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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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Assessment of FasL binding, presence of Fas in lipid rafts, and DISC formation after manipulation of Grx1. (A) Evaluation of S-glutathionylation of Fas in lipid rafts in cells stimulated with FasL + M2 and the impact of overexpression of Grx1. Cells were transfected with pcDNA3 or Flag-Grx1 and stimulated with FasL + M2 for 20 min. Lipid raft fractions (3 and 4) and soluble fraction (12) were subjected to IP with antiglutathione antibody and analyzed by immunoblotting for Fas. PSSG was decomposed with 50 mM DTT as a reagent control before IP. The middle and bottom panels reflect immunoblot assays of Fas and the raft marker caveolin1 present in the input samples. Complete fractionation is shown in Fig. S3 A (available at http://www.jcb.org/cgi/content/full/jcb.200807019/DC1). (B) Assessment of FasL binding to cells after manipulation of Grx1. Cells were subjected to control (Ctr) and Grx1 siRNA transfection. In separate experiments, cells were transfected with pcDNA3 or Grx1 plasmids. After 48 h, 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. Note that differences absolute fluorescence intensities between pcDNA3 and control siRNA–transfected cells may be a result of the different transfection procedures. Confirmation of Grx1 overexpression and knockdown is shown in Fig. S3 B and Fig. S3 C, respectively. (C) Assessment of FasL-interacting proteins in cells overexpressing Grx1. pcDNA3 or Grx1-transfected C10 cells were treated with M2 alone or FasL + M2. Cells were lysed, and 700 µg of protein was subjected to IP using protein G agarose beads to isolate DISC proteins. After SDS-PAGE, samples were analyzed by immunoblotting for Fas, FADD, procaspase 8, cleaved caspase-8, and Grx1. IP, M2 represents control IP in the absence of FasL. Note that all samples were run on the same gel. Black lines indicate that intervening lanes have been spliced out. (D) Evaluation of PSSG and Fas content in high MW complexes after IP of FasL + M2 or M2 alone via nonreducing SDS PAGE. As a control, samples were treated with DTT before electrophoresis. (E) Assessment of interaction between FasL and Fas in WT primary tracheal epithelial cells or cells lacking Glrx1. Cells were exposed to M2 alone or FasL + M2 for 30 min, lysed, and 700 µg of protein was subjected to IP using protein G agarose beads. After SDS-PAGE, samples were analyzed by immunoblotting for Fas. WCL, whole cell lysate.
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fig5: Assessment of FasL binding, presence of Fas in lipid rafts, and DISC formation after manipulation of Grx1. (A) Evaluation of S-glutathionylation of Fas in lipid rafts in cells stimulated with FasL + M2 and the impact of overexpression of Grx1. Cells were transfected with pcDNA3 or Flag-Grx1 and stimulated with FasL + M2 for 20 min. Lipid raft fractions (3 and 4) and soluble fraction (12) were subjected to IP with antiglutathione antibody and analyzed by immunoblotting for Fas. PSSG was decomposed with 50 mM DTT as a reagent control before IP. The middle and bottom panels reflect immunoblot assays of Fas and the raft marker caveolin1 present in the input samples. Complete fractionation is shown in Fig. S3 A (available at http://www.jcb.org/cgi/content/full/jcb.200807019/DC1). (B) Assessment of FasL binding to cells after manipulation of Grx1. Cells were subjected to control (Ctr) and Grx1 siRNA transfection. In separate experiments, cells were transfected with pcDNA3 or Grx1 plasmids. After 48 h, 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. Note that differences absolute fluorescence intensities between pcDNA3 and control siRNA–transfected cells may be a result of the different transfection procedures. Confirmation of Grx1 overexpression and knockdown is shown in Fig. S3 B and Fig. S3 C, respectively. (C) Assessment of FasL-interacting proteins in cells overexpressing Grx1. pcDNA3 or Grx1-transfected C10 cells were treated with M2 alone or FasL + M2. Cells were lysed, and 700 µg of protein was subjected to IP using protein G agarose beads to isolate DISC proteins. After SDS-PAGE, samples were analyzed by immunoblotting for Fas, FADD, procaspase 8, cleaved caspase-8, and Grx1. IP, M2 represents control IP in the absence of FasL. Note that all samples were run on the same gel. Black lines indicate that intervening lanes have been spliced out. (D) Evaluation of PSSG and Fas content in high MW complexes after IP of FasL + M2 or M2 alone via nonreducing SDS PAGE. As a control, samples were treated with DTT before electrophoresis. (E) Assessment of interaction between FasL and Fas in WT primary tracheal epithelial cells or cells lacking Glrx1. Cells were exposed to M2 alone or FasL + M2 for 30 min, lysed, and 700 µg of protein was subjected to IP using protein G agarose beads. After SDS-PAGE, samples were analyzed by immunoblotting for Fas. WCL, whole cell lysate.

Mentions: The localization of Fas in lipid rafts is essential for binding of FasL, assembly of DISC, and subsequently the induction of apoptosis (Hueber et al., 2002; Muppidi and Siegel, 2004). Therefore, we determined whether the extent of Fas-SSG affected these parameters. As expected, after stimulation of cells with FasL, an increase in the amount of Fas localized to the lipid rafts occurred based on its colocalization with raft marker caveolin1 (Fig. 5 A and Fig. S3 A, available at http://www.jcb.org/cgi/content/full/jcb.200807019/DC1). IP of S-glutathionylated proteins revealed that in response to FasL ligation, Fas-SSG was present within the lipid raft fractions as well as soluble fractions (Fig. 5 A), whereas Grx1 was absent in the lipid raft fraction (Fig. S3 A). In cells overexpressing Grx1, the overall content of Fas and its S-glutathionylated state were decreased in lipid rafts compared with mock-transfected cells stimulated with FasL (Fig. 5 A and Fig. S3 A). Assessment of FasL binding demonstrated dose-dependent increases in WT cells. However, in cells overexpressing Grx1, no clear increases in FasL binding occurred at a range of concentrations (Fig. 5 B and Fig. S3 B). Furthermore, IP of the DISC revealed clear associations between FasL, Fas, FADD, and procaspase-8, which were diminished in cells overexpressing Grx1 (Fig. 5 C). Grx1 was absent in DISC in all conditions evaluated (Fig. 5 C and not depicted), which may sustain the presence of Fas-SSG in these signaling platforms. IP of FasL resulted in high MW SDS-stable PSSG complexes that were attenuated in cells overexpressing Grx1 and absent in samples treated with DTT (Fig. 5 D). Evaluation of Fas in these samples confirmed its presence in the high MW complex (∼190 kD) after IP with FasL. This high MW Fas complex was sensitive to decomposition by DTT and markedly decreased in Grx1-overexpressing cells (Fig. 5 D), demonstrating that PSSG contributes to the formation of high MW Fas complexes that are known to be required for the induction of apoptosis (Feig et al., 2007). In cells lacking Grx1, a marked increase in binding of FasL occurred (Fig. 5 B and Fig. S3 C) in association with more IP of Fas (Fig. 5 E). Collectively, these observations demonstrate that the status of S-glutathionylation of Fas regulates binding of FasL, the ability of Fas to move into lipid rafts, formation of high MW Fas complexes, and assembly of DISC.


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)

Assessment of FasL binding, presence of Fas in lipid rafts, and DISC formation after manipulation of Grx1. (A) Evaluation of S-glutathionylation of Fas in lipid rafts in cells stimulated with FasL + M2 and the impact of overexpression of Grx1. Cells were transfected with pcDNA3 or Flag-Grx1 and stimulated with FasL + M2 for 20 min. Lipid raft fractions (3 and 4) and soluble fraction (12) were subjected to IP with antiglutathione antibody and analyzed by immunoblotting for Fas. PSSG was decomposed with 50 mM DTT as a reagent control before IP. The middle and bottom panels reflect immunoblot assays of Fas and the raft marker caveolin1 present in the input samples. Complete fractionation is shown in Fig. S3 A (available at http://www.jcb.org/cgi/content/full/jcb.200807019/DC1). (B) Assessment of FasL binding to cells after manipulation of Grx1. Cells were subjected to control (Ctr) and Grx1 siRNA transfection. In separate experiments, cells were transfected with pcDNA3 or Grx1 plasmids. After 48 h, 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. Note that differences absolute fluorescence intensities between pcDNA3 and control siRNA–transfected cells may be a result of the different transfection procedures. Confirmation of Grx1 overexpression and knockdown is shown in Fig. S3 B and Fig. S3 C, respectively. (C) Assessment of FasL-interacting proteins in cells overexpressing Grx1. pcDNA3 or Grx1-transfected C10 cells were treated with M2 alone or FasL + M2. Cells were lysed, and 700 µg of protein was subjected to IP using protein G agarose beads to isolate DISC proteins. After SDS-PAGE, samples were analyzed by immunoblotting for Fas, FADD, procaspase 8, cleaved caspase-8, and Grx1. IP, M2 represents control IP in the absence of FasL. Note that all samples were run on the same gel. Black lines indicate that intervening lanes have been spliced out. (D) Evaluation of PSSG and Fas content in high MW complexes after IP of FasL + M2 or M2 alone via nonreducing SDS PAGE. As a control, samples were treated with DTT before electrophoresis. (E) Assessment of interaction between FasL and Fas in WT primary tracheal epithelial cells or cells lacking Glrx1. Cells were exposed to M2 alone or FasL + M2 for 30 min, lysed, and 700 µg of protein was subjected to IP using protein G agarose beads. After SDS-PAGE, samples were analyzed by immunoblotting for Fas. WCL, whole cell lysate.
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Show All Figures
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fig5: Assessment of FasL binding, presence of Fas in lipid rafts, and DISC formation after manipulation of Grx1. (A) Evaluation of S-glutathionylation of Fas in lipid rafts in cells stimulated with FasL + M2 and the impact of overexpression of Grx1. Cells were transfected with pcDNA3 or Flag-Grx1 and stimulated with FasL + M2 for 20 min. Lipid raft fractions (3 and 4) and soluble fraction (12) were subjected to IP with antiglutathione antibody and analyzed by immunoblotting for Fas. PSSG was decomposed with 50 mM DTT as a reagent control before IP. The middle and bottom panels reflect immunoblot assays of Fas and the raft marker caveolin1 present in the input samples. Complete fractionation is shown in Fig. S3 A (available at http://www.jcb.org/cgi/content/full/jcb.200807019/DC1). (B) Assessment of FasL binding to cells after manipulation of Grx1. Cells were subjected to control (Ctr) and Grx1 siRNA transfection. In separate experiments, cells were transfected with pcDNA3 or Grx1 plasmids. After 48 h, 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. Note that differences absolute fluorescence intensities between pcDNA3 and control siRNA–transfected cells may be a result of the different transfection procedures. Confirmation of Grx1 overexpression and knockdown is shown in Fig. S3 B and Fig. S3 C, respectively. (C) Assessment of FasL-interacting proteins in cells overexpressing Grx1. pcDNA3 or Grx1-transfected C10 cells were treated with M2 alone or FasL + M2. Cells were lysed, and 700 µg of protein was subjected to IP using protein G agarose beads to isolate DISC proteins. After SDS-PAGE, samples were analyzed by immunoblotting for Fas, FADD, procaspase 8, cleaved caspase-8, and Grx1. IP, M2 represents control IP in the absence of FasL. Note that all samples were run on the same gel. Black lines indicate that intervening lanes have been spliced out. (D) Evaluation of PSSG and Fas content in high MW complexes after IP of FasL + M2 or M2 alone via nonreducing SDS PAGE. As a control, samples were treated with DTT before electrophoresis. (E) Assessment of interaction between FasL and Fas in WT primary tracheal epithelial cells or cells lacking Glrx1. Cells were exposed to M2 alone or FasL + M2 for 30 min, lysed, and 700 µg of protein was subjected to IP using protein G agarose beads. After SDS-PAGE, samples were analyzed by immunoblotting for Fas. WCL, whole cell lysate.
Mentions: The localization of Fas in lipid rafts is essential for binding of FasL, assembly of DISC, and subsequently the induction of apoptosis (Hueber et al., 2002; Muppidi and Siegel, 2004). Therefore, we determined whether the extent of Fas-SSG affected these parameters. As expected, after stimulation of cells with FasL, an increase in the amount of Fas localized to the lipid rafts occurred based on its colocalization with raft marker caveolin1 (Fig. 5 A and Fig. S3 A, available at http://www.jcb.org/cgi/content/full/jcb.200807019/DC1). IP of S-glutathionylated proteins revealed that in response to FasL ligation, Fas-SSG was present within the lipid raft fractions as well as soluble fractions (Fig. 5 A), whereas Grx1 was absent in the lipid raft fraction (Fig. S3 A). In cells overexpressing Grx1, the overall content of Fas and its S-glutathionylated state were decreased in lipid rafts compared with mock-transfected cells stimulated with FasL (Fig. 5 A and Fig. S3 A). Assessment of FasL binding demonstrated dose-dependent increases in WT cells. However, in cells overexpressing Grx1, no clear increases in FasL binding occurred at a range of concentrations (Fig. 5 B and Fig. S3 B). Furthermore, IP of the DISC revealed clear associations between FasL, Fas, FADD, and procaspase-8, which were diminished in cells overexpressing Grx1 (Fig. 5 C). Grx1 was absent in DISC in all conditions evaluated (Fig. 5 C and not depicted), which may sustain the presence of Fas-SSG in these signaling platforms. IP of FasL resulted in high MW SDS-stable PSSG complexes that were attenuated in cells overexpressing Grx1 and absent in samples treated with DTT (Fig. 5 D). Evaluation of Fas in these samples confirmed its presence in the high MW complex (∼190 kD) after IP with FasL. This high MW Fas complex was sensitive to decomposition by DTT and markedly decreased in Grx1-overexpressing cells (Fig. 5 D), demonstrating that PSSG contributes to the formation of high MW Fas complexes that are known to be required for the induction of apoptosis (Feig et al., 2007). In cells lacking Grx1, a marked increase in binding of FasL occurred (Fig. 5 B and Fig. S3 C) in association with more IP of Fas (Fig. 5 E). Collectively, these observations demonstrate that the status of S-glutathionylation of Fas regulates binding of FasL, the ability of Fas to move into lipid rafts, formation of high MW Fas complexes, and assembly of DISC.

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