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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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Abrogation of cytochrome c release in Bak-deficient mitochondria in response to recombinant GrB-cleaved Bid. (A) Purified mitochondria (100 μg) obtained from wild-type or Bak-deficient Jurkat cells were treated with his-tBid (Ser76-Asp195) at the indicated concentrations for 30 min at 30°C. The supernatants (Mit-Sup) were boiled in reducing Laemmli buffer, and assessed by immunoblotting for the presence of cytochrome c. Lysed mitochondria from each of the two cell lines tested served as positive controls. (B) Release of cytochrome c by mitochondria from either wild-type or Bak-deficient Jurkat cells is induced by recombinant Bak and inhibited by Bcl-XL. Purified mitochondria from wild-type, Bak-deficient, and Neo- or Bcl-XL–transduced cells were incubated with GST-BakΔC at the indicated concentrations for 30 min at 30°C. The presence of cytochrome c in the supernatants was assessed as described previously.
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fig4: Abrogation of cytochrome c release in Bak-deficient mitochondria in response to recombinant GrB-cleaved Bid. (A) Purified mitochondria (100 μg) obtained from wild-type or Bak-deficient Jurkat cells were treated with his-tBid (Ser76-Asp195) at the indicated concentrations for 30 min at 30°C. The supernatants (Mit-Sup) were boiled in reducing Laemmli buffer, and assessed by immunoblotting for the presence of cytochrome c. Lysed mitochondria from each of the two cell lines tested served as positive controls. (B) Release of cytochrome c by mitochondria from either wild-type or Bak-deficient Jurkat cells is induced by recombinant Bak and inhibited by Bcl-XL. Purified mitochondria from wild-type, Bak-deficient, and Neo- or Bcl-XL–transduced cells were incubated with GST-BakΔC at the indicated concentrations for 30 min at 30°C. The presence of cytochrome c in the supernatants was assessed as described previously.

Mentions: GrB-mediated cytochrome c release has recently been reported to be regulated by the Bcl-2 family member, Bid (15–17). The initiation of apoptosis by GrB has been reported to require direct cleavage of Bid, and the translocation of the cleaved product to the mitochondria, where it induces release of cytochrome c. Caspase-8–cleaved Bid has been reported to require mitochondrial Bak for cytochrome c release (25). To further explore the possibility that the block in the response of our Bak-deficient cells to GrB cytotoxicity was related to the requirement of mitochondrial Bak for Bid function, we examined the ability of a recombinant tBid that corresponds in amino acid sequence to GrB-cleaved Bid (Ser76 to Asp195) to induce cytochrome c release from purified mitochondria. In contrast to mitochondria from wild-type Jurkat cells, mitochondria obtained from Bak-deficient cells that were treated with Ser76 to Asp195 recombinant tBid did not release cytochrome c (Fig. 4 A). However, release of cytochrome c was observed when purified mitochondria from either wild-type or Bak-deficient cells were treated with recombinant Bak (GST-BakΔC, Fig. 4 B). Cytochrome c release in response to recombinant Bak was inhibited in mitochondria purified from Jurkat cells overexpressing Bcl- XL (Fig. 4 B). These results suggest that the resistance of Bak-deficient Jurkat cells to GrB-mediated apoptosis is associated with the abrogation of cytochrome c release by these mitochondria in response to GrB-cleaved Bid.


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)

Abrogation of cytochrome c release in Bak-deficient mitochondria in response to recombinant GrB-cleaved Bid. (A) Purified mitochondria (100 μg) obtained from wild-type or Bak-deficient Jurkat cells were treated with his-tBid (Ser76-Asp195) at the indicated concentrations for 30 min at 30°C. The supernatants (Mit-Sup) were boiled in reducing Laemmli buffer, and assessed by immunoblotting for the presence of cytochrome c. Lysed mitochondria from each of the two cell lines tested served as positive controls. (B) Release of cytochrome c by mitochondria from either wild-type or Bak-deficient Jurkat cells is induced by recombinant Bak and inhibited by Bcl-XL. Purified mitochondria from wild-type, Bak-deficient, and Neo- or Bcl-XL–transduced cells were incubated with GST-BakΔC at the indicated concentrations for 30 min at 30°C. The presence of cytochrome c in the supernatants was assessed as described previously.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195982&req=5

fig4: Abrogation of cytochrome c release in Bak-deficient mitochondria in response to recombinant GrB-cleaved Bid. (A) Purified mitochondria (100 μg) obtained from wild-type or Bak-deficient Jurkat cells were treated with his-tBid (Ser76-Asp195) at the indicated concentrations for 30 min at 30°C. The supernatants (Mit-Sup) were boiled in reducing Laemmli buffer, and assessed by immunoblotting for the presence of cytochrome c. Lysed mitochondria from each of the two cell lines tested served as positive controls. (B) Release of cytochrome c by mitochondria from either wild-type or Bak-deficient Jurkat cells is induced by recombinant Bak and inhibited by Bcl-XL. Purified mitochondria from wild-type, Bak-deficient, and Neo- or Bcl-XL–transduced cells were incubated with GST-BakΔC at the indicated concentrations for 30 min at 30°C. The presence of cytochrome c in the supernatants was assessed as described previously.
Mentions: GrB-mediated cytochrome c release has recently been reported to be regulated by the Bcl-2 family member, Bid (15–17). The initiation of apoptosis by GrB has been reported to require direct cleavage of Bid, and the translocation of the cleaved product to the mitochondria, where it induces release of cytochrome c. Caspase-8–cleaved Bid has been reported to require mitochondrial Bak for cytochrome c release (25). To further explore the possibility that the block in the response of our Bak-deficient cells to GrB cytotoxicity was related to the requirement of mitochondrial Bak for Bid function, we examined the ability of a recombinant tBid that corresponds in amino acid sequence to GrB-cleaved Bid (Ser76 to Asp195) to induce cytochrome c release from purified mitochondria. In contrast to mitochondria from wild-type Jurkat cells, mitochondria obtained from Bak-deficient cells that were treated with Ser76 to Asp195 recombinant tBid did not release cytochrome c (Fig. 4 A). However, release of cytochrome c was observed when purified mitochondria from either wild-type or Bak-deficient cells were treated with recombinant Bak (GST-BakΔC, Fig. 4 B). Cytochrome c release in response to recombinant Bak was inhibited in mitochondria purified from Jurkat cells overexpressing Bcl- XL (Fig. 4 B). These results suggest that the resistance of Bak-deficient Jurkat cells to GrB-mediated apoptosis is associated with the abrogation of cytochrome c release by these mitochondria in response to GrB-cleaved Bid.

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