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

Gui Qiang Wang, Eva Wieckowski, Leslie A. Goldstein, Brian R. Gastman, Asaf Rabinovitz, Andrea Gambotto, Shuchen Li, Bingliang Fang, Xiao-Ming Yin, Hannah Rabinowich

Research output: Contribution to journalArticle

62 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)1325-1337
Number of pages13
JournalJournal of Experimental Medicine
Volume194
Issue number9
DOIs
StatePublished - 2001
Externally publishedYes

Fingerprint

Granzymes
Cytochromes c
Mitochondrial Membranes
Adenoviridae
Mitochondria
Apoptosis
Caspases
Knockout Mice
Cytosol
bcl-2 Homologous Antagonist-Killer Protein
Tumor Escape
Cardiolipins
DNA Breaks
Jurkat Cells
Cell-Free System
Caspase 8
Phosphatidylserines
Serine Proteases
Substrate Specificity
Recombinant Proteins

Keywords

  • Apoptosis
  • Bak
  • Bid
  • Cytochrome c
  • Granzyme B
  • Mitochondria

ASJC Scopus subject areas

  • Immunology

Cite this

Wang, G. Q., Wieckowski, E., Goldstein, L. A., Gastman, B. R., Rabinovitz, A., Gambotto, A., ... Rabinowich, H. (2001). Resistance to granzyme B-mediated cytochrome c release in Bak-deficient cells. Journal of Experimental Medicine, 194(9), 1325-1337. https://doi.org/10.1084/jem.194.9.1325

Resistance to granzyme B-mediated cytochrome c release in Bak-deficient cells. / Wang, Gui Qiang; Wieckowski, Eva; Goldstein, Leslie A.; Gastman, Brian R.; Rabinovitz, Asaf; Gambotto, Andrea; Li, Shuchen; Fang, Bingliang; Yin, Xiao-Ming; Rabinowich, Hannah.

In: Journal of Experimental Medicine, Vol. 194, No. 9, 2001, p. 1325-1337.

Research output: Contribution to journalArticle

Wang, GQ, Wieckowski, E, Goldstein, LA, Gastman, BR, Rabinovitz, A, Gambotto, A, Li, S, Fang, B, Yin, X-M & Rabinowich, H 2001, 'Resistance to granzyme B-mediated cytochrome c release in Bak-deficient cells', Journal of Experimental Medicine, vol. 194, no. 9, pp. 1325-1337. https://doi.org/10.1084/jem.194.9.1325
Wang GQ, Wieckowski E, Goldstein LA, Gastman BR, Rabinovitz A, Gambotto A et al. Resistance to granzyme B-mediated cytochrome c release in Bak-deficient cells. Journal of Experimental Medicine. 2001;194(9):1325-1337. https://doi.org/10.1084/jem.194.9.1325
Wang, Gui Qiang ; Wieckowski, Eva ; Goldstein, Leslie A. ; Gastman, Brian R. ; Rabinovitz, Asaf ; Gambotto, Andrea ; Li, Shuchen ; Fang, Bingliang ; Yin, Xiao-Ming ; Rabinowich, Hannah. / Resistance to granzyme B-mediated cytochrome c release in Bak-deficient cells. In: Journal of Experimental Medicine. 2001 ; Vol. 194, No. 9. pp. 1325-1337.
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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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