BAP37 and Prohibitin are specifically recognized by an SV40 T antigen antibody.

We have identified two cellular proteins that are specifically immunoprecipitated by an anti-SV40 T antigen monoclonal antibody. This antibody, PAb419, recognizes an epitope contained within a region of T antigen which we have recently demonstrated is required for the initiation of immortalization by SV40 T antigen, but is not essential for maintenance of the immortal state. The two proteins were identified as BAP37 and Prohibitin. Recent results suggest Prohibitin may enhance the transcriptional inactivation of E2F by the retinoblastoma family of pocket proteins (pRb, p107, p130). BAP37 and Prohibitin are specifically recognized by PAb419 and PAb210, another anti-SV40 T antigen monoclonal antibody, which has an overlapping epitope, but not by other anti-SV40 T antigen monoclonal antibodies, demonstrating the specificity of the interaction.

Different functions are required for initiation and maintenance of immortalization of rat embryo fibroblasts by SV40 large T antigen.

We have used two different, but complementary assays to characterize functions of SV40 T antigen that are necessary for its ability to immortalize rat embryo fibroblasts. In accordance with previous work, we found that several functions were required. These include activities that map to the p53 binding domain and the amino terminal 176 amino acids which contain the J domain as well as the CR1 and CR2 domain required for binding and sequestering the RB family of pocket proteins. Moreover, we found that even though activities dependent only upon the amino terminus were sufficient for immortalization they were unable to maintain it. This suggests that immortalization by these amino terminal functions requires either additional events or immortalization of a subset of cells within the heterogeneous rat embryo fibroblast population. We further found that an activity dependent upon amino acids 17 - 27 which remove a portion of the CR1 domain and the predicted alpha-1 helix of the J domain was not necessary to maintain growth but was required for direct immortalization suggesting that at least one of the functions required initially was not required to maintain the immortal state. This represents the first demonstration that some of the functions required for maintenance of the immortal state differ from those required for initiation of immortalization.

Cytotoxic T lymphocyte-assisted suicide. Caspase 3 activation is primarily the result of the direct action of granzyme B.

Cytototoxic T lymphocyte-induced apoptosis can occur either through the directed exocytosis of granzyme B and perforin or via ligation of Fas. Both pathways involve the activation of a family of cysteine proteinases, the caspases, that cleave substrates at aspartic acid and are themselves activated by cleavage at internal aspartate residues. Fas recruits caspase 8, which initiates the death program through the subsequent activation of caspase 3. Granzyme B can process both caspase 8 and 3 in vitro, suggesting that both Fas and granzyme B access the apoptotic program in the same way. Here we demonstrate that although the two mechanisms are similar, the events that lead to activation of caspase 3 can be distinguished in vivo on the basis of their sensitivities to both pharmacological and virus-encoded caspase inhibitors. In cytotoxic T lymphocytes-mediated death the initial cleavage event on caspase 3 is insensitive to benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (zVAD-fmk) inhibition in both mouse and human systems. During Fas-mediated death, however, activation of caspase 3 is completely inhibited to zVAD-fmk. In addition, the viral serpin SPI-2, a homologue of cytokine response modifier A (crmA), is an effective inhibitor of the Fas but not the granzyme pathway. Our results demonstrate that whereas Fas-mediated activation of caspase 3 requires an upstream caspase activity that is zVAD-fmk-sensitive, the initial cleavage of caspase 3 during granule-mediated cell death is insensitive to zVAD-fmk, suggesting that caspase 3 is cleaved directly by granzyme B in vivo.

Proteases and cell-mediated cytotoxicity.

Cytotoxic T lymphocytes and natural killer cells represent the body's primary defense against viral-infected and tumorigenic cells. The classically described mechanism by which these cells induce target cell death is granule mediated: cytolytic granules within the killer cell are directionally exocytozed toward the target cell, and the granule contents inflict a "lethal hit" on the target cell. A second mechanism of cytotoxicity is now known to exist, and utilizes cell surface receptors on the target cell, for which the ligand is expressed on the killer cell. Receptor oligomerization results in the recruitment of cytoplasmic proteins to the receptors and the transduction of a death signal to the target cell. In both granule- and receptor-mediated cytotoxicity, the target cell dies through a defined series of steps, which together are termed apoptosis. Recent work on apoptosis has defined a family of cysteine proteases, the caspases, which appear to be involved in the initiation of apoptosis in response to a number of stimuli. This review focuses on studies that link these proteases to target cell death induced by cytotoxic cells.

L is for lytic granules: lysosomes that kill.

CTL are important cells in the immune system which are able to recognise and directly destroy virally infected, tumorigenic or foreign cells. The proteins which mediate this destruction are packaged into specialised secretory granules, termed lytic granules, which are secreted in response to target cell recognition. Curiously these specialised secretory granules also contain all the lysosomal hydrolases, and in CTL the lytic granules serve two separate functions: as a lysosome within the cell, and as a secretory granule when a target cell is recognised. These "secretory lysosomes", which serve important roles in both protein degradation within the cells as well as regulated secretion of proteins from the cells, are also found in other cell types, all of which are derived from the hemopoietic lineage. This observation raises the possibility that cells of the hemopoietic lineage possess specialised sorting and secretory mechanisms which allow the lysosomes to be used as secretory organelles. Studies on Chediak Higashi syndrome support this idea, since in this naturally occurring genetic mutation, cells with secretory lysosomes are unable to secrete their granules while other conventional secretory cells are able to do so. Further studies on the mechanisms which regulate secretion of lytic proteins from CTL should identify the proteins involved in this unusual secretory pathway. Some aspects of the differences between conventional and "secretory" lysosomes remain unresolved. How the biogenesis of the secretory lysosome differs from that of a conventional secretory granule is unclear. While conventional secretory cells sort proteins destined for the granule by a selective condensation in the TGN, the secretory lysosomes seem to use a combination of lysosomal and other sorting signals. Our preliminary studies suggest that haemopoietic cells possess specialised sorting mechanisms which allow the correct sorting of the secreted products to the lysosome, and that these signals are different from those found in conventional secretory (e.g. neurosecretory) cells. This finding and the observation that fibroblast lysosomes can undergo calcium-mediated exocytosis suggests that the unusual secretory system found in haemopoietic cells may be a result of specialised sorting mechanisms in these cells. In this case the Chediak lesion may turn out to be a sorting defect.

An interleukin-1beta converting enzyme-like protease is a key component of Fas-mediated apoptosis.

Cytotoxic T lymphocytes (CTLs) are able to kill target cells bearing foreign antigen through two distinct mechanisms: granule- and Fas-mediated cytotoxicity. The exact events involved in the induction of target cell apoptosis remain elusive, but research indicates a role for members of the interleukin-1beta converting enzyme (ICE)/Ced-3 family of cysteine proteases. The exact nature of the protease(s) involved is yet to be determined. Here we use activity assays and peptide inhibitors of ICE/Ced-3 proteases to study their role in Fas-mediated killing. We find that while certain inhibitors block DNA fragmentation and chromium release, others do not. Most notably, potent inhibitors of CPP32 and ICE could not inhibit DNA fragmentation during all cases of Fas-mediated cytotoxicity although an "ICE" inhibitor could suppress 51Cr release. Additionally, we find that CPP32 is not cleaved in all target cells during Fas killing. Although ICE activity (as measured by a fluorogenic substrate) is present in cell lysates from anti-Fas-treated cells, we found no pro-IL-1beta-cleaving activity in these lysates. Taken together, our results suggest that an alternate pathway to DNA fragmentation exists, which does not involve CPP32 activity, and that CPP32 and ICE activities are not essential to Fas-mediated killing.

Cleavage of CPP32 by granzyme B represents a critical role for granzyme B in the induction of target cell DNA fragmentation.

Cytotoxic T lymphocytes (CTLs) are able to recognize and destroy target cells bearing foreign antigen using one of two distinct mechanisms: granule- or Fas-mediated cytotoxicity. The exact mechanisms involved in the induction of apoptotic cell death remain elusive; however, it seems likely that a family of cysteine proteases related to interleukin-1beta converting enzyme are involved. One family member, CPP32, has been identified as an intracellular substrate for granzyme B, a CTL-specific serine protease responsible for the early induction of target cell DNA fragmentation. Here we use cytolytic cells from granzyme B-deficient mice to confirm that cleavage and activation of CPP32 represents a nonredundant role for granzyme B and that this activation plays a role in the induction of DNA fragmentation in target cells, a signature event for apoptotic cell death. A peptide inhibitor of CPP32-like proteases confirmed the function of these enzymes in fragmentation. 51Cr release was not suppressed under these conditions, suggesting that granzyme B cleavage of CPP32 is primarily involved in the induction of DNA fragmentation and not membrane damage during CTL-induced apoptosis.

Activation of the apoptotic protease CPP32 by cytotoxic T-cell-derived granzyme B.

Cytotoxic T lymphocyte (CTL)-mediated cytotoxicity represents the body's major defence against virus-infected and tumorigenic cells, and contributes to transplant rejection and autoimmune disease. During killing, CTL granules are exocytosed, releasing their contents into the intercellular space between the target cell and the effector. Perforin facilitates the entry of cytotoxic cell serine proteases, the granzymes, into the target cell, where they induce apoptotic death by an unknown pathway. Granzyme B is essential for the induction of DNA fragmentation and apoptosis in target cells, yet its substrate is unknown. Identification of the intracellular substrate for granzyme B is therefore the key to understanding the mechanism of CTL-mediated killing. Here we show that granzyme B cleaves and activates CPP32, the precursor of the protease responsible for cleavage of poly(ADP-ribose) polymerase.

The cytotoxic T cell proteinase granzyme B does not activate interleukin-1 beta-converting enzyme.

Murine granzyme B (cytotoxic cell proteinase-1 (CCP1)) is a member of a family of seven serine proteases found in cytoplasmic granules of cytotoxic T lymphocytes (CTLs). Evidence has suggested that it is involved in target cell DNA fragmentation during CTL-mediated cytotoxicity, although intracellular substrates for granzyme B have not yet been identified. The substrate specificity of granzyme B, requiring an aspartic acid residue at site P1, is unique among eukaryotic serine proteases and is shared with only one other known eukaryotic protease, interleukin-1 beta-converting enzyme (ICE). ICE is responsible for processing pro-interleukin-1 beta to produce biologically active interleukin-1 beta and is itself synthesized as an inactive precursor. Recent evidence has suggested a role for ICE in programmed cell death, which led to a model for CTL-mediated cytotoxicity. In this proposal granzyme B activates ICE in the target cell by proteolytically processing the ICE precursor, resulting in active ICE heterodimer that induces apoptosis in the target cell. We have isolated the cDNA encoding murine ICE and generated in vitro translated ICE precursor. Using lysates from COS cells expressing granzyme B we show that ICE precursor is not a substrate for granzyme B and propose an alternate mechanism for CTL-mediated cytotoxicity.