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Resistance to granzyme B-mediated cytochrome c release in Bak-deficient cells.

Wang GQ, Wieckowski E, Goldstein LA, Gastman BR, Rabinovitz A, Gambotto A, Li S, Fang B, Yin XM, Rabinowich H - J. Exp. Med. (2001)

Bottom Line: Purified mitochondria from Bid knockout mice, but not from Bax knockout mice, failed to release cytochrome c in response to autologous S-100 and GrB.Also, Bak-deficient mitochondria did not release cytochrome c in response to GrB-treated cytosol unless recombinant Bak protein was added.These results are the first to report a role for Bak in GrB-mediated mitochondrial apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, The University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, USA.

ABSTRACT
Granzyme B (GrB), a serine protease with substrate specificity similar to the caspase family, is a major component of granule-mediated cytotoxicity of T lymphocytes. Although GrB can directly activate caspases, it induces apoptosis predominantly via Bid cleavage, mitochondrial outer membrane permeabilization, and cytochrome c release. To study the molecular regulators for GrB-mediated mitochondrial apoptotic events, we used a CTL-free cytotoxicity system, wherein target cells are treated with purified GrB and replication-deficient adenovirus (Ad). We report here that the Bcl-2 proapoptotic family member, Bak, plays a dominant role in GrB-mediated mitochondrial apoptotic events. A variant of Jurkat cells, deficient in Bak expression, was resistant to GrB/Ad-mediated apoptosis, as determined by lack of membranous phosphatidylserine exposure, lack of DNA breaks, lack of mitochondrial outer membrane permeabilization, and unchanged expression of inner mitochondrial membrane cardiolipin. The resistance of Bak-deficient cells to GrB/Ad cytotoxicity was reversed by transduction of the Bak gene into these cells. The requirement for both Bid and Bak, was further demonstrated in a cell-free system using purified mitochondria and S-100 cytosol. Purified mitochondria from Bid knockout mice, but not from Bax knockout mice, failed to release cytochrome c in response to autologous S-100 and GrB. Also, Bak-deficient mitochondria did not release cytochrome c in response to GrB-treated cytosol unless recombinant Bak protein was added. These results are the first to report a role for Bak in GrB-mediated mitochondrial apoptosis. This study demonstrates that GrB-cleaved Bid, which differs in size and site of cleavage from caspase-8-cleaved Bid, utilizes Bak for cytochrome c release, and therefore, suggests that deficiency in Bak may serve as a mechanism of immune evasion for tumor or viral infected cells.

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GrB-mediated cleavage of Bid is unaltered in Bak-deficient cells and proceeds in the presence of a potent caspase inhibitor. Wild-type and Bak-deficient Jurkat cells were treated with Ad (10 PFU/ml) or GrB (1 μg/ml) for 2 h, or with a combination of GrB and Ad for various time periods as indicated. In A and B, the cells were treated in the absence of caspase inhibitors. In C and D, the cells were pretreated with Z-VAD-FMK (100 μM) for 2 h before exposure to Ad, GrB, or GrB/Ad. A DMSO control for the solvent of Z-VAD-FMK is included in C and D. At the end of the incubation periods, the cells were lysed, resolved by 15% SDS/PAGE, and immunoblotted for the presence of Bid and its cleavage products. Each of the membranes was sequentially stripped and reprobed with anticaspase-3 and anti–β-actin Ab. For the detection of PARP, the same samples were resolved by 7.5% SDS/PAGE. Please note, the presented β-actin serves as a loading control for Bid and caspase-3 (not for PARP).
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fig6: GrB-mediated cleavage of Bid is unaltered in Bak-deficient cells and proceeds in the presence of a potent caspase inhibitor. Wild-type and Bak-deficient Jurkat cells were treated with Ad (10 PFU/ml) or GrB (1 μg/ml) for 2 h, or with a combination of GrB and Ad for various time periods as indicated. In A and B, the cells were treated in the absence of caspase inhibitors. In C and D, the cells were pretreated with Z-VAD-FMK (100 μM) for 2 h before exposure to Ad, GrB, or GrB/Ad. A DMSO control for the solvent of Z-VAD-FMK is included in C and D. At the end of the incubation periods, the cells were lysed, resolved by 15% SDS/PAGE, and immunoblotted for the presence of Bid and its cleavage products. Each of the membranes was sequentially stripped and reprobed with anticaspase-3 and anti–β-actin Ab. For the detection of PARP, the same samples were resolved by 7.5% SDS/PAGE. Please note, the presented β-actin serves as a loading control for Bid and caspase-3 (not for PARP).

Mentions: A Direct, Caspase-independent Cleavage of Bid Is Unaltered in Bak-deficient Cells. Our findings suggest that the block in response of Bak-deficient Jurkat cells to GrB localizes to the cytochrome c release mechanism within the mitochondria of these cells. To ensure that the function of Bid upstream of the mitochondria is unaltered, we examined Bid expression and its translocation to the mitochondria of Bak-deficient cells. We first compared the kinetics of Bid cleavage in wild-type and Bak-deficient Jurkat cells treated with a combination of GrB and Ad. Exposure for 15 min of either wild-type or Bak-deficient cells to GrB/Ad was sufficient for processing of significant portion of full-length Bid present in these cells (Fig. 6 A and B). These results demonstrate that Bid cleavage is unaffected by Bak-deficiency. Under the conditions employed, proteolytic processing occurred more readily with Bid than with DFF45/ICAD (Fig. 2 C), reported to be a direct substrate for GrB. To ensure that the observed cleavage of Bid was directly mediated by GrB, rather than via GrB-activated caspases, cleavage of Bid was also examined in cells pretreated with the pan-caspase inhibitor, Z-VAD-FMK. Similar kinetics and levels of processing were observed in wild-type or Bak-deficient Jurkat cells in the absence or presence of Z-VAD-FMK (Fig. 6 C and D). These results suggest that GrB-mediated cleavage of Bid can proceed in a caspase-independent manner. To control for the activity of caspase-3 in GrB-treated cells, the same membranes (Fig. 6) were stripped and reprobed with anticaspase-3 Ab. In wild-type Jurkat cells, p19 and p17 caspase-3 subunits were detected after 1–2 h of exposure to GrB/Ad (Fig. 6 A). However, in the presence of Z-VAD-FMK, only the p20 caspase-3 subunit, a direct cleavage product of GrB was detected (Fig. 6 B). The absence of p19 and p17 caspase-3 subunits, suggests that the p20 subunit is unable to undergo further processing in the presence of Z-VAD-FMK. Interestingly, in Bak-deficient cells only the p20 caspase-3 subunit was detected in either the presence or the absence of Z-VAD-FMK. To assess the activity of p20 caspase-3, the same samples were examined for the presence of cleavage products of PARP. Cleavage of PARP was detected only in wild-type Jurkat cells, where full processing of caspase-3 was detected (Fig. 6 A). Caspase-3 p20 detected in wild-type cells in the presence of Z-VAD-FMK, or in Bak-deficient cells appears to be relatively inactive as it failed to cleave PARP (Fig. 6 B–D). In contrast to PARP or DFF45/ICAD whose efficient processing required the presence of both GrB and Ad (Fig. 2 C), Bid cleavage was observed also in cells treated with GrB only. These results further demonstrate that Bid is a preferred substrate for GrB, which upon cell internalization cleaves Bid even in the absence of facilitation by an Ad.


Resistance to granzyme B-mediated cytochrome c release in Bak-deficient cells.

Wang GQ, Wieckowski E, Goldstein LA, Gastman BR, Rabinovitz A, Gambotto A, Li S, Fang B, Yin XM, Rabinowich H - J. Exp. Med. (2001)

GrB-mediated cleavage of Bid is unaltered in Bak-deficient cells and proceeds in the presence of a potent caspase inhibitor. Wild-type and Bak-deficient Jurkat cells were treated with Ad (10 PFU/ml) or GrB (1 μg/ml) for 2 h, or with a combination of GrB and Ad for various time periods as indicated. In A and B, the cells were treated in the absence of caspase inhibitors. In C and D, the cells were pretreated with Z-VAD-FMK (100 μM) for 2 h before exposure to Ad, GrB, or GrB/Ad. A DMSO control for the solvent of Z-VAD-FMK is included in C and D. At the end of the incubation periods, the cells were lysed, resolved by 15% SDS/PAGE, and immunoblotted for the presence of Bid and its cleavage products. Each of the membranes was sequentially stripped and reprobed with anticaspase-3 and anti–β-actin Ab. For the detection of PARP, the same samples were resolved by 7.5% SDS/PAGE. Please note, the presented β-actin serves as a loading control for Bid and caspase-3 (not for PARP).
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Related In: Results  -  Collection

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fig6: GrB-mediated cleavage of Bid is unaltered in Bak-deficient cells and proceeds in the presence of a potent caspase inhibitor. Wild-type and Bak-deficient Jurkat cells were treated with Ad (10 PFU/ml) or GrB (1 μg/ml) for 2 h, or with a combination of GrB and Ad for various time periods as indicated. In A and B, the cells were treated in the absence of caspase inhibitors. In C and D, the cells were pretreated with Z-VAD-FMK (100 μM) for 2 h before exposure to Ad, GrB, or GrB/Ad. A DMSO control for the solvent of Z-VAD-FMK is included in C and D. At the end of the incubation periods, the cells were lysed, resolved by 15% SDS/PAGE, and immunoblotted for the presence of Bid and its cleavage products. Each of the membranes was sequentially stripped and reprobed with anticaspase-3 and anti–β-actin Ab. For the detection of PARP, the same samples were resolved by 7.5% SDS/PAGE. Please note, the presented β-actin serves as a loading control for Bid and caspase-3 (not for PARP).
Mentions: A Direct, Caspase-independent Cleavage of Bid Is Unaltered in Bak-deficient Cells. Our findings suggest that the block in response of Bak-deficient Jurkat cells to GrB localizes to the cytochrome c release mechanism within the mitochondria of these cells. To ensure that the function of Bid upstream of the mitochondria is unaltered, we examined Bid expression and its translocation to the mitochondria of Bak-deficient cells. We first compared the kinetics of Bid cleavage in wild-type and Bak-deficient Jurkat cells treated with a combination of GrB and Ad. Exposure for 15 min of either wild-type or Bak-deficient cells to GrB/Ad was sufficient for processing of significant portion of full-length Bid present in these cells (Fig. 6 A and B). These results demonstrate that Bid cleavage is unaffected by Bak-deficiency. Under the conditions employed, proteolytic processing occurred more readily with Bid than with DFF45/ICAD (Fig. 2 C), reported to be a direct substrate for GrB. To ensure that the observed cleavage of Bid was directly mediated by GrB, rather than via GrB-activated caspases, cleavage of Bid was also examined in cells pretreated with the pan-caspase inhibitor, Z-VAD-FMK. Similar kinetics and levels of processing were observed in wild-type or Bak-deficient Jurkat cells in the absence or presence of Z-VAD-FMK (Fig. 6 C and D). These results suggest that GrB-mediated cleavage of Bid can proceed in a caspase-independent manner. To control for the activity of caspase-3 in GrB-treated cells, the same membranes (Fig. 6) were stripped and reprobed with anticaspase-3 Ab. In wild-type Jurkat cells, p19 and p17 caspase-3 subunits were detected after 1–2 h of exposure to GrB/Ad (Fig. 6 A). However, in the presence of Z-VAD-FMK, only the p20 caspase-3 subunit, a direct cleavage product of GrB was detected (Fig. 6 B). The absence of p19 and p17 caspase-3 subunits, suggests that the p20 subunit is unable to undergo further processing in the presence of Z-VAD-FMK. Interestingly, in Bak-deficient cells only the p20 caspase-3 subunit was detected in either the presence or the absence of Z-VAD-FMK. To assess the activity of p20 caspase-3, the same samples were examined for the presence of cleavage products of PARP. Cleavage of PARP was detected only in wild-type Jurkat cells, where full processing of caspase-3 was detected (Fig. 6 A). Caspase-3 p20 detected in wild-type cells in the presence of Z-VAD-FMK, or in Bak-deficient cells appears to be relatively inactive as it failed to cleave PARP (Fig. 6 B–D). In contrast to PARP or DFF45/ICAD whose efficient processing required the presence of both GrB and Ad (Fig. 2 C), Bid cleavage was observed also in cells treated with GrB only. These results further demonstrate that Bid is a preferred substrate for GrB, which upon cell internalization cleaves Bid even in the absence of facilitation by an Ad.

Bottom Line: Purified mitochondria from Bid knockout mice, but not from Bax knockout mice, failed to release cytochrome c in response to autologous S-100 and GrB.Also, Bak-deficient mitochondria did not release cytochrome c in response to GrB-treated cytosol unless recombinant Bak protein was added.These results are the first to report a role for Bak in GrB-mediated mitochondrial apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, The University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, USA.

ABSTRACT
Granzyme B (GrB), a serine protease with substrate specificity similar to the caspase family, is a major component of granule-mediated cytotoxicity of T lymphocytes. Although GrB can directly activate caspases, it induces apoptosis predominantly via Bid cleavage, mitochondrial outer membrane permeabilization, and cytochrome c release. To study the molecular regulators for GrB-mediated mitochondrial apoptotic events, we used a CTL-free cytotoxicity system, wherein target cells are treated with purified GrB and replication-deficient adenovirus (Ad). We report here that the Bcl-2 proapoptotic family member, Bak, plays a dominant role in GrB-mediated mitochondrial apoptotic events. A variant of Jurkat cells, deficient in Bak expression, was resistant to GrB/Ad-mediated apoptosis, as determined by lack of membranous phosphatidylserine exposure, lack of DNA breaks, lack of mitochondrial outer membrane permeabilization, and unchanged expression of inner mitochondrial membrane cardiolipin. The resistance of Bak-deficient cells to GrB/Ad cytotoxicity was reversed by transduction of the Bak gene into these cells. The requirement for both Bid and Bak, was further demonstrated in a cell-free system using purified mitochondria and S-100 cytosol. Purified mitochondria from Bid knockout mice, but not from Bax knockout mice, failed to release cytochrome c in response to autologous S-100 and GrB. Also, Bak-deficient mitochondria did not release cytochrome c in response to GrB-treated cytosol unless recombinant Bak protein was added. These results are the first to report a role for Bak in GrB-mediated mitochondrial apoptosis. This study demonstrates that GrB-cleaved Bid, which differs in size and site of cleavage from caspase-8-cleaved Bid, utilizes Bak for cytochrome c release, and therefore, suggests that deficiency in Bak may serve as a mechanism of immune evasion for tumor or viral infected cells.

Show MeSH
Related in: MedlinePlus