Granzyme B Short-Circuits the Need for Caspase 8 Activity during Granule-Mediated Cytotoxic T-Lymphocyte Killing by Directly Cleaving Bid

Abstract

Cytotoxic T lymphocytes (CTL) can trigger an apoptotic signal through the Fas receptor or by the exocytosis of granzyme B and perforin. Caspase activation is an important component of both pathways. Granzyme B, a serine proteinase contained in granules, has been shown to proteolytically process and activate members of the caspase family in vitro. In order to gain an understanding of the contributions of caspases 8 and 3 during granule-induced apoptosis in intact cells, we have used target cells that either stably express the rabbitpox virus-encoded caspase inhibitor SPI-2 or are devoid of caspase 3. The overexpression of SPI-2 in target cells significantly inhibited DNA fragmentation, phosphatidylserine externalization, and mitochondrial disruption during Fas-mediated cell death. In contrast, SPI-2 expression in target cells provided no protection against granzyme-mediated apoptosis, mitochondrial collapse, or cytolysis, leading us to conclude that SPI-2-inhibited caspases are not an essential requirement for the granzyme pathway. Caspase 3-deficient MCF-7 cells were found to be resistant to CTL-mediated DNA fragmentation but not to CTL-mediated cytolysis and loss of the mitochondrial inner membrane potential. Furthermore, we demonstrate that granzyme B directly cleaves the proapoptotic molecule Bid, bypassing the need for caspase 8 activation of Bid. These results provide evidence for a two-pronged strategy for mediating target cell destruction and provide evidence of a direct link between granzyme B activity, Bid cleavage, and caspase 3 activation in whole cells.

Mouse Cytotoxic T Cell-derived Granzyme B Activates the Mitochondrial Cell Death Pathway in a Bim-dependent Fashion

“…gzmB may directly process procaspase-3 (10,11). Alternatively, gzmB engages the mitochondrial death pathway by converting Bid to tBid (11)(12)(13)(14)(15)(16). In turn, tBid oligomerizes Bak and Bax, which permeabilize mitochondria.…”

HSV and Glycoprotein J Inhibit Caspase Activation and Apoptosis Induced by Granzyme B or Fas

“…One mechanism by which grB may initiate apoptosis is via the cleavage of Bid, which leads, in turn, to mitochondrial disruption (38 -40). grB has also been reported to cleave some cell death substrates directly, such as inhibitor of caspase-activated DNase (ICAD) (40,41). Cleavage of ICAD leads to release of caspaseactivated DNase, resulting in DNA fragmentation of the target cell.…”

Caspase substrates

“…This site appears accessible, as it is cleaved by granzyme B, with essentially identical gain-of-function properties with respect to promoting apoptosis. 51 Possible exosite-mediated interactions preferentially guide the Asp 60 kGly site of Bid into the active site cleft of caspase-8, or conversely steer the caspase-away from the Asp 75 kSer site. Recombinant variants of Bid with mutated or swapped cleavage sites might help clarify this point.…”