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

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.

• Authors: G Q Wang, E Wieckowski, L A Goldstein, B R Gastman, A Rabinovitz, A Gambotto, S Li, B Fang, X M Yin, H Rabinowich
• Journal: The Journal of experimental medicine (J Exp Med) Vol. 194 Issue 9 Pg. 1325-37 (Nov 05 2001) ISSN: 0022-1007 [Print] United States
• PMID: 11696597 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S., Research Support, U.S. Gov't, P.H.S.)

Chemical References

• BAK1 protein, human
• BAX protein, human
• BH3 Interacting Domain Death Agonist Protein
• BID protein, human
• Carrier Proteins
• Cytochrome c Group
• Membrane Proteins
• Proto-Oncogene Proteins
• Proto-Oncogene Proteins c-bcl-2
• bcl-2 Homologous Antagonist-Killer Protein
• bcl-2-Associated X Protein
• GZMB protein, human
• Granzymes
• Serine Endopeptidases

Topics

• Adenoviridae
• Apoptosis
• BH3 Interacting Domain Death Agonist Protein
• Carrier Proteins (genetics, metabolism)
• Cytochrome c Group (metabolism)
• Genetic Vectors
• Granzymes
• Humans
• Jurkat Cells
• Membrane Proteins (metabolism)
• Mitochondria (metabolism)
• Proto-Oncogene Proteins (metabolism)
• Proto-Oncogene Proteins c-bcl-2 (metabolism)
• Serine Endopeptidases (metabolism)
• bcl-2 Homologous Antagonist-Killer Protein
• bcl-2-Associated X Protein