Monitoring yeast regulated cell death: trespassing the point of no return to loss of plasma membrane integrity.

Acetic acid and hydrogen peroxide are the most common stimuli to induce apoptosis in yeast. The initial phase of this cell death process is characterized by the maintenance of plasma membrane integrity in cells that had already lost their viability. As loss of plasma membrane integrity is typically assessed by staining with propidium iodide (PI) after exposure of cells to a stimulus and cell viability is determined 48 h after plating, the percentage of cells with compromised plasma membrane integrity and c.f.u. counts often do not correlate. Herein, we developed a simple method to explore at what point after an apoptotic stimulus and plating cells do non-viable cells die as result of plasma membrane disruption, i.e., when cells surpass the point-of-no-return and undergo a secondary necrosis. The method consisted in washing cells and re-suspending them in stimulus-free medium after acetic acid and hydrogen peroxide treatments, to mimic transfer to plating, and then assessing plasma membrane integrity through PI staining. We show that, after the stimuli are removed, cells that had lost proliferative capacity but still maintained plasma membrane integrity continue the cell death process and later lose plasma membrane integrity when progressing to secondary necrosis. After exposure to hydrogen peroxide, cells undergo secondary necrosis preceded by Nhp6Ap-GFP cytosolic localization, in contrast to acetic acid exposure, where Nhp6Ap-GFP cytosolic localization mainly occurs simultaneously with an earlier emergence of secondary necrosis. In conclusion, the developed method allows monitoring the irreversible loss of plasma membrane integrity of dying apoptotic cells after the point-of-no-return is trespassed, and better characterize the process of secondary necrosis after apoptosis.

Saccharomyces cerevisiae Cells Lacking the Zinc Vacuolar Transporter Zrt3 Display Improved Ethanol Productivity in Lignocellulosic Hydrolysates.

Yeast-based bioethanol production from lignocellulosic hydrolysates (LH) is an attractive and sustainable alternative for biofuel production. However, the presence of acetic acid (AA) in LH is still a major problem. Indeed, above certain concentrations, AA inhibits yeast fermentation and triggers a regulated cell death (RCD) process mediated by the mitochondria and vacuole. Understanding the mechanisms involved in AA-induced RCD (AA-RCD) may thus help select robust fermentative yeast strains, providing novel insights to improve lignocellulosic ethanol (LE) production. Herein, we hypothesized that zinc vacuolar transporters are involved in vacuole-mediated AA-RCD, since zinc enhances ethanol production and zinc-dependent catalase and superoxide dismutase protect from AA-RCD. In this work, zinc limitation sensitized wild-type cells to AA-RCD, while zinc supplementation resulted in a small protective effect. Cells lacking the vacuolar zinc transporter Zrt3 were highly resistant to AA-RCD, exhibiting reduced vacuolar dysfunction. Moreover, zrt3Δ cells displayed higher ethanol productivity than their wild-type counterparts, both when cultivated in rich medium with AA (0.29 g L-1 h-1 versus 0.11 g L-1 h-1) and in an LH (0.73 g L-1 h-1 versus 0.55 g L-1 h-1). Overall, the deletion of ZRT3 emerges as a promising strategy to increase strain robustness in LE industrial production.

Lactate Induces Cisplatin Resistance in S. cerevisiae through a Rad4p-Dependent Process.

Cisplatin is a widely used antineoplastic agent that has DNA as the main target, though cellular resistance hampers its therapeutic efficacy. An emerging hallmark of cancer cells is their altered metabolism, characterized by increased glycolysis even under aerobic conditions, with increased lactate production (known as the Warburg effect). Although this altered metabolism often results in increased resistance to chemotherapy, it also provides an opportunity for targeted therapeutic intervention. It has been suggested that cisplatin cytotoxicity can be affected by tumor metabolism, though with varying effects. We therefore sought to better characterize how lactate affects cisplatin sensitivity in the simplified Saccharomyces cerevisiae model. We show that lactate renders yeast cells resistant to cisplatin, independently of growth rate or respiration ability. We further show that histone acetylation is not affected, but histone phosphorylation is decreased in lactate-containing media. Finally, we show that Rad4p, essential for nucleotide excision repair, is required for the observed phenotype and thus likely underlies the mechanism responsible for lactate-mediated resistance to cisplatin. Overall, understanding how lactate modulates cisplatin sensitivity will aid in the development of new strategies to overcome drug resistance.

Pkh1p-Ypk1p and Pkh1p-Sch9p Pathways Are Activated by Acetic Acid to Induce a Mitochondrial-Dependent Regulated Cell Death.

The yeast Saccharomyces cerevisiae undergoes a mitochondrial-dependent regulated cell death (RCD) exhibiting typical markers of mammalian apoptosis. We have previously shown that ceramide production contributes to RCD induced by acetic acid and is involved in mitochondrial outer membrane permeabilization and cytochrome c release, especially through hydrolysis of complex sphingolipids catalyzed by Isc1p. Recently, we also showed that Sch9p regulates the translocation of Isc1p from the endoplasmic reticulum into mitochondria, perturbing sphingolipid balance and determining cell fate. In this study, we addressed the role of other signaling proteins in acetic acid-induced RCD. We found that single deletion of PKH1 or YPK1, as shown for SCH9 and ISC1, leads to an increase in cell survival in response to acetic acid and that Pkh1/2p-dependent phosphorylation of Ypk1p and Sch9p increases under these conditions. These results indicate that Pkh1p regulates acetic acid-induced RCD through Ypk1p and Sch9p. In addition, our results suggest that Pkh1p-Ypk1p is necessary for isc1Δ resistance to acetic acid-induced RCD. Moreover, double deletion of ISC1 and PKH1 has a drastic effect on cell survival associated with increased ROS accumulation and release of cytochrome c, which is counteracted by overexpression of the PKA pathway negative regulator PDE2. Overall, our results suggest that Pkh1p-Ypk1p and Pkh1p-Sch9p pathways contribute to RCD induced by acetic acid.

N-terminal acetylation modulates Bax targeting to mitochondria.

The pro-apoptotic Bax protein is the main effector of mitochondrial permeabilization during apoptosis. Bax is controlled at several levels, including post-translational modifications such as phosphorylation and S-palmitoylation. However, little is known about the contribution of other protein modifications to Bax activity. Here, we used heterologous expression of human Bax in yeast to study the involvement of N-terminal acetylation by yNaa20p (yNatB) on Bax function. We found that human Bax is N-terminal (Nt-)acetylated by yNaa20p and that Nt-acetylation of Bax is essential to maintain Bax in an inactive conformation in the cytosol of yeast and Mouse Embryonic Fibroblast (MEF) cells. Bax accumulates in the mitochondria of yeast naa20Δ and Naa25-/- MEF cells, but does not promote cytochrome c release, suggesting that an additional step is required for full activation of Bax. Altogether, our results show that Bax N-terminal acetylation by NatB is involved in its mitochondrial targeting.

Colorectal cancer-related mutant KRAS alleles function as positive regulators of autophagy.

The recent interest to modulate autophagy in cancer therapy has been hampered by the dual roles of this conserved catabolic process in cancer, highlighting the need for tailored approaches. Since RAS isoforms have been implicated in autophagy regulation and mutation of the KRAS oncogene is highly frequent in colorectal cancer (CRC), we questioned whether/how mutant KRAS alleles regulate autophagy in CRC and its implications. We established two original models, KRAS-humanized yeast and KRAS-non-cancer colon cells and showed that expression of mutated KRAS up-regulates starvation-induced autophagy in both. Accordingly, KRAS down-regulation inhibited autophagy in CRC-derived cells harboring KRAS mutations. We further show that KRAS-induced autophagy proceeds via up-regulation of the MEK/ERK pathway in both colon models and that KRAS and autophagy contribute to CRC cell survival during starvation. Since KRAS inhibitors have proven difficult to develop, our results suggest using autophagy inhibitors as a combined/alternative therapeutic approach in CRCs with mutant KRAS.

Modulation of mitochondrial outer membrane permeabilization and apoptosis by ceramide metabolism.

The yeast Saccharomyces cerevisiae undergoes a mitochondrial-dependent programmed cell death in response to different stimuli, such as acetic acid, with features similar to those of mammalian apoptosis. However, the upstream signaling events in this process, including those leading to mitochondrial membrane permeabilization, are still poorly characterized. Changes in sphingolipid metabolism have been linked to modulation of apoptosis in both yeast and mammalian cells, and ceramides have been detected in mitochondria upon apoptotic stimuli. In this study, we aimed to characterize the contribution of enzymes involved in ceramide metabolism to apoptotic cell death induced by acetic acid. We show that isc1Δ and lag1Δ mutants, lacking inositol phosphosphingolipid phospholipase C and ceramide synthase, respectively, exhibited a higher resistance to acetic acid that was associated with lower levels of some phytoceramide species. Consistently, these mutant cells displayed lower levels of ROS production and reduced mitochondrial alterations, such as mitochondrial fragmentation and degradation, and decreased translocation of cytochrome c into the cytosol in response to acetic acid. These results suggest that ceramide production contributes to cell death induced by acetic acid, especially through hydrolysis of complex sphingolipids catalyzed by Isc1p and de novo synthesis catalyzed by Lag1p, and provide the first in vivo indication of its involvement in mitochondrial outer membrane permeabilization in yeast.

Activation of the Hog1p kinase in Isc1p-deficient yeast cells is associated with mitochondrial dysfunction, oxidative stress sensitivity and premature aging.

The Saccharomyces cerevisiae Isc1p, an orthologue of mammalian neutral sphingomyelinase 2, plays a key role in mitochondrial function, oxidative stress resistance and chronological lifespan. Isc1p functions upstream of the ceramide-activated protein phosphatase Sit4p through the modulation of ceramide levels. Here, we show that both ceramide and loss of Isc1p lead to the activation of Hog1p, the MAPK of the high osmolarity glycerol (HOG) pathway that is functionally related to mammalian p38 and JNK. The hydrogen peroxide sensitivity and premature aging of isc1Δ cells was partially suppressed by HOG1 deletion. Notably, Hog1p activation mediated the mitochondrial dysfunction and catalase A deficiency associated with oxidative stress sensitivity and premature aging of isc1Δ cells. Downstream of Hog1p, Isc1p deficiency activated the cell wall integrity (CWI) pathway. Deletion of the SLT2 gene, which encodes for the MAPK of the CWI pathway, was lethal in isc1Δ cells and this mutant strain was hypersensitive to cell wall stress. However, the phenotypes of isc1Δ cells were not associated with cell wall defects. Our findings support a role for Hog1p in the regulation of mitochondrial function and suggest that constitutive activation of Hog1p is deleterious for isc1Δ cells under oxidative stress conditions and during chronological aging.