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Redox amplification of apoptosis by caspase-dependent cleavage of glutaredoxin 1 and S-glutathionylation of Fas.

Anathy V, Aesif SW, Guala AS, Havermans M, Reynaert NL, Ho YS, Budd RC, Janssen-Heininger YM - J. Cell Biol. (2009)

Bottom Line: In this study, we demonstrate that stimulation with Fas ligand (FasL) induces S-glutathionylation of Fas at cysteine 294 independently of nicotinamide adenine dinucleotide phosphate reduced oxidase-induced ROS.As a result, death-inducing signaling complex formation is also increased, and subsequent activation of caspase-8 and -3 is augmented.These results define a novel redox-based mechanism to propagate Fas-dependent apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Vermont, Burlington, VT 05405, USA.

ABSTRACT
Reactive oxygen species (ROS) increase ligation of Fas (CD95), a receptor important for regulation of programmed cell death. Glutathionylation of reactive cysteines represents an oxidative modification that can be reversed by glutaredoxins (Grxs). The goal of this study was to determine whether Fas is redox regulated under physiological conditions. In this study, we demonstrate that stimulation with Fas ligand (FasL) induces S-glutathionylation of Fas at cysteine 294 independently of nicotinamide adenine dinucleotide phosphate reduced oxidase-induced ROS. Instead, Fas is S-glutathionylated after caspase-dependent degradation of Grx1, increasing subsequent caspase activation and apoptosis. Conversely, overexpression of Grx1 attenuates S-glutathionylation of Fas and partially protects against FasL-induced apoptosis. Redox-mediated Fas modification promotes its aggregation and recruitment into lipid rafts and enhances binding of FasL. As a result, death-inducing signaling complex formation is also increased, and subsequent activation of caspase-8 and -3 is augmented. These results define a novel redox-based mechanism to propagate Fas-dependent apoptosis.

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S-glutathionylation of cysteine 294 of Fas promotes binding to FasL, activation of caspase-8 and -3, and cell death. (A) lpr lung fibroblasts were transfected with pcDNA3, WT Fas, or Fas mutant constructs C194A, C271A, or C294A. Cells were exposed to FasL + M2 for 2 h, and lysates were subjected to IP using antiglutathione antibody. Immunoprecipitates were subjected to SDS-PAGE, and Fas content was evaluated via immunoblotting. The middle and bottom panels show total content of Fas and β-actin in whole cell lysates (WCL) as controls. (B) Attenuation of the formation of cleaved caspase-8 and -3 fragments in cells expressing Fas C294A. Cells were transfected with pcDNA3, WT, or Fas mutant constructs before incubation with FasL + M2. Lysates were subjected to immunoblotting using anti–caspase-8 and -3 antibodies. Fas immunoblotting (top) confirms equal expression of Fas constructs. (C) Protection against death in cells expressing C294A mutant Fas. lpr lung fibroblasts were transfected and treated as described in A, and cell survival was assessed using the MTT assay. Percent survival was calculated from two independent experiments run in triplicates. Results are expressed as mean values ± SEM. *, P < 0.05 as compared with cells transfected with WT Fas (Student's t test). Confirmation that all constructs are expressed equally is demonstrated in Fig. S4 A (available at http://www.jcb.org/cgi/content/full/jcb.200807019/DC1). (D) S-glutathionylation of Fas does not affect surface expression. lpr lung fibroblasts were transfected with WT Fas or C294A mutant construct, and surface expression of Fas was evaluated 48 h after transfection using anti-Fas antibody (JO2) via flow cytometry. Log Fas immunofluorescence is shown on the x-axis, and cell counts are shown on the y-axis. pcDNA3 control (gray) reflects nonspecific background fluorescence. The dashed line represents C294A mutant Fas, and the solid line indicates WT Fas. Confirmation that both constructs are expressed equally is included in Fig. S4 B. (E) Assessment of FasL binding to lpr lung fibroblasts that express WT or C294A mutant Fas constructs. 48 h after transfection, cells were trypsinized and incubated with ascending doses of FasL + M2 for 20 min. Binding of FasL to cells was evaluated after incubation with FITC-conjugated anti–mouse antibody and evaluation of 10,000 events via flow cytometry. Binding of FasL to cells is reflected as mean fluorescence intensity (MFI), and absolute values are plotted on the y-axis. The x-axis depicts ascending concentrations of FasL. Confirmation that both constructs are expressed equally is included in Fig. S4 B. Error bars indicate ± SEM. (F) Assessment of binding of WT Fas or C294A mutant Fas in the absence of PSSG. lpr lung fibroblasts were transfected with WT or C294A mutant Fas constructs (Fig. S4 C). 48 h after transfection, whole cell lysates were prepared and incubated with 100, 300, or 1,000 ng/ml FasL + 2 µg/ml M2 at 4°C for 12–16 h. Samples were subsequently incubated with protein G agarose beads and washed several times before assessment of Fas content via Western Blot analysis. C1 represents sample from WT Fas–expressing cells subjected to IP with M2 antibody alone, whereas C2 represents sample from lpr cells transfected with pcDNA3 subjected to IP with 100 ng/ml FasL + 2 µg/ml M2. The bottom panel shows content of FasL. IgG, nonspecific reactivity. (G) Proposed model that incorporates S-glutathionylation in Fas-dependent apoptosis. In response to Fas ligation, activated caspase-8 and/or -3 degrade Grx1 either directly or potentially via indirect mechanisms. Caspase-initiated decreases in Grx1 content causes S-glutathionylation of Fas at cysteine 294 to increase. This promotes binding of FasL and enhances aggregation of Fas and its accumulation in lipid rafts and formation of the DISC, thereby further enhancing caspase activities and apoptosis. S-glutathionylation of Fas provides a mechanism whereby the extent of cell death is amplified in a feed-forward regulatory loop.
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fig6: S-glutathionylation of cysteine 294 of Fas promotes binding to FasL, activation of caspase-8 and -3, and cell death. (A) lpr lung fibroblasts were transfected with pcDNA3, WT Fas, or Fas mutant constructs C194A, C271A, or C294A. Cells were exposed to FasL + M2 for 2 h, and lysates were subjected to IP using antiglutathione antibody. Immunoprecipitates were subjected to SDS-PAGE, and Fas content was evaluated via immunoblotting. The middle and bottom panels show total content of Fas and β-actin in whole cell lysates (WCL) as controls. (B) Attenuation of the formation of cleaved caspase-8 and -3 fragments in cells expressing Fas C294A. Cells were transfected with pcDNA3, WT, or Fas mutant constructs before incubation with FasL + M2. Lysates were subjected to immunoblotting using anti–caspase-8 and -3 antibodies. Fas immunoblotting (top) confirms equal expression of Fas constructs. (C) Protection against death in cells expressing C294A mutant Fas. lpr lung fibroblasts were transfected and treated as described in A, and cell survival was assessed using the MTT assay. Percent survival was calculated from two independent experiments run in triplicates. Results are expressed as mean values ± SEM. *, P < 0.05 as compared with cells transfected with WT Fas (Student's t test). Confirmation that all constructs are expressed equally is demonstrated in Fig. S4 A (available at http://www.jcb.org/cgi/content/full/jcb.200807019/DC1). (D) S-glutathionylation of Fas does not affect surface expression. lpr lung fibroblasts were transfected with WT Fas or C294A mutant construct, and surface expression of Fas was evaluated 48 h after transfection using anti-Fas antibody (JO2) via flow cytometry. Log Fas immunofluorescence is shown on the x-axis, and cell counts are shown on the y-axis. pcDNA3 control (gray) reflects nonspecific background fluorescence. The dashed line represents C294A mutant Fas, and the solid line indicates WT Fas. Confirmation that both constructs are expressed equally is included in Fig. S4 B. (E) Assessment of FasL binding to lpr lung fibroblasts that express WT or C294A mutant Fas constructs. 48 h after transfection, cells were trypsinized and incubated with ascending doses of FasL + M2 for 20 min. Binding of FasL to cells was evaluated after incubation with FITC-conjugated anti–mouse antibody and evaluation of 10,000 events via flow cytometry. Binding of FasL to cells is reflected as mean fluorescence intensity (MFI), and absolute values are plotted on the y-axis. The x-axis depicts ascending concentrations of FasL. Confirmation that both constructs are expressed equally is included in Fig. S4 B. Error bars indicate ± SEM. (F) Assessment of binding of WT Fas or C294A mutant Fas in the absence of PSSG. lpr lung fibroblasts were transfected with WT or C294A mutant Fas constructs (Fig. S4 C). 48 h after transfection, whole cell lysates were prepared and incubated with 100, 300, or 1,000 ng/ml FasL + 2 µg/ml M2 at 4°C for 12–16 h. Samples were subsequently incubated with protein G agarose beads and washed several times before assessment of Fas content via Western Blot analysis. C1 represents sample from WT Fas–expressing cells subjected to IP with M2 antibody alone, whereas C2 represents sample from lpr cells transfected with pcDNA3 subjected to IP with 100 ng/ml FasL + 2 µg/ml M2. The bottom panel shows content of FasL. IgG, nonspecific reactivity. (G) Proposed model that incorporates S-glutathionylation in Fas-dependent apoptosis. In response to Fas ligation, activated caspase-8 and/or -3 degrade Grx1 either directly or potentially via indirect mechanisms. Caspase-initiated decreases in Grx1 content causes S-glutathionylation of Fas at cysteine 294 to increase. This promotes binding of FasL and enhances aggregation of Fas and its accumulation in lipid rafts and formation of the DISC, thereby further enhancing caspase activities and apoptosis. S-glutathionylation of Fas provides a mechanism whereby the extent of cell death is amplified in a feed-forward regulatory loop.

Mentions: Murine Fas contains a total of 24 cysteines, out of which 20 are present in the ectodomain, and four are located in the DD (GenBank accession no. ABI24113). The ectodomain cysteines are known to form intramolecular disulfide bonds and therefore do not represent likely targets for S-glutathionylation. This prompted us to search for potential cysteine targets for S-glutathionylation within the DD of Fas through the generation of constructs in which cysteines were mutated to alanines. Fas mutants, C194A, C271A, C294A, or WT Fas was transfected into fibroblasts derived from lpr mice that lack Fas (Drappa et al., 1993). After stimulation with FasL, Fas-SSG was apparent in cells transfected with WT, C194A, or C271A mutant constructs. In contrast, Fas-SSG was not apparent in cells expressing C294A mutant Fas (Fig. 6 A). In lpr fibroblasts or lung epithelial cells, expression of WT, C194A, or C271A Fas constructs resulted in enhanced formation of active caspase-8 and -3 fragments after ligation of Fas, which was not apparent in cells expressing comparable levels of C294A mutant Fas (Fig. 6 B and not depicted). Cell death in response to FasL was largely abrogated in lpr fibroblasts expressing C294A mutant Fas in comparison with cells expressing WT, C194A, or C271A mutant versions of Fas (Fig. 6 C and Fig. S4 A, available at http://www.jcb.org/cgi/content/full/jcb.200807019/DC1). Surface expression of C294A mutant Fas was indistinguishable from cells expressing WT Fas (Fig. 6 D and Fig. S4 B). However, binding of FasL was markedly decreased in cells expressing C294A Fas in comparison with cells expressing WT Fas (Fig. 6 E and Fig. S4 B), which is consistent with earlier observations, demonstrating that the extent of S-glutathionylation of Fas correlated directly with FasL binding (Fig. 5 B). To address potential intrinsic differences in binding affinities for FasL between WT or C294A mutant Fas, cells were transfected with WT or C294A mutant Fas (Fig. S4 C), lysed, and incubated with increasing amounts of FasL before IP with M2 and blotting for Fas. Results shown in Fig. 6 F demonstrate relatively comparable concentration-dependent increases in Fas associated with FasL and suggest that C294A mutant Fas is not intrinsically defective in binding to FasL. In aggregate, these findings demonstrate that S-glutathionylation of Fas at cysteine 294 is a critical regulatory event that promotes binding of FasL and subsequently strengthens apoptotic signaling.


Redox amplification of apoptosis by caspase-dependent cleavage of glutaredoxin 1 and S-glutathionylation of Fas.

Anathy V, Aesif SW, Guala AS, Havermans M, Reynaert NL, Ho YS, Budd RC, Janssen-Heininger YM - J. Cell Biol. (2009)

S-glutathionylation of cysteine 294 of Fas promotes binding to FasL, activation of caspase-8 and -3, and cell death. (A) lpr lung fibroblasts were transfected with pcDNA3, WT Fas, or Fas mutant constructs C194A, C271A, or C294A. Cells were exposed to FasL + M2 for 2 h, and lysates were subjected to IP using antiglutathione antibody. Immunoprecipitates were subjected to SDS-PAGE, and Fas content was evaluated via immunoblotting. The middle and bottom panels show total content of Fas and β-actin in whole cell lysates (WCL) as controls. (B) Attenuation of the formation of cleaved caspase-8 and -3 fragments in cells expressing Fas C294A. Cells were transfected with pcDNA3, WT, or Fas mutant constructs before incubation with FasL + M2. Lysates were subjected to immunoblotting using anti–caspase-8 and -3 antibodies. Fas immunoblotting (top) confirms equal expression of Fas constructs. (C) Protection against death in cells expressing C294A mutant Fas. lpr lung fibroblasts were transfected and treated as described in A, and cell survival was assessed using the MTT assay. Percent survival was calculated from two independent experiments run in triplicates. Results are expressed as mean values ± SEM. *, P < 0.05 as compared with cells transfected with WT Fas (Student's t test). Confirmation that all constructs are expressed equally is demonstrated in Fig. S4 A (available at http://www.jcb.org/cgi/content/full/jcb.200807019/DC1). (D) S-glutathionylation of Fas does not affect surface expression. lpr lung fibroblasts were transfected with WT Fas or C294A mutant construct, and surface expression of Fas was evaluated 48 h after transfection using anti-Fas antibody (JO2) via flow cytometry. Log Fas immunofluorescence is shown on the x-axis, and cell counts are shown on the y-axis. pcDNA3 control (gray) reflects nonspecific background fluorescence. The dashed line represents C294A mutant Fas, and the solid line indicates WT Fas. Confirmation that both constructs are expressed equally is included in Fig. S4 B. (E) Assessment of FasL binding to lpr lung fibroblasts that express WT or C294A mutant Fas constructs. 48 h after transfection, cells were trypsinized and incubated with ascending doses of FasL + M2 for 20 min. Binding of FasL to cells was evaluated after incubation with FITC-conjugated anti–mouse antibody and evaluation of 10,000 events via flow cytometry. Binding of FasL to cells is reflected as mean fluorescence intensity (MFI), and absolute values are plotted on the y-axis. The x-axis depicts ascending concentrations of FasL. Confirmation that both constructs are expressed equally is included in Fig. S4 B. Error bars indicate ± SEM. (F) Assessment of binding of WT Fas or C294A mutant Fas in the absence of PSSG. lpr lung fibroblasts were transfected with WT or C294A mutant Fas constructs (Fig. S4 C). 48 h after transfection, whole cell lysates were prepared and incubated with 100, 300, or 1,000 ng/ml FasL + 2 µg/ml M2 at 4°C for 12–16 h. Samples were subsequently incubated with protein G agarose beads and washed several times before assessment of Fas content via Western Blot analysis. C1 represents sample from WT Fas–expressing cells subjected to IP with M2 antibody alone, whereas C2 represents sample from lpr cells transfected with pcDNA3 subjected to IP with 100 ng/ml FasL + 2 µg/ml M2. The bottom panel shows content of FasL. IgG, nonspecific reactivity. (G) Proposed model that incorporates S-glutathionylation in Fas-dependent apoptosis. In response to Fas ligation, activated caspase-8 and/or -3 degrade Grx1 either directly or potentially via indirect mechanisms. Caspase-initiated decreases in Grx1 content causes S-glutathionylation of Fas at cysteine 294 to increase. This promotes binding of FasL and enhances aggregation of Fas and its accumulation in lipid rafts and formation of the DISC, thereby further enhancing caspase activities and apoptosis. S-glutathionylation of Fas provides a mechanism whereby the extent of cell death is amplified in a feed-forward regulatory loop.
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Related In: Results  -  Collection

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fig6: S-glutathionylation of cysteine 294 of Fas promotes binding to FasL, activation of caspase-8 and -3, and cell death. (A) lpr lung fibroblasts were transfected with pcDNA3, WT Fas, or Fas mutant constructs C194A, C271A, or C294A. Cells were exposed to FasL + M2 for 2 h, and lysates were subjected to IP using antiglutathione antibody. Immunoprecipitates were subjected to SDS-PAGE, and Fas content was evaluated via immunoblotting. The middle and bottom panels show total content of Fas and β-actin in whole cell lysates (WCL) as controls. (B) Attenuation of the formation of cleaved caspase-8 and -3 fragments in cells expressing Fas C294A. Cells were transfected with pcDNA3, WT, or Fas mutant constructs before incubation with FasL + M2. Lysates were subjected to immunoblotting using anti–caspase-8 and -3 antibodies. Fas immunoblotting (top) confirms equal expression of Fas constructs. (C) Protection against death in cells expressing C294A mutant Fas. lpr lung fibroblasts were transfected and treated as described in A, and cell survival was assessed using the MTT assay. Percent survival was calculated from two independent experiments run in triplicates. Results are expressed as mean values ± SEM. *, P < 0.05 as compared with cells transfected with WT Fas (Student's t test). Confirmation that all constructs are expressed equally is demonstrated in Fig. S4 A (available at http://www.jcb.org/cgi/content/full/jcb.200807019/DC1). (D) S-glutathionylation of Fas does not affect surface expression. lpr lung fibroblasts were transfected with WT Fas or C294A mutant construct, and surface expression of Fas was evaluated 48 h after transfection using anti-Fas antibody (JO2) via flow cytometry. Log Fas immunofluorescence is shown on the x-axis, and cell counts are shown on the y-axis. pcDNA3 control (gray) reflects nonspecific background fluorescence. The dashed line represents C294A mutant Fas, and the solid line indicates WT Fas. Confirmation that both constructs are expressed equally is included in Fig. S4 B. (E) Assessment of FasL binding to lpr lung fibroblasts that express WT or C294A mutant Fas constructs. 48 h after transfection, cells were trypsinized and incubated with ascending doses of FasL + M2 for 20 min. Binding of FasL to cells was evaluated after incubation with FITC-conjugated anti–mouse antibody and evaluation of 10,000 events via flow cytometry. Binding of FasL to cells is reflected as mean fluorescence intensity (MFI), and absolute values are plotted on the y-axis. The x-axis depicts ascending concentrations of FasL. Confirmation that both constructs are expressed equally is included in Fig. S4 B. Error bars indicate ± SEM. (F) Assessment of binding of WT Fas or C294A mutant Fas in the absence of PSSG. lpr lung fibroblasts were transfected with WT or C294A mutant Fas constructs (Fig. S4 C). 48 h after transfection, whole cell lysates were prepared and incubated with 100, 300, or 1,000 ng/ml FasL + 2 µg/ml M2 at 4°C for 12–16 h. Samples were subsequently incubated with protein G agarose beads and washed several times before assessment of Fas content via Western Blot analysis. C1 represents sample from WT Fas–expressing cells subjected to IP with M2 antibody alone, whereas C2 represents sample from lpr cells transfected with pcDNA3 subjected to IP with 100 ng/ml FasL + 2 µg/ml M2. The bottom panel shows content of FasL. IgG, nonspecific reactivity. (G) Proposed model that incorporates S-glutathionylation in Fas-dependent apoptosis. In response to Fas ligation, activated caspase-8 and/or -3 degrade Grx1 either directly or potentially via indirect mechanisms. Caspase-initiated decreases in Grx1 content causes S-glutathionylation of Fas at cysteine 294 to increase. This promotes binding of FasL and enhances aggregation of Fas and its accumulation in lipid rafts and formation of the DISC, thereby further enhancing caspase activities and apoptosis. S-glutathionylation of Fas provides a mechanism whereby the extent of cell death is amplified in a feed-forward regulatory loop.
Mentions: Murine Fas contains a total of 24 cysteines, out of which 20 are present in the ectodomain, and four are located in the DD (GenBank accession no. ABI24113). The ectodomain cysteines are known to form intramolecular disulfide bonds and therefore do not represent likely targets for S-glutathionylation. This prompted us to search for potential cysteine targets for S-glutathionylation within the DD of Fas through the generation of constructs in which cysteines were mutated to alanines. Fas mutants, C194A, C271A, C294A, or WT Fas was transfected into fibroblasts derived from lpr mice that lack Fas (Drappa et al., 1993). After stimulation with FasL, Fas-SSG was apparent in cells transfected with WT, C194A, or C271A mutant constructs. In contrast, Fas-SSG was not apparent in cells expressing C294A mutant Fas (Fig. 6 A). In lpr fibroblasts or lung epithelial cells, expression of WT, C194A, or C271A Fas constructs resulted in enhanced formation of active caspase-8 and -3 fragments after ligation of Fas, which was not apparent in cells expressing comparable levels of C294A mutant Fas (Fig. 6 B and not depicted). Cell death in response to FasL was largely abrogated in lpr fibroblasts expressing C294A mutant Fas in comparison with cells expressing WT, C194A, or C271A mutant versions of Fas (Fig. 6 C and Fig. S4 A, available at http://www.jcb.org/cgi/content/full/jcb.200807019/DC1). Surface expression of C294A mutant Fas was indistinguishable from cells expressing WT Fas (Fig. 6 D and Fig. S4 B). However, binding of FasL was markedly decreased in cells expressing C294A Fas in comparison with cells expressing WT Fas (Fig. 6 E and Fig. S4 B), which is consistent with earlier observations, demonstrating that the extent of S-glutathionylation of Fas correlated directly with FasL binding (Fig. 5 B). To address potential intrinsic differences in binding affinities for FasL between WT or C294A mutant Fas, cells were transfected with WT or C294A mutant Fas (Fig. S4 C), lysed, and incubated with increasing amounts of FasL before IP with M2 and blotting for Fas. Results shown in Fig. 6 F demonstrate relatively comparable concentration-dependent increases in Fas associated with FasL and suggest that C294A mutant Fas is not intrinsically defective in binding to FasL. In aggregate, these findings demonstrate that S-glutathionylation of Fas at cysteine 294 is a critical regulatory event that promotes binding of FasL and subsequently strengthens apoptotic signaling.

Bottom Line: In this study, we demonstrate that stimulation with Fas ligand (FasL) induces S-glutathionylation of Fas at cysteine 294 independently of nicotinamide adenine dinucleotide phosphate reduced oxidase-induced ROS.As a result, death-inducing signaling complex formation is also increased, and subsequent activation of caspase-8 and -3 is augmented.These results define a novel redox-based mechanism to propagate Fas-dependent apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Vermont, Burlington, VT 05405, USA.

ABSTRACT
Reactive oxygen species (ROS) increase ligation of Fas (CD95), a receptor important for regulation of programmed cell death. Glutathionylation of reactive cysteines represents an oxidative modification that can be reversed by glutaredoxins (Grxs). The goal of this study was to determine whether Fas is redox regulated under physiological conditions. In this study, we demonstrate that stimulation with Fas ligand (FasL) induces S-glutathionylation of Fas at cysteine 294 independently of nicotinamide adenine dinucleotide phosphate reduced oxidase-induced ROS. Instead, Fas is S-glutathionylated after caspase-dependent degradation of Grx1, increasing subsequent caspase activation and apoptosis. Conversely, overexpression of Grx1 attenuates S-glutathionylation of Fas and partially protects against FasL-induced apoptosis. Redox-mediated Fas modification promotes its aggregation and recruitment into lipid rafts and enhances binding of FasL. As a result, death-inducing signaling complex formation is also increased, and subsequent activation of caspase-8 and -3 is augmented. These results define a novel redox-based mechanism to propagate Fas-dependent apoptosis.

Show MeSH
Related in: MedlinePlus