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The cell death protease Kex1p is essential for hypochlorite-induced apoptosis in yeast.

Carmona-Gutierrez D, Alavian-Ghavanini A, Habernig L, Bauer MA, Hammer A, Rossmann C, Zimmermann AS, Ruckenstuhl C, Büttner S, Eisenberg T, Sattler W, Malle E, Madeo F - Cell Cycle (2013)

Bottom Line: Interestingly, HOCl cytotoxicity can be prevented by treatment with ROS scavengers, suggesting oxidative stress to mediate the lethal effect.The executing pathway involves the pro-apoptotic protease Kex1p, since its absence diminishes HOCl-induced production of ROS, apoptosis and protein modification.By characterizing HOCl-induced cell death in yeast and identifying a corresponding central executor, these results pave the way for the use of Saccharomyces cerevisiae in HOCl research, not least given that it combines both being a microorganism as well as a model for programmed cell death in higher eukaryotes.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biosciences, University of Graz, Graz, Austria.

ABSTRACT
Following microbial pathogen invasion, the human immune system of activated phagocytes generates and releases the potent oxidant hypochlorous acid (HOCl), which contributes to the killing of menacing microorganisms. Though tightly controlled, HOCl generation by the myeloperoxidase-hydrogen peroxide-chloride system of neutrophils/monocytes may occur in excess and lead to tissue damage. It is thus of marked importance to delineate the molecular pathways underlying HOCl cytotoxicity in both microbial and human cells. Here, we show that HOCl induces the generation of reactive oxygen species (ROS), apoptotic cell death and the formation of specific HOCl-modified epitopes in the budding yeast Saccharomyces cerevisiae. Interestingly, HOCl cytotoxicity can be prevented by treatment with ROS scavengers, suggesting oxidative stress to mediate the lethal effect. The executing pathway involves the pro-apoptotic protease Kex1p, since its absence diminishes HOCl-induced production of ROS, apoptosis and protein modification. By characterizing HOCl-induced cell death in yeast and identifying a corresponding central executor, these results pave the way for the use of Saccharomyces cerevisiae in HOCl research, not least given that it combines both being a microorganism as well as a model for programmed cell death in higher eukaryotes.

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Figure 2. The protease Kex1p is involved in HOCl-induced cytotoxicity. (A and B) Survival (A) and ROS production (B) of wild type, Δyca1, Δnuc1, Δndi1, Δkex1, Δnma111, Δaif1, Δpep4, Δcpl1 and Δybh3 cells after treatment with or without 300 µM HOCl for 16 h as determined via clonogenicity (A) and DHE→Ethidium conversion (B), respectively. Survival (A) was normalized to the untreated control. Experiments in (B) were quantified using a fluorescence plate reader. Data represent means ± s.e.m. (n = 8 – 16). The dashed line draws the survival (A) and ethidium fluorescence (B) levels of the HOCl-treated wild type, respectively. RFU, relative fluorescence unit. (C–F) Survival (C), ROS production (D), phosphatidylserine externalization (E), loss of membrane integrity (E) and apoptotic DNA fragmentation (F) of wild type and Δkex1 cells after treatment with or without 300 µM HOCl for 16 h as determined via clonogenicity (C), DHE→Ethidium conversion (D), Annexin V/PI co-staining (E) and TUNEL staining (F). Data represent means ± s.e.m. (n = 8; ***, p < 0.001). Survival (A) was normalized to the untreated control. In each experiment for (D–F), 30,000 cells were evaluated using flow cytometry. (G) Fluorescence intensity of wild type and Δkex1 cells after treatment with or without 300 µM HOCl for 16 h and subsequent immunofluorescence (IF) staining using mAb 2D10G9 as a primary antibody and quantified using a fluorescence plate reader. Data represent means ± s.e.m. (n = 9; **, p < 0.01). RFU, relative fluorescence unit.
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Figure 2: Figure 2. The protease Kex1p is involved in HOCl-induced cytotoxicity. (A and B) Survival (A) and ROS production (B) of wild type, Δyca1, Δnuc1, Δndi1, Δkex1, Δnma111, Δaif1, Δpep4, Δcpl1 and Δybh3 cells after treatment with or without 300 µM HOCl for 16 h as determined via clonogenicity (A) and DHE→Ethidium conversion (B), respectively. Survival (A) was normalized to the untreated control. Experiments in (B) were quantified using a fluorescence plate reader. Data represent means ± s.e.m. (n = 8 – 16). The dashed line draws the survival (A) and ethidium fluorescence (B) levels of the HOCl-treated wild type, respectively. RFU, relative fluorescence unit. (C–F) Survival (C), ROS production (D), phosphatidylserine externalization (E), loss of membrane integrity (E) and apoptotic DNA fragmentation (F) of wild type and Δkex1 cells after treatment with or without 300 µM HOCl for 16 h as determined via clonogenicity (C), DHE→Ethidium conversion (D), Annexin V/PI co-staining (E) and TUNEL staining (F). Data represent means ± s.e.m. (n = 8; ***, p < 0.001). Survival (A) was normalized to the untreated control. In each experiment for (D–F), 30,000 cells were evaluated using flow cytometry. (G) Fluorescence intensity of wild type and Δkex1 cells after treatment with or without 300 µM HOCl for 16 h and subsequent immunofluorescence (IF) staining using mAb 2D10G9 as a primary antibody and quantified using a fluorescence plate reader. Data represent means ± s.e.m. (n = 9; **, p < 0.01). RFU, relative fluorescence unit.

Mentions: It should be noted that there exists a quantitative difference between the survival levels upon HOCl treatment (300 µM) displayed in Figure 1A vs. Figure 1C (compare also Fig. 2A–C). In fact, the degree of HOCl cytotoxicity at a given concentration differed depending on the batch of SMD growth medium used, though a priori all batches should have had exactly the same composition. Conversely, the range of concentrations in which HOCl killed wild type yeast cells at a comparable quantitative level varied between 260–300 µM, depending on the specific medium batch (note the use of different HOCl concentrations in Fig. 1E and F vs. Fig. 2E and F). Within a batch and at a determined concentration, the qualitative and quantitative results remained stable. For this reason, we assume that some compound(s) in the medium cross-reacting with HOCl and slightly modifying its potency to kill yeast cells are sensitive to external factors that are difficult to control and might slightly differ from medium preparation to preparation. One of these compounds might be photosensitive, since the medium needed to be kept in the dark to maintain constant HOCl cytotoxic performance within one medium batch over time. Interestingly, it has been reported that ammonium ions, which are a component in the medium used in this study, influence HOCl cytotoxicity.38 Also, moderate modifications in extracellular pH interfere with HOCl apoptotic induction.39 These examples suggest that, indeed, slight changes in media composition may have an impact on the quantitative extension of HOCl-induced cytotoxicity, and that the corresponding concentration range must be assessed prior to the use of a specific medium batch. However, the actual nature of the described medium batch-dependent variance remains undetermined.


The cell death protease Kex1p is essential for hypochlorite-induced apoptosis in yeast.

Carmona-Gutierrez D, Alavian-Ghavanini A, Habernig L, Bauer MA, Hammer A, Rossmann C, Zimmermann AS, Ruckenstuhl C, Büttner S, Eisenberg T, Sattler W, Malle E, Madeo F - Cell Cycle (2013)

Figure 2. The protease Kex1p is involved in HOCl-induced cytotoxicity. (A and B) Survival (A) and ROS production (B) of wild type, Δyca1, Δnuc1, Δndi1, Δkex1, Δnma111, Δaif1, Δpep4, Δcpl1 and Δybh3 cells after treatment with or without 300 µM HOCl for 16 h as determined via clonogenicity (A) and DHE→Ethidium conversion (B), respectively. Survival (A) was normalized to the untreated control. Experiments in (B) were quantified using a fluorescence plate reader. Data represent means ± s.e.m. (n = 8 – 16). The dashed line draws the survival (A) and ethidium fluorescence (B) levels of the HOCl-treated wild type, respectively. RFU, relative fluorescence unit. (C–F) Survival (C), ROS production (D), phosphatidylserine externalization (E), loss of membrane integrity (E) and apoptotic DNA fragmentation (F) of wild type and Δkex1 cells after treatment with or without 300 µM HOCl for 16 h as determined via clonogenicity (C), DHE→Ethidium conversion (D), Annexin V/PI co-staining (E) and TUNEL staining (F). Data represent means ± s.e.m. (n = 8; ***, p < 0.001). Survival (A) was normalized to the untreated control. In each experiment for (D–F), 30,000 cells were evaluated using flow cytometry. (G) Fluorescence intensity of wild type and Δkex1 cells after treatment with or without 300 µM HOCl for 16 h and subsequent immunofluorescence (IF) staining using mAb 2D10G9 as a primary antibody and quantified using a fluorescence plate reader. Data represent means ± s.e.m. (n = 9; **, p < 0.01). RFU, relative fluorescence unit.
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Related In: Results  -  Collection

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Figure 2: Figure 2. The protease Kex1p is involved in HOCl-induced cytotoxicity. (A and B) Survival (A) and ROS production (B) of wild type, Δyca1, Δnuc1, Δndi1, Δkex1, Δnma111, Δaif1, Δpep4, Δcpl1 and Δybh3 cells after treatment with or without 300 µM HOCl for 16 h as determined via clonogenicity (A) and DHE→Ethidium conversion (B), respectively. Survival (A) was normalized to the untreated control. Experiments in (B) were quantified using a fluorescence plate reader. Data represent means ± s.e.m. (n = 8 – 16). The dashed line draws the survival (A) and ethidium fluorescence (B) levels of the HOCl-treated wild type, respectively. RFU, relative fluorescence unit. (C–F) Survival (C), ROS production (D), phosphatidylserine externalization (E), loss of membrane integrity (E) and apoptotic DNA fragmentation (F) of wild type and Δkex1 cells after treatment with or without 300 µM HOCl for 16 h as determined via clonogenicity (C), DHE→Ethidium conversion (D), Annexin V/PI co-staining (E) and TUNEL staining (F). Data represent means ± s.e.m. (n = 8; ***, p < 0.001). Survival (A) was normalized to the untreated control. In each experiment for (D–F), 30,000 cells were evaluated using flow cytometry. (G) Fluorescence intensity of wild type and Δkex1 cells after treatment with or without 300 µM HOCl for 16 h and subsequent immunofluorescence (IF) staining using mAb 2D10G9 as a primary antibody and quantified using a fluorescence plate reader. Data represent means ± s.e.m. (n = 9; **, p < 0.01). RFU, relative fluorescence unit.
Mentions: It should be noted that there exists a quantitative difference between the survival levels upon HOCl treatment (300 µM) displayed in Figure 1A vs. Figure 1C (compare also Fig. 2A–C). In fact, the degree of HOCl cytotoxicity at a given concentration differed depending on the batch of SMD growth medium used, though a priori all batches should have had exactly the same composition. Conversely, the range of concentrations in which HOCl killed wild type yeast cells at a comparable quantitative level varied between 260–300 µM, depending on the specific medium batch (note the use of different HOCl concentrations in Fig. 1E and F vs. Fig. 2E and F). Within a batch and at a determined concentration, the qualitative and quantitative results remained stable. For this reason, we assume that some compound(s) in the medium cross-reacting with HOCl and slightly modifying its potency to kill yeast cells are sensitive to external factors that are difficult to control and might slightly differ from medium preparation to preparation. One of these compounds might be photosensitive, since the medium needed to be kept in the dark to maintain constant HOCl cytotoxic performance within one medium batch over time. Interestingly, it has been reported that ammonium ions, which are a component in the medium used in this study, influence HOCl cytotoxicity.38 Also, moderate modifications in extracellular pH interfere with HOCl apoptotic induction.39 These examples suggest that, indeed, slight changes in media composition may have an impact on the quantitative extension of HOCl-induced cytotoxicity, and that the corresponding concentration range must be assessed prior to the use of a specific medium batch. However, the actual nature of the described medium batch-dependent variance remains undetermined.

Bottom Line: Interestingly, HOCl cytotoxicity can be prevented by treatment with ROS scavengers, suggesting oxidative stress to mediate the lethal effect.The executing pathway involves the pro-apoptotic protease Kex1p, since its absence diminishes HOCl-induced production of ROS, apoptosis and protein modification.By characterizing HOCl-induced cell death in yeast and identifying a corresponding central executor, these results pave the way for the use of Saccharomyces cerevisiae in HOCl research, not least given that it combines both being a microorganism as well as a model for programmed cell death in higher eukaryotes.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biosciences, University of Graz, Graz, Austria.

ABSTRACT
Following microbial pathogen invasion, the human immune system of activated phagocytes generates and releases the potent oxidant hypochlorous acid (HOCl), which contributes to the killing of menacing microorganisms. Though tightly controlled, HOCl generation by the myeloperoxidase-hydrogen peroxide-chloride system of neutrophils/monocytes may occur in excess and lead to tissue damage. It is thus of marked importance to delineate the molecular pathways underlying HOCl cytotoxicity in both microbial and human cells. Here, we show that HOCl induces the generation of reactive oxygen species (ROS), apoptotic cell death and the formation of specific HOCl-modified epitopes in the budding yeast Saccharomyces cerevisiae. Interestingly, HOCl cytotoxicity can be prevented by treatment with ROS scavengers, suggesting oxidative stress to mediate the lethal effect. The executing pathway involves the pro-apoptotic protease Kex1p, since its absence diminishes HOCl-induced production of ROS, apoptosis and protein modification. By characterizing HOCl-induced cell death in yeast and identifying a corresponding central executor, these results pave the way for the use of Saccharomyces cerevisiae in HOCl research, not least given that it combines both being a microorganism as well as a model for programmed cell death in higher eukaryotes.

Show MeSH
Related in: MedlinePlus