Nicoletta Guaragnella

YCA1 participates in the acetic acid induced yeast programmed cell death also in a manner unrelated to its caspase-like activity

Yeast cells lacking the metacaspase‐encoding gene YCA1 (Δyca1) were compared with wild‐type (WT) cells with respect to the occurrence, nature and time course of acetic‐acid triggered death. We show that Δyca1 cells undergo programmed cell death (PCD) with a rate lower than that of the WT and that PCD in WT cells is caused at least in part by the caspase activity of Yca1p. Since in Δyca1 cells this effect is lost, but z‐VAD‐fmk does not prevent both WT and Δyca1 cell death, PCD in WT cells occurs via a Yca1p caspase and a non‐caspase route with similar characteristics.

D-Lactate transport and metabolism in rat liver mitochondria

In the present study we investigated whether isolated rat liver mitochondria can take up and metabolize D-lactate. We found the following: (1) externally added D-lactate causes oxygen uptake by mitochondria [P/O ratio (the ratio of mol of ATP synthesized to mol of oxygen atoms reduced to water during oxidative phosphorylation)=2] and membrane potential (Delta(psi)) generation in processes that are rotenone-insensitive, but inhibited by antimycin A and cyanide, and proton release from coupled mitochondria inhibited by alpha-cyanocinnamate, but not by phenylsuccinate; (2) the activity of the putative flavoprotein (D-lactate dehydrogenase) was detected in inside-out submitochondrial particles, but not in mitochondria and mitoplasts, as it is localized in the matrix phase of the mitochondrial inner membrane; (3) three novel separate translocators exist to mediate D-lactate traffic across the mitochondrial inner membrane: the D-lactate/H(+) symporter, which was investigated by measuring fluorimetrically the rate of endogenous flavin reduction, the D-lactate/oxoacid antiporter (which mediates both the D-lactate/pyruvate and D-lactate/oxaloacetate exchanges) and D-lactate/malate antiporter studied by monitoring photometrically the appearance of the D-lactate counteranions outside mitochondria. The D-lactate translocators, in the light of their different inhibition profiles separate from the monocarboxylate carrier, were found to differ from each other in the V(max) values and in the inhibition and pH profiles and were shown to regulate mitochondrial D-lactate metabolism in vitro. The D-lactate translocators and the D-lactate dehydrogenase could account for the removal of the toxic methylglyoxal from cytosol, as well as for D-lactate-dependent gluconeogenesis.

Molecular mechanisms of Saccharomyces cerevisiae stress adaptation and programmed cell death in response to acetic acid.

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.

Yeast acetic acid-induced programmed cell death can occur without cytochrome c release which requires metacaspase YCA1

To investigate the role of cytochrome c (cyt c) release in yeast acetic acid‐induced programmed cell death (AA‐PCD), wild type (wt) and cells lacking metacaspase (Δyca1), cytochrome c (Δcyc1,7) and both (Δcyc1,7Δyca1) were compared for AA‐PCD occurrence, hydrogen peroxide (H2O2) production and caspase activity. AA‐PCD occurs in Δcyc1,7 and Δcyc1,7Δyca1 cells slower than in wt, but similar to that in Δyca1 cells, in which no cytochrome c release occurs. Both H2O2 production and caspase activation occur in these cells with early and extra‐activation in Δcyc1,7 cells. We conclude that alternative death pathways can be activated in yeast AA‐PCD, one dependent on cyt c release, which requires YCA1, and the other(s) independent on it.

Catalase T and Cu,Zn-superoxide dismutase in the acetic acid-induced programmed cell death in Saccharomyces cerevisiae.

To investigate the role of catalase and superoxide dismutase (SOD) in the acetic acid (AA) induced yeast programmed cell death (AA‐PCD), we compared Saccharomyces cerevisiae cells (C‐Y) and cells individually over‐expressing catalase T (CTT1‐Y) and Cu, Zn‐SOD (SOD1‐Y) with respect to cell survival, hydrogen peroxide (H2O2) levels and enzyme activity as measured up to 200 min after AA treatment. AA‐PCD does not occur in CTT1‐Y, where H2O2 levels were lower than in C‐Y and the over‐expressed catalase activity decreased with time. In SOD1‐Y, AA‐PCD was exacerbated; high H2O2 levels were found, SOD activity increased early, remaining constant en route to AA‐PCD, but catalase activity was strongly reduced.

Cytochrome c is released from coupled mitochondria of yeast en route to acetic acid-induced programmed cell death and can work as an electron donor and a ROS scavenger

To gain insight into the processes by which acetic acid‐induced programmed cell death (AA‐PCD) takes place in yeast, we investigated both cytochrome c release from yeast mitochondria and mitochondrial coupling over the time course of AA‐PCD. We show that the majority of cytochrome c release occurs early in AA‐PCD from intact coupled mitochondria which undergo only gradual impairment. The released cytochrome c can be reduced both by ascorbate and by superoxide anion and in turn be oxidized via cytochrome c oxidase, thus working both as a ROS scavenger and a respiratory substrate. Late in AA‐PCD, the released cytochrome c is degraded.

Hydrogen Peroxide and Superoxide Anion Production during Acetic Acid-Induced Yeast Programmed Cell Death*

Hydrogen peroxide production in yeast cells undergoing programmed cell death in response to acetic acid occurred in the majority of live cells 15 min after death induction and was no longer detectable after 60 min. Superoxide anion production was found later, 60 and 90 min after death induction when cells viability was 60 and 30 %, respectively. In cells protected from death due to acid stress adaptation neither hydrogen peroxide nor superoxide anion could be observed after acetic acid treatment. The early production of hydrogen peroxide in cells in which survival was 100 % could play a major role in acetic acid-induced programmed cell death signaling. Superoxide anion is assumed to be generated in cells alreadyen route to acetic acid-induced programmed cell death.

The role of mitochondria in yeast programmed cell death

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.

Achievements and perspectives in yeast acetic acid-induced programmed cell death pathways

The use of non-mammalian model organisms, including yeast Saccharomyces cerevisiae, can provide new insights into eukaryotic PCD (programmed cell death) pathways. In the present paper, we report recent achievements in the elucidation of the events leading to PCD that occur as a response to yeast treatment with AA (acetic acid). In particular, ROS (reactive oxygen species) generation, cyt c (cytochrome c) release and mitochondrial function and proteolytic activity will be dealt with as they vary along the AA-PCD time course by using both wild-type and mutant yeast cells. Two AA-PCD pathways are described sharing common features, but distinct from one another with respect to the role of ROS and mitochondria, the former in which YCA1 acts upstream of cyt c release and caspase-like activation in a ROS-dependent manner and the latter in which cyt c release does not occur, but caspase-like activity increases, in a ROS-independent manner.

Mitochondrial dysfunction in cancer chemoresistance.

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.