How does granzyme B activate caspases and lead to the destruction of virus-infected or transformed cells? This is clearly an area of considerable interest at present — and some controversy — as in addition to The Journal of Cell Biology paper described on page 355, work from the labs of Chris Bleackley and Joe Trapani has also just been published on this subject in Immunity.

Cytotoxic lymphocytes mainly destroy target cells by exocytosis of the contents of secretory granules, which include serine proteases, such as granzyme B, and the pore-forming protein perforin. The enzymatic activity of granzyme B is thought to be central to its ability to induce cell death through the activation of caspases, but how it does this remains controversial. Previous work has shown that overexpression of BCL-2 — an anti-apoptotic protein, which acts by blocking death effectors that target mitochondria — protects cells against granzyme-B-mediated destruction. This indicates that granzyme B is involved not only in the direct activation of caspases, including caspase-3, but also in the mitochondrial death pathway. Until now, the molecular mechanisms for this were not known.

To investigate this further, Bleackley and colleagues analysed the caspase-3 cleavage profile of BCL-2-overexpressing cells after treatment with granzyme B and adenovirus, which functions as a pore-forming protein to facilitate the release of granzyme B into the target cell. Caspase-3 activation occurs in two steps: an initial cleavage event, followed by autoactivation that is controlled by the self-catalytic activity of the caspase. In BCL-2-overexpressing cells, only partial cleavage of caspase-3 occurred, showing that high levels of BCL-2 prevented subsequent caspase autoactivation.

The authors reasoned that inhibitor of apoptosis proteins (IAPs) might be responsible for this block in caspase activation, and so they tested the effect of overexpressing XIAP on granzyme-induced cell death. Caspase-3 activation was blocked in cells overexpressing XIAP and these cells were protected against the toxic effects of granzyme B. Expression of SMAC/DIABLO, a mitochondrial protein that is released from apoptotic cells and has been shown to displace IAPs from pro-caspases and promote caspase autoactivation, overcame this blockade in both the XIAP-overexpressing cells and BCL-2-overexpressing cells and rendered them susceptible to granzyme-B-mediated destruction.

Data from Trapani and colleagues investigating the effects of granzyme B and perforin on Jurkat T cells also highlight the importance of the mitochondrial pathway in granzyme-B-induced apoptosis. First, they showed that the pro-apoptotic BH3-interacting domain protein BID is cleaved by granzyme B and then translocates to the mitochondria. Next, they showed that exposure of Jurkat cells to granzyme B and perforin resulted in release of the pro-apoptotic factors cytochrome c, SMAC/DIABLO and HtrA2/OMI from mitochondria. BCL-2 overexpression blocked the release of these pro-apoptotic factors and blocked caspase autoactivation, which resulted in inhibition of granzyme-B-mediated apoptosis of these cells. As above, overexpression of XIAP also resulted in the inhibition of caspase-3 autoactivation and protected these cells from the toxic effects of granzyme B.

Taken together, these results indicate the importance of the mitochondrial pathway in granzyme-B-mediated cell death. Although granzyme B can directly cause the initial cleavage of caspase-3, for full caspase activation, pro-apoptotic mitochondrial proteins, including SMAC/DIABLO and HtrA2/OMI, are required to block the effects of IAPs, thereby allowing granzyme-mediated apoptosis to occur.