Apoptosis: A lethal injection

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Research suggests that the predominant granzyme B death pathway starts with the activation of pro-caspase-3, and that the death signal is maximized by mitochondria through caspase-mediated engagement of the BH3-only/BAX/BAK pathway.

The stealthy assassin creeps up to the victim and quickly injects a dose of deadly poison. No, it's not a scene from a spy novel, but one of the ways in which the immune system's natural assassins — cytotoxic T lymphocytes and natural killer cells — can kill their target cells. The deadly poison in this case is a serine protease called granzyme B (GrB), and the killing mechanism is discussed by Christopher Froelich and colleagues in The Journal of Cell Biology.

Much is known about GrB — there's evidence that it kills with or without caspase activation, and that it causes mitochondrial permeabilization with or without members of the Bcl-2 family. So Froelich and co-workers set out to assess the relative contributions of these pathways, to come up with a probable scheme for how GrB normally acts. To do this, they measured several different hallmarks of apoptosis — loss of mitochondrial membrane potential ( psi _m), DNA fragmentation and activation of procaspase precursors.

Most previous studies have been done on cell lysates, after GrB has been delivered to its target. By contrast, Froelich and colleagues studied whole cells after delivery of the physiological form of GrB, a GrB– Serglycin (SG) complex. Previous studies with lysates had indicated that GrB-treated cells rapidly acquire cleaved (and presumably active) procaspase-3. Mature caspase-3 and GrB then process caspase-7 to complete the initial phase of apoptosis. The authors therefore asked how the absence of procaspase-3 might alter the pattern of apoptosis in whole cells after delivery of GrB–SG.

The authors compared MCF-7 cells that lacked procaspase-3 (MCF-7_vec) with cells that contained a stable transfectant that expressed this zymogen (MCF-7_casp-3), after delivery of GrB–SG. They saw DNA fragmentation and a loss of psi _m in the MCF-7_casp-3 cells, but not in MCF-7_vec cells, indicating that caspase-3 is essential for starting apoptosis. The authors also showed that the requirement for caspase-3 was not due simply to the fact that caspase-7 could no longer be activated in the caspase-3-deficient cells.

If caspase-3 is the crucial initiator, what's the contribution of the Bcl-2-family members? First, the pro-apoptotic members — such as Bax, Bak and the so-called 'BH3-only' protein BID — might be involved in mitochondrial permeabilization by forming large ion channels in the outer mitochondrial membrane. Froelich and colleagues looked into this by studying the sensitivity of Bax/Bak (-/-) mouse embryo fibroblasts (MEFs) to GrB-mediated killing. The Bax/Bak (-/-) MEFs showed lower levels of active caspase-3 intracellularly and lower rates of psi _m loss than wild-type MEFs, which indicates that these proteins are required for the response to GrB. The authors also studied whether BID is cleaved in response to GrB. Unlike previous studies, however, they could not detect the truncated (active) form of BID when precautions were taken to minimize proteolysis during processing of cells for immunoblots.

A second role for certain members of the Bcl-2 family is in preventing cell death. The eponymous Bcl-2 has been shown to block GrB-mediated apoptosis, and the authors wondered whether it might do this by acting directly on caspase-3. They therefore looked at the effects of GrB–SG in a Jurkat cell line that overexpressed Bcl-2. In contrast to wild-type cells, the Jurkat_Bcl-2 cells showed no caspase-3 activity, loss of psi _m or DNA fragmentation. Bcl-2 did not inhibit isolated active caspase-3, and processed caspase-3 was observed by immunoblot, leading the authors to conclude that Bcl-2 might suppress apoptosis by blocking the assembly of processed caspase-3.

On the basis of these and several other observations, Froelich and colleagues suggest that the predominant GrB death pathway starts with the activation of procaspase-3, and that the death signal is maximized by mitochondria through caspase-mediated engagement of the BH3-only/Bax/Bak pathway. The BH3-only protein that's cleaved by caspase-3 might be BID, but Froelich and colleagues' results indicate that there might be other participants too.

Alison Mitchell

References

Metkar, S. S. et al. Granzyme B activates procaspase-3 which signals a mitochondrial amplification loop for maximal apoptosis. J. Cell Biol. 160, 875–885 (2003) | Article | PubMed |