Granzyme B–Mediated Cytochrome C Release Is Regulated by the Bcl-2 Family Members Bid and Bax

Abstract

Cytotoxic T lymphocytes (CTLs) destroy target cells through a mechanism involving the exocytosis of cytolytic granule components including granzyme B (grB) and perforin, which have been shown to induce apoptosis through caspase activation. However, grB has also been linked with caspase-independent disruption of mitochondrial function. We show here that cytochrome c release requires the direct proteolytic cleavage of Bid by grB to generate a 14-kD grB-truncated product (gtBid) that translocates to mitochondria. In turn, gtBid recruits Bax to mitochondria through a caspase-independent mechanism where it becomes integrated into the membrane and induces cytochrome c release. Our results provide evidence for a new pathway by which CTLs inflict damage and explain the caspase-independent mechanism of mitochondrial dysfunction.

Granzyme F induces a novel death pathway characterized by Bid-independent cytochrome c release without caspase activation

“…Our observations are consistent with previous reports. 25,26 We further showed that Bid was not processed to tBid in GzmF-loaded cells, whereas Bid was activated to tBid by GzmB treatment (Figure 6b). Caspase-3 was probed as a positive control for GzmB.…”

“…Cyt c release induced by GzmB or GzmK is through the processing of Bid to tBid. 22,25 However, GzmF cannot activate Bid to tBid in the induction of death. Cyt c was still released in Bid À/À and Bax/Bak DKO MEF cells with GzmF treatment but was not released with GzmB treatment.…”

Death by a Thousand Cuts: Granzyme Pathways of Programmed Cell Death

“…It induces target cell apoptosis by activating the caspases, particularly the key executioner caspase, caspase-3 (143,144) (Figure 3). Human GzmB, but not the mouse enzyme, also activates cell death by directly cleaving the key caspase substrates, bid and ICAD, to activate the same mitochondrial and DNA damage pathways, respectively, as the caspases (53,(145)(146)(147)(148)(149)(150)(151) (Figure 3). As a consequence, caspase inhibitors have little effect on human GzmB-mediated cell death and DNA fragmentation, whereas the same inhibitors significantly block the action of the mouse enzyme.…”

A quarter century of granzymes

“…18,28 These experiments demonstrated that human GrB proteolytically activated Bid at aspartate 75 , resulting in its translocation to the mitochondrial outer membrane, and the facilitation of oligomerization of Bax/Bak molecules. [29][30][31] It was initially thought that this resulted in the release of cytochrome C and apoptosome formation. This proved not to be the case, but rather involved inhibitor of apoptosis proteins (IAPs) that are potent antagonists of caspase activity.…”