphatases Pph21p and Pph22p but not Sit4p. Altogether, our results indicate that GAAC and TOR pathways (Tor1p) are involved in the signaling of acetic acid‐induced apoptosis.

Cdc42p controls yeast-cell shape and virulence of Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is characterized by a multiple budding phenotype and a polymorphic cell growth, leading to the formation of cells with extreme variations in shape and size. Since Cdc42 is a pivotal molecule in establishing and maintaining polarized growth for diverse cell types, as well as during pathogenesis of certain fungi, we evaluated its role during cell growth and virulence of the yeast-form of P. brasiliensis. We used antisense technology to knock-down PbCDC42s expression in P. brasiliensis yeast cells, promoting a decrease in cell size and more homogenous cell growth, altering the typical polymorphism of wild-type cells. Reduced expression levels also lead to increased phagocytosis and decreased virulence in a mouse model of infection. We provide genetic evidences underlying Pbcdc42p as an important protein during host-pathogen interaction and the relevance of the polymorphic nature and cell size in the pathogenesis of P. brasiliensis.

Sugar Metabolism in Yeasts: an Overview of Aerobic and Anaerobic Glucose Catabolism

Yeasts are ubiquitous unicellular fungi widespread in natural environments colonizing from terrestrial, to aerial to aquatic environments, where the successful colonization is intimately related to their physiological adaptability to a highly variable environment. The metabolic pathways of the central carbon metabolism are basically identical between different yeast species, suggesting that these microorganisms might constitute a metabolic homogenous group. Nonetheless, the mechanisms for nutrient uptake, the number of different isoenzymes and most importantly the regulation of fermentation and respiration differ substantially (Flores et al. 2000) and make yeasts a highly heterogeneous and complex metabolic group. In yeasts, like other heterotrophic organisms, the energy and carbon metabolism are intimately interconnected, i.e., anabolism is coupled with catabolism. ATP is provided by the oxidation of organic molecules that also act as carbon sources for biosynthesis, and ultimately it is used as energetic currency for all kinds of cellular work. In the natural environment yeast species have a broad set of carbon sources (e.g., polyols, alcohols, organic acids and amino acids) that can support their growth but preferentially they metabolize sugars. The information related to the metabolism of different carbon sources is huge, the most widely studied being sugars such as hexoses (glucose, fructose, galactose or mannose) and disaccharides (maltose or sucrose) as well as compounds with two carbons (ethanol or acetate). The metabolic networks employed for the metabolism of hexoses and disaccharides share the same pathways (most metabolic building blocks are derived from intermediaries of glycolysis, the tricarboxylic acid cycle (TCA), and the pentose phosphate pathway) and differ only in the initial basic steps of metabolism. However, significant changes could be observed when the metabolism of sugars is compared with that of the Chapter 6