Ras/PKA signal transduction pathway participates in the regulation of Saccharomyces cerevisiae cell apoptosis in an acidic environment
Egle˙ Lastauskiene˙ 1∗ ˇ Aukse˙ Zinkevicien e˙ 2 ˇ ˇ 1 Donaldas Citavi cius
of Microbiology and Biotechnology, Faculty of Natural Sciences, Vilnius University, Vilnius, Lithuania
of Diagnosis and Treatment of Allergic Diseases, Vilnius, Lithuania
Abstract The acidification of the medium is observed during yeast cell growth. This process contributes to the emission of organic acids, mainly acetic acid. Acetic acid is known as the inducer of apoptosis in the yeast Saccharomyces cerevisiae. In this study, we showed that hydrochloric acid can also induce apoptosis in yeast cells, and the apoptotic phenotype triggered by treating yeast cells with hydrochloric acid is modulated by the Ras/PKA pathway. The Ras/PKA pathway is highly conserved between all eukaryotic organisms, as well as cell processes that are related to apoptosis and aging. In this research, we demonstrated that the activation of the Ras/PKA pathway by insertion of Ras2Val19 allele or deletion of PDE2
gene increases cell death, displaying the markers of apoptosis in an acidic environment. Downregulation of the pathway by deletion of RAS2, RAS1, PDE1, and insertion of the Ha-ras gene increases the cell viability and diminishes cell death with the apoptotic phenotypes. The deletion of PDE1 gene and double deletion of both phosphodiesterase genes prevent the induction of apoptosis in the cells. Modulations in the Ras/PKA pathway affect cell viability and apoptosis during natural gradual acidification of the medium as well as in acid stress conditions. C 2013 International Union of Biochemistry and Molecular Biology, Inc. Volume 61, Number 1, Pages 3–10, 2014
Keywords: Hydrochloric acid, apoptosis, medium acidification, Ras/PKA, signal transduction, yeast
1. Introduction In multicellular organisms, apoptosis plays an important role during the development and response to different environmental conditions. Many features of aging and apoptosis are conserved between yeast and multicellular organisms, and this
Abbreviations: CFU, colony-forming unit; CLS, chronological lifespan; DAPI, 4 ,6-diamino-2-phenylindole; OD, optical density; PI, propidium iodide; PS, phosphatidylserine; TUNEL, terminal deoxynucleotidyltransferase (TdT)-mediated dUTP nick end labeling. ∗ Address for correspondence: Egle ˙ Lastauskiene, ˙ PhD, Department of Microbiology and Biotechnology, Faculty of Natural Sciences, Vilnius ˇ University, M. K. Ciurlionio 21/27, Vilnius LT-03101, Lithuania. Tel.: +37 06124191; Fax: +37 052398204; e-mail: [email protected]
Supporting Information is available in the online issue at wileyonlinelibrary.com. Received 11 April 2013; accepted 12 November 2013 DOI: 10.1002/bab.1183 Published online 3 February 2014 in Wiley Online Library (wileyonlinelibrary.com)
makes yeast Saccharomyces cerevisiae suitable as a model organism; it also exhibits fine replicative capacity . The ability of yeast to survive stress conditions such as freezing, thawing, osmotic shock, or low pH, as well as the metabolic capacity of the cells, plays a key role in the usage of yeast in bakery, brewery, and other industrial purposes. Apoptosis of the yeast cell is the source of molecules such as amino acids and peptides [2, 3]. One of the determined inducers of apoptosis in S. cerevisiae cells is acetic acid. After the treatment of yeast cells with acetic acid, concentration-dependent changes (chromatin condensation along the nuclear envelope, externalization of phosphatidylserine (PS), and formation of the DNA strand breaks) can be observed . Acidification of the medium during the growth of yeast in glucose-containing surroundings is generally related to accumulation of acetic acid, which is a normal end product of alcoholic fermentation produced by growing yeast . Acetic acid easily enters cells by simple diffusion. If the extracellular pH is lower than the intracellular pH, this will lead to intracellular acidification and anion accumulation
Biotechnology and Applied Biochemistry . Buffering of the medium can increase yeast cell viability during a stationary phase [6, 7]. Additionally, Burtner et al.  showed that buffering of the medium could increase yeast chronological lifespan (CLS). Thus, the concentration of acetic acid, achieved naturally during cell growth, is toxic to the yeast cells only in combination with low pH . The Ras/PKA signal transduction pathway regulates both the acidification of the medium, by regulating cell metabolism intensity, and the induction of apoptosis. The Ras/PKA pathway gives cell information about the presence of nutrients and various stress conditions in the surrounding environment and is the main determinant of yeast longevity . Yeast cells respond to environmental stimuli by increasing the level of the secondary messenger cAMP, which activates PKA. It has been shown that apoptosis can be prevented by downregulation of RAS signaling or by overexpression of the PDE2 gene . Constitutive activation of the RAS signaling causes yeast cell death, displaying markers of apoptosis [11, 12]. In this study, we analyzed how alterations affecting the Ras/PKA pathway activity regulate the induction of apoptosis during natural gradual acidification of the medium as well as under acid stress conditions. We demonstrated that hydrochloric acid, as well as acetic acid, is able to induce apoptosis in yeast, and the Ras/PKA pathway contributes to the regulation of apoptosis under these conditions.
2. Materials and Methods Yeast strains were selected on the genetic background of SP1 strain. SP1 genotype—MATα his3 leu2 ura3 trp1 ade8 canR . TK161R2V contains (MATα his3 leu2 ura3 trp1 ade8 canR Ras2Val19 ) Ras2Val19 mutation, which leads to constitutive activation of the Ras/PKA pathway. ras1 (MATα his3 leu2 ura3 trp1 ade8 canR ras1::URA3), ras2 (MATα his3 leu2 ura3 trp1 ade8 canR ras2::LEU2), ras1/2 (MATα his3 leu2 ura3 trp1 ade8 canR ras2::LEU2, ras1::URA3 (pHa-ras, TRP1). Phosphodiesterase gene mutation containing strains pde1 (MATa his3 leu2 ura3 trp1 ade8 canR pde1::LEU2), pde2 (MATa his3 leu2 ura3 trp1 ade8 canR pde2::HIS3), pde1/2 (MATa his3 leu2 ura3 trp1 ade8 canR pde1::URA3 pde2::HIS3). All strains were the kind gift of Professor Thevelein, Katholieke Universiteit, Leuven, Belgium .
2.1. Growth conditions Yeast strains were grown in YPD medium (2% glucose, 2% peptone, and 1% yeast extract) and in SC medium (0.67% yeast nitrogen base [without amino acids, with ammonium sulfate], 2% glucose and supplemented with the appropriate amino acids). The incubation was performed aerobically on a rotary shaker at 30 ◦ C for 78 H. Optical density (OD) and pH of the medium were measured every 6 H. 2-Morpholinoethanesulfonic acid (MES) was used to buffer the medium. For colony-forming unit (CFU) analysis, yeast strains were grown in SC medium for 78 H. A known number
of cells were plated on YPD plates, and the colonies formed by viable cells were counted after 48 H of growth. Yeast cells were grown overnight for acid stress induction in 10 mL YPD medium, until the stationary phase. Cells were harvested by centrifugation and resuspended in 1 M sorbitol buffer (MES buffer supplemented with 1 M of sorbitol) (pH 2.1) to a final density of 105 –106 CFU/mL. Cells were incubated for 4 and 6 H at 30 ◦ C. A 1 M Sorbitol buffer (pH 5.4) was used as the control. The pH of the buffer was adjusted to the required values using a 9 mM concentration of HCl.
2.2. Nucleus staining with 4 ,6-diamino-2-phenylindole Cells (1 × 106 ) were washed twice with phosphate-buffered saline (PBS) buffer, added onto polylysine-coated slides, and left to air dry at room temperature. Four microliters of 4 ,6diamino-2-phenylindole (DAPI) (1 μg/mL) was added to the immobilized cells. The cells were stained for 10 Min at room temperature and washed with PBS. Samples were covered with coverslips using mounting media and immediately analyzed by fluorescence microscopy and confocal laser scanning microscopy using a wavelength of 330–380 nm.
2.3. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining For the terminal deoxynucleotidyltransferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, the Fluorescein FragELTM DNA Fragmentation Detection Kit (Calbiochem, USA) was used. Briefly, yeast cells were fixed in 4% PBS-buffered formaldehyde for 10 Min at room temperature. Formaldehyde was removed by centrifugation at 2,348g for 5 Min at 4 ◦ C. The cells were resuspended in 200 μL TBS (Tris-buffered saline) buffer and incubated for 10 Min at room temperature. For the permeabilization of the specimen 100 μL proteinase K (20 μg/mL) was used. For a positive control, cells were additionally treated with DNaseI. After the removal of proteinase K, cells were stained with a Fluorescein-FregELTM reaction mixture and analyzed by fluorescence microscopy using a wavelength of 450–500 nm.
2.4. Caspase detection The FITC-VAD-FMK (CaspACETM In Situ Marker Kit; Promega, USA) kit was used for caspase detection. First, 1 × 106 cells were washed with PBS buffer three times. A CaspACETM FITCVAD-FMK in situ marker was added to the final concentration of 10 μM. The mixture was incubated at 30 ◦ C for 20 min, in the dark. Stained cells (1 × 106 cells/mL) were added onto the polylysine-coated slides. The cells were fixed with 10% buffered formaldehyde for 30 Min at room temperature. Formaldehyde was removed by washing immobilized cells with PBS. Coverslips were added using mounting media. Slides were immediately analyzed by fluorescence microscopy using a wavelength of 490–525 nm.
2.5. Externalization of the phosphatidylserine PS exposure at the outer layer of the cytoplasmic membrane was assayed using FITC/annexin-V (Roche, Germany) . Briefly, 1 × 106 cells were washed twice with 200 μL digestion
Ras/PKA Influence on Yeast Apoptosis
buffer (1.2 M sorbitol, 0.5 mM MgCl2 , 35 mM K2 HPO4 , pH 6.8) and digested with 15 U/mL lyticase and 5.5% glusulase. The cells were incubated at 30 ◦ C for 2 H and washed twice with 200 μL annexin-V binding buffer (10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2 ) containing 1.2 M sorbitol. Two microliters FITC/annexin-V (Roche) and 2 μL propidium iodide (PI) were added to 38 μL cell suspension and incubated at room temperature for 30 Min. The cells were harvested by centrifugation, added onto the polylysine-coated slides, and analyzed by fluorescence microscopy (400–500 nm and >560 nm) and flow cytometry. All apoptosis experiments were repeated three times. In each microscopy analysis, 300–3,000 cells were analyzed. In total, 30,000 cells were analyzed for flow cytometry. Standard deviations represent results of three independent experiments. A one-way analysis of variance (ANOVA) and the Student’s two-side t-test were used for evaluation of the occurrence of apoptotic phenotypes. P < 0.05 was considered as the level of significance.
3. Results We have analyzed early stationary phase cells (control group, 46 H of growth) and late stationary phase cells (78 H of growth) for the apoptosis assay. The OD of the culture and pH of the medium were measured every 6 H during cell growth. When yeast cells were grown in YPD medium, a slight acidification, ranging from 6.2 (start medium pH) to 4.81 (after 78 H), was observed. OD of all analyzed strains was over 8.52 ± 0.75%. The pH of the medium has changed from 5.4 (start medium pH) to 2.1 (after 78 H) when yeast cells were grown in SC medium. OD of the cultures varied in the strains with different genotypes. The yeast strains were grown in SC medium for the apoptotic phenotype analysis. A known number of cells were plated on YPD medium and incubated for 48 H for CFU analysis. CFU results are presented in Fig. 1. The cells were harvested by centrifugation and stained for nucleus morphology, PS externalization, DNA fragmentation, and active caspase detection analysis for apoptotic phenotype detection. Changes in nucleus morphology were detected by staining yeast cells with DAPI and performing fluorescence and confocal laser scanning microscopy. Less than 1% of cells with nucleus morphology changes were detected in samples after 78 H of growth in YPD and YPD–MES media. CFU at this point was over 92% (data not shown). After 78 H of growth in SC medium, various changes in the nucleus morphology were detected: fragmented nucleus, double nucleus, no visible nucleus, and others (Fig. 2A). The highest percentage of the cells with abnormal nucleus was detected in the strains with the lowest cell viability (Fig. 2B). Costaining with Annexin-V and PI allowed the discrimination between early apoptotic cells exhibiting PS externalization (AnnexinV+/PI−), cells with ruptured membrane indicative of primary necrosis (AnnexinV−/PI+), and late apop-
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CFU count after 78 H of growth in SC medium.
totic/secondary necrotic cells, which showed both PS exposure and membrane permeability (AnnexinV+/PI+) (Supporting Information Fig. 1). Flow-cytometry analysis has showed that after 78 H of growth in SC medium, 22.47 ± 5.26% of SP1 cells displayed markers of early apoptosis and 7.32 ± 2.17% of late apoptosis (Fig. 3). Meanwhile, 36.04 ± 2.05% of SP1 cells were stained Annexin−/PI+ indicating necrotic phenotype. An increased percentage of cells with the markers of early and late apoptosis (AnnexinV+/PI−) was registered in the Ras2Val19 strain (69.95 ± 3.56% AnnexinV+/PI− and 11.56 ± 1.69% AnnexinV+/PI+) and in the pde2 strain (58.04 ± 6.69% AnnexinV+/PI− and 8.99 ± 1.53%). In ras1, ras2, and ras1/2 strains, the percentage of the cells with early and late apoptosis phenotypes was less than 4%. Disruption of PDE1 activity has also led to a decreased percentage of apoptotic cells in the population: only 8.91 ± 1.79% of the cells displayed markers of early apoptosis and 3.79 ± 0.98% of late apoptosis. In pde1/2, 4.75 ± 1.46% of AnnexinV+/PI− cells and 3.56 ± 1.02% AnnexinV+/PI+ cells were counted. Flow-cytometry analysis results were also confirmed by microscopy analysis (data not shown). The percentage of annexin-positive cells counted by flow cytometry was about 5% higher as compared with the microscopy results. These differences could appear due to the distinct sensitivities of the methods. During flow-cytometry analysis, all spectra of the green-emitted light signals are registered, whereas during microscopy analysis, only fully stained cells are registered as early or late apoptotic signals. DNA fragmentation analysis has revealed that 17.58 ± 1.54% of SP1 cells have displayed TUNEL-positive phenotype (Fig. 4A, Supporting Information Fig. 2). DNA fragmentation was detected in 79.52 ± 1.84% cells of the Ras2Val19 strain and 65.52 ± 1.97% of the pde2 strain. Less than 4% of TUNEL+ cells were registered in ras1, ras2, and ras1/2
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(A) Confocal laser scanning microscopy picture of Ras2Val19 cells after 78 H of growth in SC medium shows various morphological changes in the nucleus structure: 1, normal nucleus; 2, fragmented nucleus; 3, no visible nucleus (diffused nucleus); 4, double nucleus. (B) Percentage of the cells with abnormal nucleus after 78 H of Ras2Val19 cell growth in SC medium.
strains. In pde1 and pde1/2 strains, the percentage of TUNEL+ cells was 5.56 ± 1.64% and 5.33 ± 1.36%, respectively. Buffering of the medium has increased the viability of the cells and diminished cell death displaying the markers of apoptosis (Fig. 4A). The percentage of the apoptotic cells in the population after growth in SC-MES medium was less than 6%. CFU in all the strains was over 85.31 ± 3.14% (data not shown). Similar results were obtained using active caspase detection staining (Fig. 4B, Supporting Information Fig. 3). In the SP1 strain, 17.62 ± 1.54% of cells displayed active caspase (Caspase+/PI−). In the Ras2Val19 strain, active caspases were detected in 66.43 ± 1.84% of cells. In the pde2 strain, 60.61 ± 1.97% of cells showed caspase-positive phenotype. In Ras2Val19 and pde2 strains, unspecific stained cells (Caspase+/PI+) were 16.91 ± 1.53% and 13.59 ± 0.87%, respectively. In ras1, ras2, ras1/2, pde1, and pde1/2, active caspases were detected in less than 5% of the total cells
Externalization of PS was detected by staining yeast cells with AnnexinV and PI and performing flow-cytometry analysis. AnnexinV+/PI+, AnnexinV+/PI−, AnnexinV−/PI− (nonstained cells) phenotypes were registered in yeast strains after 78 H of growth in SC medium (P < 0.05).
and the percentage of unspecifically stained cells was less than 4%. The apoptosis marker analysis was in agreement with the CFU count (Fig. 1). Strains with the highest cell viability in SC medium after 78 H of growth have not displayed markers of early and late apoptosis. The pH of the buffer was chosen for acid stress induction according to the pH values reached naturally during yeast cell growth in SC medium. Incubation in the pH 5.4 buffer for 4 or 6 H had no effect on the appearance of apoptotic phenotypes in the cell cultures. The percentage of apoptotic cells was less than 2%. In the Ras2Val19 strain, 79.52 ± 1.98% of cells displayed markers of early apoptosis and 9.75 ± 0.94% of late apoptosis after 6 H of incubation in pH 2.1 buffer (Fig. 5). A high percentage of apoptotic cells was also registered in pde2 strain—78.7 ± 2.46% and 7.81 ± 0.47%, respectively. Only 1.1 ± 0.33% of Ras2Val19 cells and 0.38 ± 0.29% of pde2 cells were undergoing necrosis after treatment with HCl. Meanwhile, in SP1, the percentage of AnnexinV+/PI− cells was 24.62 ± 2.56% and AnnexinV+/PI+ 6.26 ± 1.76%. Downregulation of the Ras/PKA pathway (ras1, ras2) and insertion of the pHa-Ras have increased the resistance of the cells to acid stress induced by HCl and prevented apoptosis. In the ras2 strain, 14.9 ± 0.49% of cells underwent necrosis and only 6.27 ± 1.59% apoptosis. In pde1 and pde1/2 strains, the percentage of early apoptotic cells was 16.78 ± 2.15% and 7.44 ± 2.01%. Similar results were obtained in the TUNEL test. The highest percentage of cells with fragmented DNA was registered in Ras2Val19 and pde2 strains (89.27 ± 1.85% and 74.89 ± 1.86%, respectively) (Fig. 6A).
Ras/PKA Influence on Yeast Apoptosis
AnnexinV/PI staining results after 6 H of incubation of yeast cells in pH 2.1 buffer. Appearance of early and late apoptotic phenotypes in yeast cells was triggered by treatment with HCl and regulated by alterations in Ras/PKA pathway (P < 0.05).
(A) Detection of the DNA strand breaks was assayed by the TUNEL test. The highest percentage of TUNEL+ cells after 78 H of growth in SC medium was registered in Ras2Val19 and pde2− strains. Buffering of the medium (SC-MES) prevented the formation of the DNA strand breaks in yeast cells. (B) The highest percentage of cells with active caspase was detected in Ras2Val19 and pde2− strains. Apoptosis induced by gradual acidification of medium was dependent on activation of the yeast caspase.
Active caspase detection assay revealed that apoptosis in yeast cells proceeded in a caspase-dependent way after treatment with HCl (Fig. 7B). In the Ras2Val19 strain, 72.13 ± 1.87% of cells had active caspase, as did 67.24 ± 1.72% of pde2 cells. The apoptosis marker analysis results were confirmed by CFU analysis (Fig. 7). Yeast strains maintaining the highest cell viability under acid stress conditions failed to commit apoptosis, and the percentage of necrotic cells found in those strains was higher than that of apoptotic cells.
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The ability of yeast cells to grow and survive in a particular environment is limited by the pH of the medium . The pH of the medium affects the replicative and chronological lifespan of the cell as well as apoptosis in yeast . The Ras/PKA pathway regulates many apoptosis-related features of the cell: it regulates apoptosis related to actin reorganization [15, 16]; the increased activity of the pathway is related to accumulation of ROS and cellular respiration . The Ras/PKA pathway also regulates cell death in an acidic environment. The increased pathway activity in combination with low pH and acetic acid kills yeast cells while exhibiting the markers of apoptosis . Acetic acid-induced acidification of medium leads to intracellular acidification , which causes activation of the Ras/PKA signal transduction pathway . During cell growth in a glucose-containing environment, malic, citric, pyruvic, and other organic acids are also emitted into growth medium. They enhance the toxicity of the acetic acids by contributing to the general acidity of the medium and play a secondary role in cell aging and death . In this study, we have shown that buffering of the medium to pH 5.4 leads to increased cell viability and prevents apoptosis (Fig. 5). These findings are in agreement with those of other authors. Burtner et al.  showed that in the medium buffered to pH 6.0, CLS of the cell is increased. Acetic acid is not toxic for yeast at slightly acidic pH. In the buffered medium, acetate anions are unable to go to the protonated state, cross the plasma membrane, and induce intracellular acidification [17, 18]. Low doses of acetic acid induce cell death accompanied by DNA strand breaks, chromatin condensation, and externalization of PS, whereas high doses induce necrosis . During growth in SC medium, yeast strains containing deletion of the RAS gene reach about seven times higher OD as compared with SP1
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Apoptotic phenotypes detected in yeast cells after 6 H of incubation in pH 2.1 buffer. Acid stress induced by HCl was accompanied by (A) DNA strand break appearance and (B) activation of the yeast caspase. Ras/PKA pathway regulates both the resistance of yeast cells to the acid stress and the induction of apoptosis in these conditions.
and Ras2Val19 strains; thus, the amount of emitted acetic acid is also higher. It is possible that during the growth of ras1, ras2, ras1/2 or pde1, and pde1/2 strains, the amount of emitted acetic acid, in combination with low pH values, reaches a level high enough to induce necrosis. In the present study we showed that HCl is also able to induce apoptosis in yeast cells. Apoptosis induced by hydrochloric acid is modulated through the Ras/PKA pathway. After incubation of yeast cells in pH 2.1 buffer, the CFU value was generally lower in all the strains as compared with the results after 78 H of growth in SC medium (Fig. 1). During growth, the cells had time for adaptation to the constantly changing environment, and in the stationary phase of growth they were the most resistant to various stresses. We harvested cells from the exponential phase of growth for HCl treatment and incubation in a pH 2.1 buffer. Consequently, cell viability
CFU count after 6 H of incubation in pH 2.1 buffer supplemented with HCl.
was decreased (Fig. 6) and apoptotic or necrotic phenotypes were registered. The nucleus morphology analysis showed that the percentage of the cells with an abnormal nucleus, as compared with other markers of apoptosis, was higher in all analyzed strains. Part of the changes was not lethal to the cells and could be repaired after the change in growth conditions. Thus, changes in the nucleus structure could only predict possible apoptosis in yeast cells, but were not able to show the real number of the cells dying because of apoptosis. The main part of nucleus abnormalities found in ras1, ras2, ras1/2 and pde1, pde1/2 strains was a double nucleus (Fig. 2). Appearance of the double nucleus in yeast cells could be related to endomitosis and also could be a result of symmetric nucleus fragmentation . In this discussion, we support the proposition of endomitosis. The size of the double nucleus was the same as in the cell with a normal nucleus, and after the fragmentation, the size of the two fragments was usually smaller than the normal nucleus. The high resistance of the yeast cells to stress conditions could be related to the presence of a double genome in one single cell. Two methods of apoptosis are present in the yeast: caspase dependent and caspase independent . We showed that in acidic conditions, caspase-dependent cell apoptosis was induced (Fig. 5B). Deletion of the RAS genes abolished formation of the apoptotic phenotype in yeast and led to the necrotic pathway of death. Therefore, constitutive activation of the pathway led to increased cell death due to apoptosis and prevented the cells from the appearance of necrotic phenotype. Resistance/sensitivity of the yeast to stress conditions contributes to the cAMP level inside the cell. The constitutive activation of the Ras2p (Ras2Val19 allele) leads to increased levels of cAMP inside the cell, and this causes sensitivity to stress conditions in general, as well as a low amount of
Ras/PKA Influence on Yeast Apoptosis
accumulated storage carbohydrates and an inability to arrest in G1 after the depletion of nutrient sources. The basal cAMP level inside the cells is controlled at the level of synthesis by RAS genes or degradation by Pde2p . We have determined that the sensitivity of the yeast to acidic conditions and an increased percentage of the cells displaying markers of apoptosis were increased after the deletion of PDE2 gene. In contrast, the deletion of the PDE1 gene caused a significant increase in cell viability and abolished cell death while exhibiting the markers of apoptosis. Ma et al.  showed that the deletion of PDE1 results in a much higher glucose cAMP accumulation compared with the deletion of PDE2. The overexpression of PDE1 abolished cAMP accumulation induced by glucose and acidification. The overexpression of PDE2 enhanced the basal heat stress resistance of the cell by controlling the basic level of cAMP in the stationary phase  and protected the cell from the extracellular cAMP . It seems that PDE1 has a specific role in controlling increased cAMP levels in a glucose-containing environment and PDE2 controls the basal level of cAMP. During natural gradual acidification of the medium and after the treatment with HCl, PDE2 performs the main function in the cAMP level control. The deletion of PDE2 leads to an increased basal cAMP level inside the cell, mimicking the phenotype of Ras2Val19 . The deletion of both phosphodiesterase genes in pde1/2 increased the resistance of the cells to acidic conditions during gradual acidification of the medium and to treatment with HCl. In acidic conditions, the prevention of cAMP accumulation is controlled by high feedback of PKA on cAMP synthesis. There are two ways for PKA to inhibit accumulation of cAMP. One of them is the activation of Pde1p by phosphorylation presumably in the Pde1ser252 position . The second one is feedback inhibition on the cAMP synthesis. After the deletion of both phosphodiesterases genes, PKA presumably downregulates cAMP at the synthesis stage. Similar results were obtained by Ma et al. . They showed that after addition of glucose to the strains with the double deletion of both PDE genes, the cAMP level did not increase. The deletion of both PDE genes in cells displayed an increased PKA activity phenotype . Nikawa et al.  demonstrated that stains lacking both phosphodiesterases showed only a slight increase in the basal level of cAMP, indicating that most of the feedback inhibition was independent of phosphodiesterases. Our results showed that the deletion of PDE2 causes an increase in cell death and apoptosis. The deletion of PDE1 and both PDE genes has an opposite effect. It seems that acidic conditions after the deletion of PDE1 PKA dramatically downregulates cAMP synthesis and increases the resistance of cells to acidic conditions and prevents apoptosis. The Ras/PKA signal transduction pathway regulates apoptosis in different eukaryotic organisms. Constitutive activation of the Ras/PKA pathway causes apoptosis in Candida albicans . Philips et al.  demonstrated that Ras-cAMP
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signaling plays an important role in programmed cell death in C. albicans. In higher mammals, low pH is known as a stimulator of Ras/PKA . In the rat thyroid cell line, activation of the pathway triggers the massive wave of apoptosis . The introduction of oncogenic Ha-ras to the yeast cells served as a motivation for studying aging processes in yeast . In this study, we showed that insertion of the Ha-ras to the RAS deleted yeast cells, increased cell viability, and diminished cell death with markers of apoptosis during gradual acidification of the medium and in HCl-modulated acid stress conditions. Insertion of the human Ha-ras led to an increased number of dying cells in acidic conditions, but had no effect on the percentage of apoptotic cells as compared with ras2. It can be concluded that the insertion of the Ha-ras to ras1/2 was partially sufficient to complement the deletion of RAS genes in viability-related processes, but had no effect on the formation of an apoptotic phenotype in yeast. To confirm this assumption, additional experiments are required. Detailed analysis of the pH influence on the apoptosis of the yeast cells through the Ras/PKA signal transduction pathway can help in understanding the progression of human diseases related to aging and acidosis and the treatment options.
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