Granzyme B activates procaspase-3 which signals a mitochondrial amplification loop for maximal apoptosis

Abstract

Granzyme B (GrB), acting similar to an apical caspase, efficiently activates a proteolytic cascade after intracellular delivery by perforin. Studies here were designed to learn whether the physiologic effector, GrB–serglycin, initiates apoptosis primarily through caspase-3 or through BH3-only proteins with subsequent mitochondrial permeabilization and apoptosis. Using four separate cell lines that were either genetically lacking the zymogen or rendered deficient in active caspase-3, we measured apoptotic indices within whole cells (active caspase-3, mitochondrial depolarization [ΔΨm] and TUNEL). Adhering to these conditions, the following were observed in targets after GrB delivery: (a) procaspase-3–deficient cells fail to display a reduced ΔΨm and DNA fragmentation; (b) Bax/Bak is required for optimal ΔΨm reduction, caspase-3 activation, and DNA fragmentation, whereas BID cleavage is undetected by immunoblot; (c) Bcl-2 inhibits GrB-mediated apoptosis (reduced ΔΨm and TUNEL reactivity) by blocking oligomerization of caspase-3; and (d) in procaspase-3–deficient cells a mitochondrial-independent pathway was identified which involved procaspase-7 activation, PARP cleavage, and nuclear condensation. The data therefore support the existence of a fully implemented apoptotic pathway initiated by GrB, propagated by caspase-3, and perpetuated by a mitochondrial amplification loop but also emphasize the presence of an ancillary caspase-dependent, mitochondria-independent pathway.

Granzyme B-induced cell death exerted by ex vivo CTL: discriminating requirements for cell death and some of its signs

“…These data indicate that Bid and Bak/Bax are critical for gzmB-induced cytochrome c release, but not for ROS production, suppression of DC m , perturbation of the plasma membrane and, most surprisingly, cell death. Our finding supports some previous work where purified gzmB was used to measure plasma and mitochondrial membrane perturbation, 7,21 but apparently contradicts those data obtained with Bid-deficient cells where resistance to gzmBmediated cell death was shown. 23 The role of Bid and Caspase-3 and -7 are activated by gzmB þ CTL, independently of Bid, Bax and Bak but neither of the two enzymes are required for gzmB þ CTL-mediated target cell death.…”

“…17 Moreover, for the concomitant induction of AIF and endoG release from mitochondria by gzmB and a sustained DC m loss, additional activation of caspases seems to be required. 12 Finally, others have suggested that neither Bid nor Bak and Bax are necessary for initiating mitochondrial perturbation, 7,21 but that caspases 21 or other cytosolic factors 7 are needed. Recent evidence from caspase-3/-7 double knockout cells has lent support to the notion that effector caspases can be critical mediators of mitochondrial events during apoptosis, 22 but it has remained enigmatic to what extent they play a role in gzmB-initiated mitochondrial signaling leading to cell death.…”

“…Recent evidence from caspase-3/-7 double knockout cells has lent support to the notion that effector caspases can be critical mediators of mitochondrial events during apoptosis, 22 but it has remained enigmatic to what extent they play a role in gzmB-initiated mitochondrial signaling leading to cell death. 17,21 The major hurdle for dissecting the molecular actions of gzmB during CTL-mediated cell death has been the use of artificial in vitro or semi-intact cellular systems with purified gzmB, gzmA and perf preparations rather than cell-based CTL assays. Here, we have used physiological conditions namely ex vivo virus-immune CTLs that express perf and gzmB, but not gzmA (gzmB þ CTL) or any other functionally active gzm to reevaluate the pro-apoptotic potential of gzmB under more physiological conditions.…”

Granzyme B-mediated apoptosis – the elephant and the blind men?

“…Relying on measurement of apoptotic indices (active caspase-3, DCm and TUNEL) in cells lacking procaspases-3 (CASP-3 (À/À) MEFs) and deficient MCF-7 cells with a paired line overexpressing the zymogen (MCF-7 7casp-3 ), the presence of procaspase-3 appeared to be necessary for mitochondrial disruption. 16 Coupled with the observation that DCm was substantially reduced in a Bax/ Bak-and caspase-dependent manner, the results suggested that the predominant GrB death pathway starts with intracellular delivery of the granzyme, continues with activation of procaspase-3 and is followed by caspase-mediated engagement of a BH3-only/Bax/Bak pathway. The BH3-only protein that is cleaved by caspase-3 remains unclear.…”

“…The BH3-only protein that is cleaved by caspase-3 remains unclear. If adventitious proteolysis is controlled, then BID cleavage was not readily detected in MCF-7 casp-3 cells by immunoblot but processing is detectable after the line is exposed to TRAIL 16 or staurosporine. 17 More sensitive in situ assays may reveal that truncated BID (or related protein) is generated to amplify GrB-mediated apoptosis through mitochondria.…”