A Central Role for Bid in Granzyme B-induced Apoptosis

Abstract

Granzyme B, a protease released from cytotoxic lymphocytes, has been proposed to induce target cell death by cleaving and activating the pro-apoptotic Bcl-2 family member Bid. It has also been proposed that granzyme B can induce target cell death by activating caspases directly, by cleaving caspase substrates, and/or by cleaving several non-caspase substrates. The relative importance of Bid in granzyme B-induced cell death has therefore remained unclear. Here we report that cells isolated from various tissues of Bid-deficient mice were resistant to granzyme B-induced cell death. Consistent with the proposed role of Bid in regulating mitochondrial outer membrane permeabilization, cytochrome c remained in the mitochondria of Bid-deficient cells treated with granzyme B. Unlike wild type cells, Biddeficient cells survived and were then able to proliferate normally, demonstrating the critical role for Bid in mediating granzyme B-induced apoptosis.Granzyme B is a serine protease contained within the granules of cytotoxic lymphocytes (CLs).1 Upon conjugation with their targets, CLs release their granule contents into the synaptic cleft. Granzyme B then enters the target cell by endocytosis and induces apoptotic death via a perforin-dependent mechanism. The importance of CL-mediated killing in the immune response to various pathogens has made it imperative to understand the mechanism of action of granzyme B.Overexpression of the oncogene Bcl-2 renders cells resistant to granzyme B-induced apoptosis (1, 2), and the cells maintain their ability to proliferate (2, 3). Bcl-2 is one of a large family of proteins that regulate mitochondrial outer membrane permeabilization (MOMP) during apoptosis (4). Pro-apoptotic Bcl-2 family members (such as Bid, Bax, and Bak) induce MOMP (5), whereas anti-apoptotic members (e.g. Bcl-2 and Bcl-XL) prevent MOMP (6). Following MOMP, several pro-apoptotic proteins are released from the mitochondrial intermembrane space. In the cytosol, these proteins facilitate the activation of caspases, proteases that orchestrate the death of a cell by apoptosis. One of these proteins, cytochrome c, initiates a complex with dATP, apoptotic protease-activating factor (APAF-1), and pro-caspase-9. This results in the activation of caspase-9, which in turn activates caspase-3 (7). A second protein SMAC/ Diablo that is also released from the mitochondrial intermembrane space displaces inhibitor of apoptosis proteins (IAPs) from caspases, allowing them to become activated by autoprocessing (8, 9).Granzyme B has been reported to induce MOMP by cleaving and activating the pro-apoptotic Bcl-2 family member Bid after residue [10][11][12]. Granzyme B has also been shown to cleave caspase-3 directly when mixed with cytosolic lysates (13, 14); however, in intact cells, granzyme B only appears to be capable of partially processing procaspase-3 to a p20 form that shows little activity in a cellular context (15). SMAC/Diablo, released following MOMP, then displaces the IAPs from the p20 form of caspase-3, allowing auto-proc...

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

“…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. Next, we determined whether effector caspase-3 and -7 were activated and/or required for gzmB þ CTL-mediated cell death of gp33-pulsed MEFs.…”

Granzyme B-induced mitochondrial ROS are required for apoptosis

“…Experiments in (e-i) were repeated at least three times with similar results apoptogenic factors release was followed to assess MOMP. 4,[6][7][8][9][10] GB induced Cyt c and Endo G release only in the presence of S100 (Figure 3a), whereas cleavage of NDUFS1 occurred in the absence of S100 (Figure 3a). Interestingly, NDUFS1 cleavage was increased in the presence of S100, most likely as the result of the combined action of GB and caspase-3.…”

“…2 GB causes reactive oxygen species (ROS) production, dissipation of the mitochondrial transmembrane potential (ΔΨm) and MOMP, which leads to the release of apoptogenic factors such as cytochrome c (Cyt c), HtrA2/Omi, endonuclease G (Endo G), Smac/Diablo and apoptosis-inducing factor, from the mitochondrial intermembrane space to the cytosol. [4][5][6][7][8][9][10][11] Interestingly, cells deficient for Bid, Bax and Bak are still sensitive to human GB-induced cell death, 5,[11][12][13] suggesting that human GB targets the mitochondria in another way that needs to be characterized. Altogether, much attention has been focused on the importance of MOMP in the execution of GB-mediated cell death, leaving unclear whether ROS production is a bystander effect or essential to the execution of GBinduced apoptosis.…”

Targeted induction of apoptosis by chimeric granzyme B fusion proteins carrying antibody and growth factor domains for cell recognition

“…7 Importantly, even in the presence of the caspase inhibitor A431 cells were killed by GrB-5, albeit to a lesser extent. This ability of GrB fusion proteins to also initiate caspase-independent cell death pathways, possibly through cleavage of Bid or ICAD, 13,14 could be relevant for elimination of tumor cells with a block in caspase-dependent apoptosis.…”

“…5 Once inside the cytosol of a target cell, GrB induces apoptosis by mimicking caspase activity and activating caspase-3 and other caspases. [6][7][8][9][10] In addition, GrB was found to cleave central caspase substrates, 11 such as the BH3-only protein Bid 12,13 and the inhibitor of the caspase-activated DNase (ICAD). 14 Here, we have employed recombinant human GrB to selectively eliminate tumor cells that express the epidermal growth factor receptor (EGFR) or the closely related ErbB2 (HER2) receptor tyrosine kinase.…”