Death caspase-1

EVOLUTIONARY HOMOLOGS

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Bcl-2 family members act upstream of caspases and Apaf-1

Examining the effects of overexpressing cell-death-related genes in specific C. elegans neurons that normally live, it was demonstrated that the cell-death genes ced-3 (a caspase), ced-4 (an Apaf-1 homolog), and ced-9 (a BCL-2 homolog) all can act cell autonomously to control programmed cell death. Not only the protective activity of ced-9 but also the killer activities of ced-3 and ced-4 are likely to be present in cells that normally live. Killing by overexpression of ced-3 does not require endogenous ced-4 function, whereas killing by overexpression of ced-4 is at least in part dependent on endogenous ced-3 function. These results suggest either that ced-4 acts upstream of ced-3 and ced-4 function can be bypassed by high levels of ced-3 activity or that ced-3 and ced-4 act in parallel, with ced-3 perhaps having a greater ability to kill. The finding that ced-4 appears to facilitate the inhibition of ced-3 by ced-9 suggests that ced-9 acts to negatively regulate ced-4. It is proposed that both in C. elegans and in other organisms a competition between antagonistic protective and killer activities determines whether specific cells will live or die. These results suggest a genetic pathway for programmed cell death in C. elegans in which ced-4 acts upstream of or in parallel withced-3, and ced-9 negatively regulates the activity of ced-4 (Shaham, 1996a).

ced-4 encodes two transcripts; whereas the major transcript can cause programmed cell death, the minor transcript can act oppositely and prevent programmed cell death, thus defining a novel class of cell death inhibitors. That ced-4 has both cell-killing and cell-protective functions is consistent with previous genetic studies. The dual protective and killer functions of the C. elegans bcl-2-like gene ced-9 are mediated by inhibition of the killer and protective ced-4 functions, respectively. It is proposed that a balance between opposing ced-4 functions influences the decision of a cell to live or to die by programmed cell death and that both ced-9 and ced-4 protective functions are required to prevent programmed cell death (Shaham, 1996b).

Three principal genes are involved in developmental programmed cell death in C. elegans: ced-3 and ced-4 genes are both required for PCD, whereas ced-9 acts to prevent the death-promoting actions of these genes. ced-9 is homologous to the Bcl-2 family, whose role in protecting PCD is illusive; no vertebrate homolog of ced-4 is known. This paper describes the effect of expression of C. elegans ced-4 in yeast. Induction of wild type ced-4 results in rapid focal chromatin condensation and lethality. Mutation of a putative nucleotide binding P-loop motif of CED-4 eliminates the lethal phenotype. Immunolocalization of CED-4 to the condensed chromatin suggests that the phenotype may result from an intrinsic ability of CED-4 to interact with chromatin. Co-expression of ced-9 prevents CED-4-induced chromatin condensation and lethality, and causes the relocalization of CED-4 to endoplasmic reticulum and outer mitochondrial membranes. A direct interaction between CED-4 and CED-9 was confirmed by yeast two-hybrid analysis. It is concluded that CED-4 has a direct role in chromatin condensation. Chromatin condensation is a ubiquitous feature of metazoan apoptosis that has yet to be linked to an effector. Further studies are required to establish whether the CED-9/CED-4 interaction is required for the activation of CED-3, the Caspase cysteine protease (James, 1997).

Genetic studies suggest that ced-9 controls programmed cell death by regulating ced-4 and ced-3. However, the mechanism by which CED-9 controls the activities of CED-4 and the cysteine protease CED-3, the effector arm of the cell-death pathway, remains poorly understood. Immunoprecipitation analysis demonstrates that in vivo CED-9 forms a multimeric protein complex with CED-4 and CED-3. Expression of wild-type CED-4 promotes the ability of CED-3 in mammalian cells to induce apoptosis otherwise inhibited by CED-9. The pro-apoptotic activity of CED-4 requires the expression of a functional CED-3 protease. Significantly, loss-of-function CED-4 mutants are impaired in their ability to promote CED-3-mediated apoptosis. Expression of CED-4 enhances the proteolytic activation of CED-3. CED-9 inhibits the formation of p13 and p15, two cleavage products of CED-3 associated with its proteolytic activation in vivo. Moreover, CED-9 inhibits the enzymatic activity of CED-3 promoted by CED-4. Thus, these results provide evidence that CED-4 and CED-9 regulate the activity of CED-3 through physical interactions, which may provide a molecular basis for the control of programmed cell death in C. elegans (Wu, 1997).

In the initiation of apoptosis, Apaf-1, homologous to C. elegans CED-4, functions downstream of bcl-2 but upstream of caspase-3. Bcl-2 may function upstream of Apaf-1 by regulating the release of cytochrome c from mitochondria. Cytochrome c is a required cofactor for Apaf-1. Another protein factor, Apaf-3, has been identified that participates in caspase-3 activation in vitro. Apaf-3 was identified as a member of the caspase family, caspase-9. Caspase-9 and Apaf-1 bind to each other via their respective NH2-terminal CED-3 homologous domains in the presence of cytochrome c and dATP, an event that leads to caspase-9 activation. This N-terminal region of caspase-9 is termed a caspase recruitment domain (CARD). Activated caspase-9 in turn cleaves and activates caspase-3. Depletion of caspase-9 from S-100 extracts diminished caspase-3 activation. Mutation of the active site of caspase-9 attenuates the activation of caspase-3 and cellular apoptotic response in vivo, indicating that caspase-9 is the most upstream member of the apoptotic protease cascade that is triggered by cytochrome c and dATP (P. Li, 1997).

Caspases are intracellular proteases that cleave substrates involved in apoptosis or inflammation. In C. elegans, a paradigm for caspase regulation exists in which caspase CED-3 is activated by nucleotide-binding protein CED-4, which is suppressed by Bcl-2-family protein CED-9. A mammalian analog of this caspase-regulatory system has been identified in the NLR-family protein NALP1, a nucleotide-dependent activator of cytokine-processing protease caspase-1, which responds to bacterial ligand muramyl-dipeptide (MDP). Antiapoptotic proteins Bcl-2 and Bcl-XL bind and suppress NALP1, reducing caspase-1 activation and interleukin-1β (IL-1β) production. When exposed to MDP, Bcl-2-deficient macrophages exhibit more caspase-1 processing and IL-1β production, whereas Bcl-2-overexpressing macrophages demonstrate less caspase-1 processing and IL-1β production. The findings reveal an interaction of host defense and apoptosis machinery (Bruey, 2007).

Caspases act upstream of Bcl-2 family members to promote cell death

The proapoptotic protein BAX contains a single predicted transmembrane domain at its COOH terminus. In unstimulated cells, BAX is located in the cytosol and in peripheral association with intracellular membranes including mitochondria, but inserts into mitochondrial membranes after a death signal. This failure to insert into mitochondrial membrane in the absence of a death signal correlates with repression of the transmembrane signal-anchor function of BAX by the NH2-terminal domain. Targeting can be instated by deleting the domain or by replacing the BAX transmembrane segment with that of BCL-2. In stimulated cells, the contribution of the NH2 terminus of BAX correlates with further exposure of this domain after membrane insertion of the protein. The peptidyl caspase inhibitor zVAD-fmk partly blocks the stimulated mitochondrial membrane insertion of BAX in vivo, which is consistent with the ability of apoptotic cell extracts to support mitochondrial targeting of BAX in vitro, dependent on activation of caspase(s). Taken together, these results suggest that regulated targeting of BAX to mitochondria in response to a death signal is mediated by discrete domains within the BAX polypeptide. The contribution of one or more caspases may reflect an initiation and/or amplification of this regulated targeting (Goping, 1998).

There is a direct interaction between caspases and Bcl-xL. The loop domain of Bcl-xL is cleaved by caspases in vitro and in cells induced to undergo apoptotic death after Sindbis virus infection or interleukin 3 withdrawal. Mutation of the caspase cleavage site in Bcl-xL in conjunction with a mutation in the BH1 homology domain impairs the death-inhibitory activity of Bcl-xL, suggesting that interaction of Bcl-xL with caspases may be an important mechanism for inhibiting cell death. The BH1 and BH2 domains of Bcl-2 and Bcl-xL are important for heterodimerization with other Bcl-2 family members. Once Bcl-xL is cleaved, the C-terminal fragment of Bcl-xL potently induces apoptosis. Taken together, these findings indicate that the recognition/cleavage site of Bcl-xL may facilitate protection against cell death by acting at the level of caspase activation and that cleavage of Bcl-xL during the execution phase of cell death converts Bcl-xL from a protective to a lethal protein (Clem, 1998).

The mechanism of cytochrome c release in response to apoptotic stimuli and its regulation by the Bcl2 family of proteins is unclear. Inasmuch as the structure of Bcl-xL is reminiscent of pore-forming proteins of bacterial toxins such as diphtheria toxin and colicins, it has been hypothesized that Bcl-xL may function as an ion channel that regulates the permeability of mitochondria. Such an ion channel could minimize osmotic stress, and in doing so, the release of cytochrome c would be prevented due to mitochondrial matrix swelling and outer membrane disruption. Indeed, both swelling of the mitochondrial matrix and bursting of the outer membrane were observed in cells treated with agonistic antibody against Fas. However, whether such a phenomenon is the cause of cytochrome c release or an effect of the apoptotic program is unclear. Activation of cell surface receptor Fas leads to rapid inactivation of the electron transfer activity of cytochrome c and subsequent release of cytochrome c from mitochondria. The inactivation and release of cytochrome c induced by Fas activation is sensitive to z-VAD-fmk, a broad range caspase inhibitor. Since activation of cell surface death receptor leads to rapid activation of caspase-8, the apical caspase in the Fas-induced apoptotic pathway, the loss of cytochrome c from mitochondria is likely a result of caspase-8 activation. Indeed, addition of active caspase-8 to a Xenopus cell-free system induces rapid cytochrome c release from mitochondria. The activation of caspase-8, therefore, initiates two pathways leading to the activation of downstream caspases. Caspase-8 can activate downstream caspases (like caspase-3, caspase-6, and caspase-7) by directly cleaving them. Caspase-8 activates these downstream caspases indirectly by causing cytochrome c release from mitochondria that triggers caspase activation through Apaf1. The latter pathway is regulated by Bcl2 or Bcl-xL while a caspase-8 inhibitor like CrmA blocks both pathways. The contributions of these two pathways to Fas-induced cell death vary between different cell types, presumably due to different levels of activity (Luo, 1998 and references).

The target of Caspase-8, the apical caspase activated by cell surface death receptors such as Fas and TNF, has now been identifed. A cytosolic protein has been purified that induces cytochrome c release from mitochondria in response to caspase-8. Peptide mass fingerprinting identified this protein as Bid, a BH3 domain-containing protein known to interact with both Bcl2 and Bax. Caspase-8 cleaves Bid, and the COOH-terminal part translocates to mitochondria where it triggers cytochrome c release. Immunodepletion of Bid from cell extracts eliminates the cytochrome c releasing activity. The cytochrome c releasing activity of Bid is antagonized by Bcl2. A mutation at the BH3 domain diminishes its cytochrome c releasing activity. Bid, therefore, relays an apoptotic signal from the cell surface to mitochondria (Luo, 1998).

Bcl-2 family proteins and ICE/CED-3 family proteases (caspases) are regarded as the basic regulators of apoptotic cell death. They are evolutionarily conserved and implicated in a variety of apoptosis. However, the precise mechanism by which these two families interact to regulate cell death is not yet known. Overexpression of the Bcl-2 family member Bax induces apoptotic cell death in COS-7 cells through the activation of CPP32 (caspase-3)-like proteases that cleave the DEVD tetrapeptide. This apoptotic cell death is suppressed by the viral proteins CrmA and p35, as well as by the chemically synthesized caspase inhibitors Z-Asp-CH2-DCB and zVAD-fmk. The Bax-induced apoptosis of COS-7 cells is suppressed by Bcl-xL and Bcl-2, though both Bcl-xL and Bcl-2 similarly prevent etoposide-induced apoptosis in COS-7 cells. Bcl-xL inhibits the activation of caspase-3-like proteases accompanying Bax-induced COS-7 cell death, but Bcl-2 does not. These results indicate that the caspase activation is essential for Bax-induced apoptosis, and that the ability of Bcl-2 and Bcl-xL to prevent the Bax-induced caspase activation and apoptosis in COS-7 cells can be differentially regulated. These results also suggest that Bcl-2 family proteins function upstream of caspase activation and control apoptosis through the regulation of caspase activity (Kitanaka, 1997).

Stimulation of the Fas or tumor necrosis factor receptor 1 (TNFR1) cell surface receptors leads to the activation of the death effector protease, caspase-8, and subsequent apoptosis. In some cells, Bcl-xL overexpression can inhibit anti-Fas- and tumor necrosis factor (TNF)-alpha-induced apoptosis. To address the effect of Bcl-xL on caspase-8 processing, Fas- and TNFR1-mediated apoptosis were studied in the MCF7 breast carcinoma cell line stably transfected with human Fas cDNA (MCF7/F) or transfected with both Fas and human Bcl-xL cDNAs (MCF7/FB). Bcl-xL strongly inhibits apoptosis induced by either anti-Fas or TNF-alpha. Bcl-xL prevents the change in cytochrome c immunolocalization induced by anti-Fas or TNF-alpha treatment. Using antibodies that recognize the p20 and p10 subunits of active caspase-8, proteolytic processing of caspase-8 was detected in MCF7/F cells following anti-Fas or TNF-alpha, but not during UV-induced apoptosis. In MCF7/FB cells, caspase-8 is processed normally, while processing of the downstream caspase-7 is markedly attenuated. Apoptosis induced by direct microinjection of recombinant, active caspase-8 is completely inhibited by Bcl-xL. These data demonstrate that Bcl-xL can exert an anti-apoptotic function in cells in which caspase-8 is activated. Thus, at least in some cells, caspase-8 signaling in response to Fas or TNFR1 stimulation is regulated by a Bcl-xL-inhibitable step (Srinivasan, 1998).

Caspases comprise a family of cysteine proteases implicated in the biochemical and morphological changes that occur during apoptosis (programmed cell death). The loop domain of Bcl-2 is cleaved at Asp34 by caspase-3 (CPP32) in vitro, in cells overexpressing caspase-3, and after induction of apoptosis by Fas ligation and interleukin-3 withdrawal. The carboxyl-terminal Bcl-2 cleavage product triggers cell death and accelerates Sindbis virus-induced apoptosis, which is dependent on the BH3 homology and transmembrane domains of Bcl-2. Inhibitor studies indicate that cleavage of Bcl-2 may further activate downstream caspases and contribute to amplification of the caspase cascade. Cleavage-resistant mutants of Bcl-2 have increased protection from interleukin-3 withdrawal and Sindbis virus-induced apoptosis. Thus, cleavage of Bcl-2 by caspases may ensure the inevitability of cell death (Cheng, 1997).

Apoptotic cell death is driven by ICE family proteases (caspases) and negatively regulated by Bcl-2 family proteins. Although it has been shown that Bcl-2 exerts anti-apoptotic activity by blocking a step(s) leading to the activation of caspases, a role for Bcl-2 and Bcl-xL downstream of the caspase cascade has remained unclear. Purified active caspase-3 (CPP32/Yama/apopain) and caspase-1 (ICE) induce apoptosis when microinjected into the cytoplasm of cells; the apoptosis is not at all prevented by Bcl-2 and Bcl-xL, which are overexpressed more than sufficiently to prevent Fas-mediated and overexpressed procaspase-1-mediated apoptosis. Thus, Bcl-2 and Bcl-xL do not act downstream of the caspase cascade (Yasuhara, 1997).

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