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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 and its translocation to the mitochondria in extracts of Bak-deficient cells. (A) Jurkat cell lines, including wild-type, Bak-deficient, Neo-, or Bcl-XL–transduced cells were Dounce homogenized, and then treated with GrB (1 μg/ml) for 1 h at 30°C. The extracts were then separated into S-100 cytosol and mitochondria fractions. Loss of full-length Bid was detected in cytosols of all Jurkat cell variants treated with GrB. The anti-BID Ab used for this blot detects only full length Bid. (B) GrB-mediated cleavage of Bid proceeds in the presence of Z-VAD-FMK. Extracts of Bak-deficient Jurkat cells were incubated with Z-VAD-FMK (100 μM) for 20 min before the addition of GrB (1 μg/m) for 1 h at 30°C. The extracts were then fractionated into S-100 and mitochondrial fractions, which were assessed by immunoblotting and sequential probing for the presence of Bid, caspase-3, and β-actin. (C) Translocation of GrB-cleaved Bid to the mitochondria of Bak-deficient Jurkat cells. The mitochondrial fraction of extracts of Bak-deficient cells treated with GrB in the presence or absence of Z-VAD-FMK was assessed by immunoblotting for the presence of tBid. The membrane was stripped and reprobed with anti-Cox IV mAb, as a mitochondrial marker and to demonstrate equal loading.
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fig7: GrB-mediated cleavage of Bid and its translocation to the mitochondria in extracts of Bak-deficient cells. (A) Jurkat cell lines, including wild-type, Bak-deficient, Neo-, or Bcl-XL–transduced cells were Dounce homogenized, and then treated with GrB (1 μg/ml) for 1 h at 30°C. The extracts were then separated into S-100 cytosol and mitochondria fractions. Loss of full-length Bid was detected in cytosols of all Jurkat cell variants treated with GrB. The anti-BID Ab used for this blot detects only full length Bid. (B) GrB-mediated cleavage of Bid proceeds in the presence of Z-VAD-FMK. Extracts of Bak-deficient Jurkat cells were incubated with Z-VAD-FMK (100 μM) for 20 min before the addition of GrB (1 μg/m) for 1 h at 30°C. The extracts were then fractionated into S-100 and mitochondrial fractions, which were assessed by immunoblotting and sequential probing for the presence of Bid, caspase-3, and β-actin. (C) Translocation of GrB-cleaved Bid to the mitochondria of Bak-deficient Jurkat cells. The mitochondrial fraction of extracts of Bak-deficient cells treated with GrB in the presence or absence of Z-VAD-FMK was assessed by immunoblotting for the presence of tBid. The membrane was stripped and reprobed with anti-Cox IV mAb, as a mitochondrial marker and to demonstrate equal loading.

Mentions: Next, we treated Dounce homogenized extracts of wild-type, Bak-deficient, Neo, and BCl-XL transduced Jurkat cells with GrB (1 μg/ml, 1 h, 30°C). The extracts were then separated into cytosol S-100 and mitochondria fractions. GrB treatment resulted in complete processing of Bid, as full-length Bid was not detected in the cytosols obtained from GrB-treated extracts of all the variants of Jurkat cells examined (Fig. 7 A). Thus, Bak-deficiency or overexpression of Bcl-XL do not affect GrB-mediated processing of Bid. Interestingly, full processing of caspase-3 was detected in the cytosol fraction of Bak-deficient cell extracts treated with GrB (Fig. 7 B). To ensure that Bid cleavage was not mediated by GrB-activated caspase-3, the extracts were treated with Z-VAD-FMK (100 μM, 20 min) before the addition of GrB. Similar processing of Bid was also observed in the presence of the caspase inhibitor (Fig. 7 B). In the presence of Z-VAD-FMK, only p20 caspase-3 was detected in extracts of Bak-deficient cells, whereas both p19 and p17 caspase-3 subunits were detected in the absence of the inhibitor. Thus, in contrast to GrB-treated Bak-deficient cells, in treated extracts full processing of caspase-3 was observed. It appears that the application of GrB directly into the extracts results in a higher dose than would have been internalized via the cell membrane. Such an augmented dose of GrB seems to overcome the lack of mitochondrial contribution to caspase-3 processing in Bak-deficient cells. The cleavage product of Bid was detected in the mitochondria obtained from extracts of Bak-deficient cells treated in the presence or the absence of Z-VAD-FMK (Fig. 7 C). These results suggest that GrB-cleaved Bid targets the mitochondria within the cell extract. Together, the results shown in Figs. 6 and 7 suggest that the block in GrB-mediated cytochrome c release in Bak-deficient cells is downstream of GrB-cleaved Bid translocation to the mitochondria.


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 and its translocation to the mitochondria in extracts of Bak-deficient cells. (A) Jurkat cell lines, including wild-type, Bak-deficient, Neo-, or Bcl-XL–transduced cells were Dounce homogenized, and then treated with GrB (1 μg/ml) for 1 h at 30°C. The extracts were then separated into S-100 cytosol and mitochondria fractions. Loss of full-length Bid was detected in cytosols of all Jurkat cell variants treated with GrB. The anti-BID Ab used for this blot detects only full length Bid. (B) GrB-mediated cleavage of Bid proceeds in the presence of Z-VAD-FMK. Extracts of Bak-deficient Jurkat cells were incubated with Z-VAD-FMK (100 μM) for 20 min before the addition of GrB (1 μg/m) for 1 h at 30°C. The extracts were then fractionated into S-100 and mitochondrial fractions, which were assessed by immunoblotting and sequential probing for the presence of Bid, caspase-3, and β-actin. (C) Translocation of GrB-cleaved Bid to the mitochondria of Bak-deficient Jurkat cells. The mitochondrial fraction of extracts of Bak-deficient cells treated with GrB in the presence or absence of Z-VAD-FMK was assessed by immunoblotting for the presence of tBid. The membrane was stripped and reprobed with anti-Cox IV mAb, as a mitochondrial marker and to demonstrate equal loading.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2195982&req=5

fig7: GrB-mediated cleavage of Bid and its translocation to the mitochondria in extracts of Bak-deficient cells. (A) Jurkat cell lines, including wild-type, Bak-deficient, Neo-, or Bcl-XL–transduced cells were Dounce homogenized, and then treated with GrB (1 μg/ml) for 1 h at 30°C. The extracts were then separated into S-100 cytosol and mitochondria fractions. Loss of full-length Bid was detected in cytosols of all Jurkat cell variants treated with GrB. The anti-BID Ab used for this blot detects only full length Bid. (B) GrB-mediated cleavage of Bid proceeds in the presence of Z-VAD-FMK. Extracts of Bak-deficient Jurkat cells were incubated with Z-VAD-FMK (100 μM) for 20 min before the addition of GrB (1 μg/m) for 1 h at 30°C. The extracts were then fractionated into S-100 and mitochondrial fractions, which were assessed by immunoblotting and sequential probing for the presence of Bid, caspase-3, and β-actin. (C) Translocation of GrB-cleaved Bid to the mitochondria of Bak-deficient Jurkat cells. The mitochondrial fraction of extracts of Bak-deficient cells treated with GrB in the presence or absence of Z-VAD-FMK was assessed by immunoblotting for the presence of tBid. The membrane was stripped and reprobed with anti-Cox IV mAb, as a mitochondrial marker and to demonstrate equal loading.
Mentions: Next, we treated Dounce homogenized extracts of wild-type, Bak-deficient, Neo, and BCl-XL transduced Jurkat cells with GrB (1 μg/ml, 1 h, 30°C). The extracts were then separated into cytosol S-100 and mitochondria fractions. GrB treatment resulted in complete processing of Bid, as full-length Bid was not detected in the cytosols obtained from GrB-treated extracts of all the variants of Jurkat cells examined (Fig. 7 A). Thus, Bak-deficiency or overexpression of Bcl-XL do not affect GrB-mediated processing of Bid. Interestingly, full processing of caspase-3 was detected in the cytosol fraction of Bak-deficient cell extracts treated with GrB (Fig. 7 B). To ensure that Bid cleavage was not mediated by GrB-activated caspase-3, the extracts were treated with Z-VAD-FMK (100 μM, 20 min) before the addition of GrB. Similar processing of Bid was also observed in the presence of the caspase inhibitor (Fig. 7 B). In the presence of Z-VAD-FMK, only p20 caspase-3 was detected in extracts of Bak-deficient cells, whereas both p19 and p17 caspase-3 subunits were detected in the absence of the inhibitor. Thus, in contrast to GrB-treated Bak-deficient cells, in treated extracts full processing of caspase-3 was observed. It appears that the application of GrB directly into the extracts results in a higher dose than would have been internalized via the cell membrane. Such an augmented dose of GrB seems to overcome the lack of mitochondrial contribution to caspase-3 processing in Bak-deficient cells. The cleavage product of Bid was detected in the mitochondria obtained from extracts of Bak-deficient cells treated in the presence or the absence of Z-VAD-FMK (Fig. 7 C). These results suggest that GrB-cleaved Bid targets the mitochondria within the cell extract. Together, the results shown in Figs. 6 and 7 suggest that the block in GrB-mediated cytochrome c release in Bak-deficient cells is downstream of GrB-cleaved Bid translocation to the mitochondria.

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