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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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Deficient expression of Bak in a clonal Jurkat cell line. (A) Wild-type or the variant Jurkat cell line, Bak−, were incubated in 1% NP-40 lysis buffer for 30 min at 4°C. The resultant lysates which contained both cytoplasm and mitochondria, were resolved by SDS/PAGE and assessed by immunoblotting for the presence of Bak. Four different anti–human Bak Ab were used for blotting. The membranes were stripped and reprobed for β-actin to demonstrate equal loading. (B) Expression of Bak in mitochondria of wild-type, but not in mitochondria of Bak-deficient Jurkat cells. Expression of Bak was examined in cytosol (S-100), purified mitochondria, or purified mitochondria treated with alkali to remove nonspecifically attached proteins. These cell fractions were resolved by SDS/PAGE and immunoblotted sequentially by Bak-specific Ab-1 and Ab-2. After additional stripping, the membranes were probed with anti–Cox IV Ab, as a marker for mitochondrial fractions, and with anti–β-actin as a marker for cytosolic proteins.
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fig1: Deficient expression of Bak in a clonal Jurkat cell line. (A) Wild-type or the variant Jurkat cell line, Bak−, were incubated in 1% NP-40 lysis buffer for 30 min at 4°C. The resultant lysates which contained both cytoplasm and mitochondria, were resolved by SDS/PAGE and assessed by immunoblotting for the presence of Bak. Four different anti–human Bak Ab were used for blotting. The membranes were stripped and reprobed for β-actin to demonstrate equal loading. (B) Expression of Bak in mitochondria of wild-type, but not in mitochondria of Bak-deficient Jurkat cells. Expression of Bak was examined in cytosol (S-100), purified mitochondria, or purified mitochondria treated with alkali to remove nonspecifically attached proteins. These cell fractions were resolved by SDS/PAGE and immunoblotted sequentially by Bak-specific Ab-1 and Ab-2. After additional stripping, the membranes were probed with anti–Cox IV Ab, as a marker for mitochondrial fractions, and with anti–β-actin as a marker for cytosolic proteins.

Mentions: A clonal cell line isolated from the ATCC wild-type Jurkat cell line was found to be Bak-deficient. The deficiency was determined by immunoblotting of whole cell lysates by four different anti-Bak Abs (Fig. 1 A). To further analyze the expression of Bak, lysates of wild-type or the clonal Jurkat cells were fractionated to yield S-100 cytosol or purified mitochondria. These protein fractions were assessed for the expression of Bak by Western blot analyses. Whereas expression of Bak was detected in mitochondria of wild-type Jurkat cells, only minor expression of Bak was observed in a similar quantity of purified mitochondria from Bak-deficient cells (Fig. 1 B). To assess the mitochondrial localization of Bak, purified mitochondria from either wild-type or Bak-deficient cells were treated with alkali to remove proteins nonspecifically attached to the mitochondria (34). In wild-type Jurkat cells, Bak was found to be a mitochondrial integral membrane protein, as it was detected in the pellet of alkali-treated mitochondria (Fig. 1 B). Levels of protein expression of other Bcl-2 family members in Bak-deficient cells, including Bcl-2, BCl-XL, or Bax, were similar to those of the wild-type cell line (data not shown).


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)

Deficient expression of Bak in a clonal Jurkat cell line. (A) Wild-type or the variant Jurkat cell line, Bak−, were incubated in 1% NP-40 lysis buffer for 30 min at 4°C. The resultant lysates which contained both cytoplasm and mitochondria, were resolved by SDS/PAGE and assessed by immunoblotting for the presence of Bak. Four different anti–human Bak Ab were used for blotting. The membranes were stripped and reprobed for β-actin to demonstrate equal loading. (B) Expression of Bak in mitochondria of wild-type, but not in mitochondria of Bak-deficient Jurkat cells. Expression of Bak was examined in cytosol (S-100), purified mitochondria, or purified mitochondria treated with alkali to remove nonspecifically attached proteins. These cell fractions were resolved by SDS/PAGE and immunoblotted sequentially by Bak-specific Ab-1 and Ab-2. After additional stripping, the membranes were probed with anti–Cox IV Ab, as a marker for mitochondrial fractions, and with anti–β-actin as a marker for cytosolic proteins.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Deficient expression of Bak in a clonal Jurkat cell line. (A) Wild-type or the variant Jurkat cell line, Bak−, were incubated in 1% NP-40 lysis buffer for 30 min at 4°C. The resultant lysates which contained both cytoplasm and mitochondria, were resolved by SDS/PAGE and assessed by immunoblotting for the presence of Bak. Four different anti–human Bak Ab were used for blotting. The membranes were stripped and reprobed for β-actin to demonstrate equal loading. (B) Expression of Bak in mitochondria of wild-type, but not in mitochondria of Bak-deficient Jurkat cells. Expression of Bak was examined in cytosol (S-100), purified mitochondria, or purified mitochondria treated with alkali to remove nonspecifically attached proteins. These cell fractions were resolved by SDS/PAGE and immunoblotted sequentially by Bak-specific Ab-1 and Ab-2. After additional stripping, the membranes were probed with anti–Cox IV Ab, as a marker for mitochondrial fractions, and with anti–β-actin as a marker for cytosolic proteins.
Mentions: A clonal cell line isolated from the ATCC wild-type Jurkat cell line was found to be Bak-deficient. The deficiency was determined by immunoblotting of whole cell lysates by four different anti-Bak Abs (Fig. 1 A). To further analyze the expression of Bak, lysates of wild-type or the clonal Jurkat cells were fractionated to yield S-100 cytosol or purified mitochondria. These protein fractions were assessed for the expression of Bak by Western blot analyses. Whereas expression of Bak was detected in mitochondria of wild-type Jurkat cells, only minor expression of Bak was observed in a similar quantity of purified mitochondria from Bak-deficient cells (Fig. 1 B). To assess the mitochondrial localization of Bak, purified mitochondria from either wild-type or Bak-deficient cells were treated with alkali to remove proteins nonspecifically attached to the mitochondria (34). In wild-type Jurkat cells, Bak was found to be a mitochondrial integral membrane protein, as it was detected in the pellet of alkali-treated mitochondria (Fig. 1 B). Levels of protein expression of other Bcl-2 family members in Bak-deficient cells, including Bcl-2, BCl-XL, or Bax, were similar to those of the wild-type cell line (data not shown).

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