Interdimer processing and linearity of procaspase-3 activation. A unifying mechanism for the activation of initiator and effector caspases

Caspase activation during apoptosis occurs in a cascade from the initiator caspase(s) (e.g. caspase-8) to the effector caspases (e.g. caspase-3), which ensures the generation of large amounts of active caspases to dismantle cells. However, the mechanism that safeguards against inadvertent caspase activation is not well understood. Previous studies have suggested that the activation of procaspase-8 is mediated by cross-cleavage of precursor dimers, formed upon apoptosis induction, which are not only enzymatically competent but also highly susceptible to cleavage, and that procaspase-8 activation is a linear process without self-amplification. Effector procaspases constitutively exist as dimers and their activation is started by trans-cleavage by an initiator caspase followed by autocleavage of effector caspases. Here we show that the dimerization of caspase-3 molecules through their protease domains is required for their processing by initiator caspases. The subsequent autoprocessing takes place through cleavage between the dimeric intermediates. Moreover, mature caspase-3 fails to process its own precursor. Thus, despite a marked difference in the generation of active intermediates, the activation of initiator and effector caspases shares the features of interdimer cleavage and lack of self-amplification. These features may be important in preventing accidental cell death.     

Oligomerization is a general mechanism for the activation of apoptosis initiator and inflammatory procaspases

Proteolytic activation of initiator procaspases is a crucial step in the cellular commitment to apoptosis. Alternative models have been postulated for the activation mechanism, namely the oligomerization or induced proximity model and the allosteric regulation model. While the former holds that procaspases become activated upon proper oligomerization by an adaptor protein, the latter states that the adaptor is an allosteric regulator for procaspases. The allosteric regulation model has been applied for the activation of procaspase-9 by apoptotic protease-activating factor (Apaf-1) in an oligomeric complex known as the apoptosome. Using approaches that allow for controlled oligomerization, we show here that aggregation of multiple procaspase-9 molecules can induce their activation independent of the apoptosome. Oligomerization-induced procaspase-9 activation, both within the apoptosome and in artificial systems, requires stable homophilic association of the protease domains, raising the possibility that the function of Apaf-1 is not only to oligomerize procaspase-9 but also to maintain the interaction of the caspase-9 protease domain after processing. In addition, we provide biochemical evidence that other apoptosis initiator caspases (caspase-2 and -10) as well as a procaspase involved in inflammation (murine caspase-11) are also activated by oligomerization. Thus, oligomerization of precursor molecules appears to be a general mechanism for the activation of both apoptosis initiator and inflammatory procaspases.     

Kallikrein-related Peptidase-7 Regulates Caspase-14 Maturation during Keratinocyte Terminal Differentiation by Generating an Intermediate Form

The maturation and activation mechanisms of caspases are generally well understood, except for those of caspase-14, which is activated at the onset of keratinocyte terminal differentiation. We investigated the possible involvement of epidermal proteases expressed in the late stage of differentiation, and found that the chymotrypsin-like serine protease kallikrein-related peptidase-7 (KLK7) cleaved procaspase-14 at Tyr(178), generating an intermediate form that consists of a large (20 kDa) and a small subunit (8 kDa). We prepared an antibody directed to this cleavage site (h14Y178 Ab), and confirmed that it recognized a 20-kDa band formed when procaspase-14 was incubated with chymotrypsin or KLK7. We then constructed a constitutively active form of the intermediate, revC14-Y178. The substrate specificity of revC14-Y178 was completely different from that of caspase-14, showing broad specificity for various caspase substrates except WEHD-7-amino-4-trifluoromethylcoumarin (AFC), the preferred substrate of active, mature caspase-14. K(m) values for VEID-AFC, DEVD-AFC, LEVD-AFC, and LEHD-AFC were 0.172, 0.261, 0.504, and 0.847 μm, respectively. We confirmed that the mature form of caspase-14 was generated when procaspase-14 was incubated with KLK7 or revC14-Y178. Expression of constitutively active KLK7 in cultured keratinocytes resulted in generation of both the intermediate form and the mature form of caspase-14. Immunohistochemical analysis demonstrated that the intermediate form was localized at the granular layer. Our results indicate that regulation of procaspase-14 maturation during terminal differentiation is a unique two-step process involving KLK7 and an activation intermediate of caspase-14.     

Crystal structure of a procaspase-7 zymogen: mechanisms of activation and substrate binding.

Apoptosis is primarily executed by active caspases, which are derived from the inactive procaspase zymogens through proteolytic cleavage. Here we report the crystal structures of a caspase zymogen, procaspase-7, and an active caspase-7 without any bound inhibitors. Compared to the inhibitor-bound caspase-7, procaspase-7 zymogen exhibits significant structural differences surrounding the catalytic cleft, which precludes the formation of a productive conformation. Proteolytic cleavage between the large and small subunits allows rearrangement of essential loops in the active site, priming active caspase-7 for inhibitor/substrate binding. Strikingly, binding by inhibitors causes a 180 degrees flipping of the N terminus in the small subunit, which interacts with and stabilizes the catalytic cleft. These analyses reveal the structural mechanisms of caspase activation and demonstrate that the inhibitor/substrate binding is a process of induced fit.     

Caspase inhibitor P35 and inhibitor of apoptosis Op-IAP block in vivo proteolytic activation of an effector caspase at different steps

Signal-induced activation of caspases, the critical protease effectors of apoptosis, requires proteolytic processing of their inactive proenzymes. Consequently, regulation of procaspase processing is critical to apoptotic execution. We report here that baculovirus pancaspase inhibitor P35 and inhibitor of apoptosis Op-IAP prevent caspase activation in vivo, but at different steps. By monitoring proteolytic processing of endogenous Sf-caspase-1, an insect group II effector caspase, we show that Op-IAP blocked the first activation cleavage at TETD downward arrowG between the large and small caspase subunits. In contrast, P35 failed to affect this cleavage, but functioned downstream to block maturation cleavages (DXXD downward arrow(G/A)) of the large subunit. Substitution of P35's reactive site residues with TETDG failed to increase its effectiveness for blocking TETD downward arrowG processing of pro-Sf-caspase-1, despite wild-type function for suppressing apoptosis. These data are consistent with the involvement of a novel initiator caspase that is resistant to P35, but directly or indirectly inhibitable by Op-IAP. The conservation of TETD downward arrowG processing sites among insect effector caspases, including Drosophila drICE and DCP-1, suggests that in vivo activation of these group II caspases involves a P35-insensitive caspase and supports a model wherein apical and effector caspases function through a proteolytic cascade to execute apoptosis in insects.     

Caspase-8 can be activated by interchain proteolysis without receptor-triggered dimerization during drug-induced apoptosis

Proteases of the caspase family are thought to be activated by proteolytic processing of their inactive zymogens. However, although proteolytic cleavage is sufficient for executioner caspases, a different mechanism has been recently proposed for initiator caspases, such as caspase-8, which are believed to be activated by proximity-induced dimerization. According to this model, dimerization rather than proteolytic processing is considered as the critical event for caspase-8 activation. Such a mechanism would suggest that in the absence of a dimerization platform such as the death-inducing signaling complex, caspase-8 proteolytic cleavage would result in an inactive enzyme. As several studies have described caspase-8 cleavage during mitochondrial apoptosis, we now investigated whether caspase-8 becomes indeed catalytically active in this pathway. Using an in vivo affinity labeling approach, we demonstrate that caspase-8 is activated in etoposide-treated cells in vivo in the absence of the receptor-induced death-inducing signaling complex formation. Furthermore, we show that both caspase-3 and -6 are required for the efficient activation of caspase-8. Our data therefore indicate that interchain cleavage of caspase-8 in the mitochondrial pathway is sufficient to produce an active enzyme even in the absence of receptor-driven procaspase-8 dimerization.     

Galectin-1 induced activation of the mitochondrial apoptotic pathway: evidence for a connection between death-receptor and mitochondrial pathways in human Jurkat T lymphocytes

Galectin-1 (gal-1) triggers T cell death by several distinct intracellular pathways including the activation of the death-receptor pathway. The aim of this study was to investigate whether gal-1 induced activation of the death-receptor pathway in Jurkat T lymphocytes mediates apoptosis via the mitochondrial pathway linked by truncated Bid (tBid). We demonstrate that gal-1 induced proteolytic cleavage of the death agonist Bid, a member of the Bcl-2/Bcl-xL family and a substrate of activated caspase-8, was inhibited by caspase-8 inhibitor II (Z-IETD-FMK). Downstream of Bid, gal-1 stimulated mitochondrial cytochrome c release as well as the activation and proteolytic processing of initiator procaspase-9 were effectively decreased by caspase-8 inhibitor II. Blocking of gal-1 induced cleavage of effector procaspase-3 by caspase-8 inhibitor II as well as by caspase-9 inhibitors I (Z-LEHD-FMK) and III (Ac-LEHD-CMK) indicates that receptor and mitochondrial pathways converged in procaspase-3 activation and contribute to proteolytic processing of effector procaspase-6 and -7. Western blot analyses and immunofluorescence staining revealed that exposure of Jurkat T cells to gal-1 resulted in the cleavage of the DNA-repair enzyme poly (ADP-ribose) polymerase, cytoskeletal α-fodrin, and nuclear lamin A as substrates of activated caspases. Our data demonstrate that Bid provides a connection between the death receptor and the mitochondrial pathway of gal-1 induced apoptosis in human Jurkat T lymphocytes.     

The proteolytic procaspase activation network: an in vitro analysis

In general, apoptotic stimuli lead to activation of caspases. Once activated, a caspase can induce intracellular signaling pathways involving proteolytic activation of other caspase family members. We report the in vitro processing of eight murine procaspases by their enzymatically active counterparts. Caspase-8 processed all procaspases examined. Caspase-1 and -11 processed the effector caspases procaspase-3 and -7, and to a lesser extent procaspase-6. However, vice versa, none of the caspase-1-like procaspases was activated by the effector caspases. This suggests that the caspase-1 subfamily members either act upstream of the apoptosis effector caspases or else are part of a totally separate activation pathway. Procaspase-2 was maturated by caspase-8 and -3, and to a lesser extent by caspase-7, while the active caspase-2 did not process any of the procaspases examined, except its own precursor. Hence, caspase-2 might not be able to initiate a wide proteolytic signaling cascade. Additionally, cleavage data reveal not only proteolytic amplification between caspase-3 and -8, caspase-6 and -3, and caspase-6 and -7, but also positive feedback loops involving multiple activated caspases. Our results suggest the existence of a hierarchic proteolytic procaspase activation network, which would lead to a dramatic increase in multiple caspase activities once key caspases are activated. The proteolytic procaspase activation network might allow that different apoptotic stimuli result in specific cleavage of substrates responsible for typical processes at the cell membrane, the cytosol, the organelles, and the nucleus, which characterize a cell dying by apoptosis.     

Activation of a cysteine protease in MCF-7 and T47D breast cancer cells during beta-lapachone-mediated apoptosis

beta-Lapachone (beta-lap) effectively killed MCF-7 and T47D cell lines via apoptosis in a cell-cycle-independent manner. However, the mechanism by which this compound activated downstream proteolytic execution processes were studied. At low concentrations, beta-lap activated the caspase-mediated pathway, similar to the topoisomerase I poison, topotecan; apoptotic reactions caused by both agents at these doses were inhibited by zVAD-fmk. However at higher doses of beta-lap, a novel non-caspase-mediated "atypical" cleavage of PARP (i.e., an approximately 60-kDa cleavage fragment) was observed. Atypical PARP cleavage directly correlated with apoptosis in MCF-7 cells and was inhibited by the global cysteine protease inhibitors iodoacetamide and N-ethylmaleimide. This cleavage was insensitive to inhibitors of caspases, granzyme B, cathepsins B and L, trypsin, and chymotrypsin-like proteases. The protease responsible appears to be calcium-dependent and the concomitant cleavage of PARP and p53 was consistent with a beta-lap-mediated activation of calpain. beta-Lap exposure also stimulated the cleavage of lamin B, a putative caspase 6 substrate. Reexpression of procaspase-3 into caspase-3-null MCF-7 cells did not affect this atypical PARP proteolytic pathway. These findings demonstrate that beta-lap kills cells through the cell-cycle-independent activation of a noncaspase proteolytic pathway.     

Processing of native caspase-14 occurs at an atypical cleavage site in normal epidermal differentiation

Caspase-14, a cysteinyl aspartate-specific protease expressed during epidermal differentiation, is detected exclusively in the cytosolic fraction of epidermis as a complex of procaspase-14 together with caspase-14 large and small subunits. On non-denaturing protein gels, native caspase-14 has a relative electrophoretic mobility of approximately 80kDa, which resolves into caspase-14 proform, large and small subunit in SDS-polyacrylamide. Purification of caspase-14 from native skin with subsequent N-terminal sequencing of the small subunit and tryptic digest analysis of the large subunit revealed an atypical processing site between Ile152 and Lys153, which distinguishes it from other caspases described to date that are processed at aspartate residues. Expression of caspase-14 in heterologous systems results in unprocessed procaspase-14 without generation of the large and small subunits that characterize this protein family. However, addition of cellular extracts to purified recombinant human caspase-14 generated immunoreactive peptides indistinguishable from large and small subunits in skin. These data provide evidence for novel processing of caspase-14 suggesting that this enzyme has unique mechanisms of regulation during epidermal differentiation.     

Apoptosis in motion. An apical, P35-insensitive caspase mediates programmed cell death in insect cells

Activation of caspases by proteolytic processing is a critical step during apoptosis in metazoans. Here we use high resolution time lapse microscopy to show a tight link between caspase activation and the morphological events delineating apoptosis in cultured SF21 cells from the moth Spodoptera frugiperda, a model insect system. The principal effector caspase, Sf-caspase-1, is proteolytically activated during SF21 apoptosis. To define the potential role of initiator caspases in vivo, we tested the effect of cell-permeable peptide inhibitors on pro-Sf-caspase-1 processing. Anti-caspase peptide analogues prevented apoptosis induced by diverse signals, including UV radiation and baculovirus infection. IETD-fmk potently inhibited the initial processing of pro-Sf-caspase-1 at the junction (TETD-G) of the large and small subunit, a cleavage that is blocked by inhibitor of apoptosis Op-IAP but not pancaspase inhibitor P35. Because Sf-caspase-1 was inhibited poorly by IETD-CHO, our data indicated that the protease responsible for the first step in pro-Sf-caspase-1 activation is a distinct apical caspase. Thus, Sf-caspase-1 activation is mediated by a novel, P35-resistant caspase. These findings support the hypothesis that apoptosis in insects, like that in mammals, involves a cascade of caspase activations.     

S-Nitrosation regulates the activation of endogenous procaspase-9 in HT-29 human colon carcinoma cells

Nitric oxide-mediated signals have been suggested to regulate the activity of caspases negatively, yet literature has provided little direct evidence. We show in this paper that cytokines and nitric-oxide synthase (NOS) inhibitors regulate S-nitrosation of an initiator caspase, procaspase-9, in a human colon adenocarcinoma cell line, HT-29. A NOS inhibitor, N(G)-methyl-l-arginine, enhanced the tumor necrosis factor-alpha (TNF-alpha)-induced cleavage of procaspase-9, procaspase-3, and poly-(ADP-ribose) polymerase, as well as the level of apoptosis. N(G)-Methyl-l-arginine, however, did not affect the cleavage of procaspase-8. These results suggest that nitric oxide regulates the cleavage of procaspase-9 and its downstream proteins and, subsequently, apoptosis in HT-29 cells. Labeling S-nitrosated cysteines with a biotin tag enabled us to reveal S-nitrosation of endogenous procaspase-9 that was immunoprecipitated from the HT-29 cell extracts. Furthermore, the treatment with TNF-alpha, as well as NOS inhibitors, decreased interferon-gamma-induced S-nitrosation in procaspase-9. Our results show that S-nitrosation of endogenous procaspase-9 occurs in the HT-29 cells under normal conditions and that denitrosation of procaspase-9 enhances its cleavage and consequent apoptosis. We, therefore, suggest that S-nitrosation regulates activation of endogenous procaspase-9 in HT-29 cells.     

The three-dimensional structure of caspase-8: an initiator enzyme in apoptosis.

BACKGROUND: In the initial stages of Fas-mediated apoptosis the cysteine protease caspase-8 is recruited to the cell receptor as a zymogen (procaspase-8) and is incorporated into the death-signalling complex. Procaspase-8 is subsequently activated leading to a cascade of proteolytic events, one of them being the activation of caspase-3, and ultimately resulting in cell destruction. Variations in the substrate specificity of different caspases have been reported. RESULTS: We report here the crystal structure of a complex of the activated human caspase-8 (proteolytic domain) with the irreversible peptidic inhibitor Z-Glu-Val-Asp-dichloromethylketone at 2.8 A resolution. This is the first structure of a representative of the long prodomain initiator caspases and of the group III substrate specificity class. The overall protein architecture resembles the caspase-1 and caspase-3 folds, but shows distinct structural differences in regions forming the active site. In particular, differences observed in subsites S(3), S(4) and the loops involved in inhibitor interactions explain the preference of caspase-8 for substrates with the sequence (Leu/Val)-Glu-X-Asp. CONCLUSIONS: The structural differences could be correlated with the observed substrate specificities of caspase-1, caspase-3 and caspase-8, as determined from kinetic experiments. This information will help us to understand the role of the various caspases in the propagation of the apoptotic signal. The information gained from this investigation should be useful for the design of specific inhibitors.     

Cytochrome c and dATP-mediated oligomerization of Apaf-1 is a prerequisite for procaspase-9 activation.

To elucidate the mechanism of activation of procaspase-9 by Apaf-1, we produced recombinant full-length Apaf-1 and purified it to complete homogeneity. Here we show using gel filtration that full-length Apaf-1 exists as a monomer that can be transformed to an oligomeric complex made of at least eight subunits after binding to cytochrome c and dATP. Apaf-1 binds to cytochrome c in the absence of dATP but does not form the oligomeric complex. However, when dATP is added to the cytochrome c-bound Apaf-1 complex, complete oligomerization occurs, suggesting that oligomerization is driven by hydrolysis of dATP. This was supported by the observation that ATP, but not the nonhydrolyzable adenosine 5'-O-(thiotriphosphate), can induce oligomerization of the Apaf-1-cytochrome c complex. Like the spontaneously oligomerizing Apaf-530, which lacks its WD-40 domain, the oligomeric full-length Apaf-1-cytochrome c complex can bind and process procaspase-9 in the absence of additional dATP or cytochrome c. However, unlike the truncated Apaf-530 complex, the full-length Apaf-1 complex can release the mature caspase-9 after processing. Once released, mature caspase-9 can process procaspase-3, setting into motion the caspase cascade. These observations indicate that cytochrome c and dATP are required for oligomerization of Apaf-1 and suggest that the WD-40 domain plays an important role in oligomerization of full-length Apaf-1 and the release of mature caspase-9 from the Apaf-1 oligomeric complex.     

Teratogen-induced activation of caspase-6 and caspase-7 in early postimplantation mouse embryos

BACKGROUND: Previous work has shown that teratogens such as hyperthermia (HS), 4-hydroperoxycyclophosphamide (4CP), and staurosporine (ST) induce cell death in day 9 mouse embryos by activating the mitochondrial apoptotic pathway. Key to the activation of this pathway is the activation of a caspase cascade involving the cleavage-induced activation of an initiator procaspase, caspase-9, and the downstream effector procaspase, caspase-3. For example, procaspase-3, an inactive proenzyme of 32 kDa is cleaved by activated caspase-9 to generate a large subunit of approximately 17 kDa and a small subunit of approximately 10 kDa. In turn, caspase-3 is known to target a variety of cellular proteins for proteolytic cleavage as part of the process by which dying cells are eliminated. Previous work has also shown that neuroepithelial cells are sensitive to teratogen-induced activation of this pathway and subsequent cell death whereas cells of the heart are resistant. Although caspase-3 is a key effector caspase activated by teratogens, two other effector caspases, caspase-6 and caspase-7, are known; however, their role in teratogen-induced cell death is unknown. METHODS: Because cleavage-induced generation of specific subunits is the most specific assay for activation of caspases, we have used antibodies that recognize the procaspase and one of its active subunits and a Western blot approach to assess the activation of caspase-6 and caspase-7 in day 9 mouse embryos (or heads, hearts and trunks isolated from whole embryos) exposed to HS, 4CP, and ST. To probe the relationship between teratogen-induced activation of caspase-9/caspase-3 and the activation of caspase-6/caspase-7, we used a mitochondrial-free embryo lysate with or without the addition of cytochrome c, recombinant active caspase-3, or recombinant active caspase-9. RESULTS: Western blot analyses show that these three teratogens, HS, 4CP, and ST, induce the activation of procaspase-6 (appearance of the 13 kDa subunit, p13) and caspase-7 (appearance of the 19 kDa subunit, p19) in day 9 mouse embryos. In vitro studies showed that both caspase-6 and caspase-7 could be activated by the addition of cytochrome c to a lysate prepared from untreated embryos. In addition, caspase-6 could be activated by the addition of either recombinant caspase-3 or caspase-9 to a lysate prepared from untreated embryos. In contrast, caspase-7 could be activated by addition of recombinant caspase-3 but only minimally by recombinant caspase-9. Like caspase-9/caspase-3, caspase-6 and caspase-7 were not activated in hearts isolated from embryos exposed to these three teratogens. CONCLUSIONS: HS, 4CP and ST induce the cleavage-dependent activation of caspase-6 and caspase-7 in day 9 mouse embryos. Results using DEVD-CHO, a caspase-3 inhibitor, suggest that teratogen-induced activation of caspase-6 is mediated by caspase-3. In addition, our data suggest that caspase-7 is activated primarily by caspase-3; however, we cannot rule out the possibility that this caspase is also activated by caspase-9. Finally, we also show that teratogen-induced activation of caspase-6 and caspase-7 are blocked in the heart, a tissue resistant to teratogen-induced cell death.     

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-induced apoptosis is dependent on activation of cysteine and serine proteases

We examined the role of caspases and serine protease(s) in cell death induced by tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). After incubation of adenocarcinoma cells with TRAIL, caspase-3, -8 were activated and the cleavage of Bid induced the release of cytochrome c, from the mitochondria to the cytosol. Tetrapeptide inhibitors of caspase-1, -2, -3, and -8 suppressed DNA fragmentation and attenuated the release of cytochrome c, whereas inhibitors of caspase-5 did not. Interestingly, the general serine protease(s) inhibitor 4-(2-aminoethyl)benzylsulfonyl fluoride (AEBSF) resulted in the arrest of apoptosis. However, the AEBSF did not prevent the release of mitochondrial cytochrome c during TRAIL-induced apoptosis. From these results, we postulate that serine protease(s) may be involved in post-mitochondrial apoptotic events, that lead to the activation of the initiator, caspase-9.     

An APAF-1.cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9

We report here the reconstitution of the de novo procaspase-9 activation pathway using highly purified cytochrome c, recombinant APAF-1, and recombinant procaspase-9. APAF-1 binds and hydrolyzes ATP or dATP to ADP or dADP, respectively. The hydrolysis of ATP/dATP and the binding of cytochrome c promote APAF-1 oligomerization, forming a large multimeric APAF-1.cytochrome c complex. Such a complex can be isolated using gel filtration chromatography and is by itself sufficient to recruit and activate procaspase-9. The stoichiometric ratio of procaspase-9 to APAF-1 is approximately 1 to 1 in the complex. Once activated, caspase-9 disassociates from the complex and becomes available to cleave and activate downstream caspases such as caspase-3.     

TRAF1 is a substrate of caspases activated during tumor necrosis factor receptor-alpha-induced apoptosis

TRAF family proteins are signal-transducing adapter proteins that interact with the cytosolic domains of tumor necrosis factor (TNF) family receptors. Here we show that TRAF1 (but not TRAF2-6) is cleaved by certain caspases in vitro and during TNF-alpha- and Fas-induced apoptosis in vivo. (160)LEVD(163) was identified as the caspase cleavage site within TRAF1, generating two distinct fragments. Significant enhancement of TNF receptor-1 (CD120a)- and, to a lesser extent, Fas (CD95)-mediated apoptosis was observed when overexpressing the C-terminal TRAF1 fragment in HEK293T and HT1080 cells. The same fragment was capable of potently suppressing TNF receptor-1- and TRAF2-mediated nuclear factor-kappaB activation in reporter gene assays, providing a potential mechanism for the enhancement of TNF-mediated apoptosis. Cell death induced by other death receptor-independent stimuli such as cisplatin, staurosporine, and UV irradiation did not result in cleavage of TRAF1, and overexpression of the C-terminal TRAF1 fragment did not enhance cell death in these cases. TRAF1 cleavage was markedly reduced in cells that contain little procaspase-8 protein, suggesting that this apical protease in the TNF/Fas death receptor pathway is largely responsible. These data identify TRAF1 as a specific target of caspases activated during TNF- and Fas-induced apoptosis and illustrate differences in the repertoire of protease substrates cleaved during activation of different apoptotic pathways.     

Mechanisms of caspase activation

The core effectors of apoptosis encompass proteolytic enzymes of the caspase family, which reside as latent precursors in most nucleated metazoan cells. A majority of studies on apoptosis are based on the assumption that caspase precursors are activated by cleavage, a common mechanism for most protease zymogen activations. Although this appears to be true for the executioner caspases, recent research points to a distinct activation mechanism for the initiator caspases that trigger the apoptotic pathways. This mechanism is proximity-induced dimerization without cleavage, and its elucidation has led to the revision of concepts of feedback regulation of apoptosis.     

Glucocorticoid modulatory element-binding protein 1 binds to initiator procaspases and inhibits ischemia-induced apoptosis and neuronal injury

Caspases are divided into two classes: initiator caspases, which include caspase-8 and -9 and possess long prodomains, and effector caspases, which include caspase-3 and -7 and possess short prodomains. Recently, we demonstrated that glucocorticoid modulatory element-binding protein 1 (GMEB1) interacts with the prodomain of procaspase-2, thereby disrupting its autoactivation and the induction of apoptosis. Here we show that GMEB1 is also capable of binding to procaspase-8 and -9. GMEB1 attenuated the Fas-mediated activation of these caspases and the subsequent apoptosis. The knockdown of endogenous GMEB1 using RNA interference revealed that cells with decreased GMEB1 expression are more sensitive to stress and undergo accelerated apoptosis. Transgenic mice expressing a neurospecific GMEB1 had smaller cerebral infarcts and less brain swelling than wild-type mice in response to transient focal ischemia. These results suggest that GMEB1 is an endogenous regulator that selectively binds to initiator procaspases and inhibits caspase-induced apoptosis.