Interactive Fly, Drosophila

Caspase 8 and Fas activation of apoptosis

The pivotal discovery that Fas-associated death domain protein (FADD) interleukin-1beta-converting enzyme (FLICE)/MACH is recruited to the CD95 signaling complex by virtue of CD95's ability to bind the adapter molecule FADD establishes that this protease has a role in initiating the death pathway. A new member of the caspase family has been cloned, a homologue of FLICE/MACH (Caspase 8), and Mch4. Since the overall architecture and function of this molecule is similar to that of FLICE, it has been designated FLICE2. Importantly, the carboxyl-terminal half of the small catalytic subunit that includes amino acids predicted to be involved in substrate binding is distinct. The pro-domain of FLICE2 encodes a functional death effector domain that binds to the corresponding domain in the adapter molecule FADD. Consistent with this finding, FLICE2 is recruited to both the CD95 and p55 tumor necrosis factor receptor signaling complexes in a FADD-dependent manner. A functional role for FLICE2 is suggested by the finding that an active site mutant of FLICE2 inhibits CD95 and tumor necrosis factor receptor-mediated apoptosis. FLICE2 is therefore involved in CD95 and p55 signal transduction (Vincenz, 1997).

Human CLARP, a caspase-like apoptosis-regulatory protein, contains two amino-terminal death effector domains fused to a carboxyl-terminal caspase-like domain. The structure and amino acid sequence of CLARP resemble those of caspase-8, caspase-10, and DCP2, a Drosophila melanogaster protein identified in this study. Unlike caspase-8, caspase-10, and DCP2, however, two important residues predicted to be involved in catalysis were lost in the caspase-like domain of CLARP. Analysis with fluorogenic substrates for caspase activity confirms that CLARP is catalytically inactive. CLARP interacts with caspase-8 but not with FADD/MORT-1, an upstream death effector domain-containing protein of the Fas and tumor necrosis factor receptor 1 signaling pathway. Expression of CLARP induces apoptosis, which is blocked by the viral caspase inhibitor p35, dominant negative mutant caspase-8, and the synthetic caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethylketone (zVAD-fmk). Moreover, CLARP augments the killing ability of caspase-8 and FADD/MORT-1 in mammalian cells. The human clarp gene maps to 2q33. Thus, CLARP represents a regulator of the upstream caspase-8, which may play a role in apoptosis during tissue development and homeostasis (Inohara, 1997).

Caspase 9 and Apaf: Apaf is a homolog of C. elegans CED-4

CED-4 protein plays an important role in the induction of programmed cell death in Caenorhabditis elegans through the activation of caspases. CED-4 acts as a positive regulator of caspases by enhancing the processing of procaspases to their mature forms. In mammalian 293 cells and insect SF21 cells, the processing of the proform of caspase CED-3 to its mature form is facilitated by CED-4, resulting in the acceleration of CED-3-induced cell death. CED-4 can directly activate CED-3 and ATP hydrolysis associated with the ATP binding site (P-loop) of CED-4 is required for CED-3 processing. Additionally, Apaf-1, a mammalian homolog of CED-4 that also has a P-loop motif, activates caspase-9 in the presence of cytochrome c and dATP, resulting in the sequential activation of caspase-3 in vitro. The CED-3/CED-4 complex has been shown to be activated by the oligomerization of CED-4. To investigate the conservation of CED-4 function in evolution, transgenic Drosophila lines that express CED-4 in the compound eye were generated. Ectopic expression of CED-4 in the eyes induces massive apoptotic cell death through caspase activation. An ATP-binding site (P-loop) mutation in CED-4 (K165R) causes a loss of function in its ability to activate Drosophila caspase, and an ATPase inhibitor blocks the CED-4-dependent caspase activity in Drosophila S2 cells. Immunoprecipitation analysis has shown that in S2 cells, both CED-4 and CED-4 (K165R) bind directly to Drosophila caspase drICE, and the overexpression of CED-4 (K165R) inhibits CED-4-, ecdysone-, or cycloheximide-dependent caspase activation. Furthermore, CED-4 (K165R) partially preventes cell death induced by CED-4 in Drosophila compound eyes. Thus, CED-4 function is evolutionarily conserved in Drosophila, and the molecular mechanisms by which CED-4 activates caspases might require ATP binding and direct interaction with the caspases (Kanuka, 1999).

The exit of cytochrome c from mitochondria into the cytosol has been implicated as an important step in apoptosis. In the cytosol, cytochrome c binds to the CED-4 homolog, Apaf-1, thereby triggering Apaf-1-mediated activation of caspase-9. Caspase-9 is thought to propagate the death signal by triggering other caspase activation events, the details of which remain obscure. Six additional caspases (caspases-2, -3, -6, -7, -8, and -10) are processed in cell-free extracts in response to cytochrome c, and three others (caspases-1, -4, and -5) fail to be activated under the same conditions. In vitro association assays confirm that caspase-9 selectively binds to Apaf-1, whereas caspases-1, -2, -3, -6, -7, -8, and -10 do not. Depletion of caspase-9 from cell extracts abrogates cytochrome c-inducible activation of caspases-2, -3, -6, -7, -8, and -10, suggesting that caspase-9 is required for all of these downstream caspase activation events. Immunodepletion of caspases-3, -6, and -7 from cell extracts enables an ordering of the sequence of caspase activation events downstream of caspase-9 and reveals the presence of a branched caspase cascade. Caspase-3 is required for the activation of four other caspases (-2, -6, -8, and -10) in this pathway and also participates in a feedback amplification loop involving caspase-9 (Slee, 1999).

Caspase 9 (Casp9)/Apaf3, a 45 kDa protein (also known as ICE-LAP-6 or Mch6) forms a multiprotein complex containing Apaf1 and cytochrome c. It has been proposed that cytochrome c initiates apoptosis by inducing the formation of the Casp9/Apaf1 complex. Physical association of Casp9 and Apaf1 is mediated by the interaction of their respective caspase recruitment domains (CARDs). CARDs are also found in other caspases with large prodomains, such as Casp4 and Casp8, that can associate with Apaf1 in mammalian cells. The antiapoptotic protein Bcl-XL has also been shown to interact with Casp9 and Apaf1, resulting in the inhibition of Casp9 activation. The association of Casp9 with antiapoptotic as well as proapoptotic proteins suggests a major role for Casp9 in the control of apoptosis in vivo. Mutation of Caspase 9 (Casp9) results in embryonic lethality and defective brain development associated with decreased apoptosis. The absence of Casp9 leads to a dramatic disturbance of telencephalic development that apparently results from decreased apoptosis in this region. The gross morphological features observed in Casp9-/- mice are remarkably similar to those observed in mice lacking Casp3. Both mutants exhibit a profound disturbance of cortical morphology, an expanded germinal zone, and hydrocephaly, suggesting that these mutations affect a common cellular apoptotic pathway dependent on both Casp9 and Casp3. Casp9-/- embryonic stem cells and embryonic fibroblasts are resistant to several apoptotic stimuli, including UV and gamma irradiation. Casp9-/- thymocytes are also resistant to dexamethasone- and gamma irradiation-induced apoptosis, but are surprisingly sensitive to apoptosis induced by UV irradiation or anti-CD95. Resistance to apoptosis is accompanied by retention of the mitochondrial membrane potential in mutant cells. In addition, cytochrome c is translocated to the cytosol of Casp9-/- ES cells upon UV stimulation, suggesting that Casp9 acts downstream of cytochrome c. The Casp9-dependent, Casp3-independent apoptotic pathway is preferentially triggered in thymocytes in response to dexamethasone. However, the fact that Casp3 is still processed in dexamethasone-treated Casp9-/- thymocytes suggests that dexamethasone also activates a Casp9-independent and Casp3-dependent apoptotic pathway in these cells. Comparison of the requirement for Casp9 and Casp3 in different apoptotic settings indicates the existence of multiple apoptotic pathways in mammalian cells (Hakem,1998).

The hypothesis that Caspase 9 (Casp9) is a critical upstream activator of caspases was tested through gene targeting in mice. The majority of Casp9 knockout mice die perinatally with a markedly enlarged and malformed cerebrum caused by reduced apoptosis during brain development. Casp9 deletion prevents activation of Casp3 in embryonic brains in vivo, and Casp9-deficient thymocytes show resistance to a subset of apoptotic stimuli, including absence of Casp3-like cleavage and delayed DNA fragmentation. Moreover, the cytochrome c-mediated cleavage of Casp3 is absent in the cytosolic extracts of Casp9-deficient cells but is restored after addition of in vitro-translated Casp9. Together, these results indicate that Casp9 is a critical upstream activator of the caspase cascade in vivo (Kuida, 1998).

Apaf-1 (apoptotic protease activating factor-1), a novel 130 kd protein from HeLa cell cytosol, participates in the cytochrome c-dependent activation of caspase-3. The release of cytochrome c from mitochondria in response to apoptotic stimuli is blocked in cells overexpresssing Bcl-2. The NH2-terminal 85 amino acids of Apaf-1 show 21% identity and 53% similarity to the NH2-terminal prodomain of the Caenorhabditis elegans caspase, CED-3. However, Apaf-1 does not seem to be a caspase, since the conserved active site pentapeptide that is present in all identified caspases is not present in Apaf-1. The caspase homologous region is followed by 320 amino acids that show 22% identity and 48% similarity to CED-4, a protein that is believed to initiate apoptosis in C. elegans. The COOH-terminal region of Apaf-1 comprises multiple WD repeats, which are proposed to mediate protein-protein interactions. Cytochrome c binds to Apaf-1, an event that may trigger the activation of caspase-3, leading to apoptosis (Zou, 1997).

This paper reports the purification of Apaf-3, a third protein factor that participates in caspase-3 activation in vitro. Apaf-3 is a member of the caspase family: specifically, 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. In turn, activated caspase-9 cleaves and activates caspase-3. Depletion of caspase-9 from S-100 extracts diminishes 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 triggered by cytochrome c and dATP. Several caspases, including caspase-9, have long prodomains at their NH2 termini. This domain has been proposed to function as a caspase recruitment domain (CARD), allowing proteins with such domains to interact with each other. Although Apaf-1 is not a caspase, its NH2-terminal region contains a CARD, suggesting that Apaf-1 may recruit caspase-9 through their respective CARDs. The data suggest that, within the context of full-length Apaf-1, the CARD is not accessible for caspase-9 binding. Cytochrome c and dATP may induce a conformational change in Apaf-1 that exposes its CARD (P. Li, 1997).

Recent studies indicate that Caenorhabditis elegans CED-4 interacts with and promotes the activation of the death protease CED-3, and that this activation is inhibited by CED-9. A mammalian homolog of CED-4, Apaf-1, can associate with several death proteases in mammalian cells, including caspase-4, caspase-8, caspase-9, and nematode CED-3. The interaction with caspase-9 was mediated by the N-terminal CED-4-like domain of Apaf-1. Expression of Apaf-1 enhances the killing activity of caspase-9, which requires the CED-4-like domain of Apaf-1. Furthermore, Apaf-1 promotes the processing and activation of caspase-9 in vivo. Bcl-XL, an antiapoptotic member of the Bcl-2 family, has been shown to physically interact with Apaf-1 and caspase-9 in mammalian cells. The association of Apaf-1 with Bcl-XL is mediated through both domains of Apaf-1: the CED-4-like domain and the C-terminal domain containing WD-40 repeats. Expression of Bcl-XL inhibits the association of Apaf-1 with caspase-9 in mammalian cells. Significantly, recombinant Bcl-XL purified from Escherichia coli or insect cells inhibits Apaf-1-dependent processing of caspase-9. Furthermore, Bcl-XL fails to inhibit caspase-9 processing mediated by a constitutively active Apaf-1 mutant, suggesting that Bcl-XL regulates caspase-9 through Apaf-1. These experiments demonstrate that Bcl-XL associates with caspase-9 and Apaf-1, and show that Bcl-XL inhibits the maturation of caspase-9 mediated by Apaf-1, a process that is evolutionarily conserved from nematodes to humans (Hu, 1998).

Apoptosis is essential for the precise regulation of cellular homeostasis and development. The role in vivo of Apaf1, a mammalian homolog of C. elegans CED-4, was investigated in gene-targeted Apaf1-/- mice. Apaf1-deficient mice exhibit reduced apoptosis in the brain and striking craniofacial abnormalities with hyperproliferation of neuronal cells. Apaf1-deficient cells are resistant to a variety of apoptotic stimuli, and the processing of Caspases 2, 3, and 8 is impaired. However, both Apaf1-/- thymocytes and activated T lymphocytes are sensitive to Fas-induced killing, showing that Fas-mediated apoptosis in these cells is independent of Apaf1. These data indicate that Apaf1 plays a central role in the common events of mitochondria-dependent apoptosis in most death pathways and that this role is critical for normal development (Yoshida, 1998).

The ability of p53 to promote apoptosis in response to mitogenic oncogenes appears to be critical for its tumor suppressor function. Caspase-9 and its cofactor Apaf-1 are essential downstream components of p53 in Myc-induced apoptosis. Like p53 null cells, mouse embryo fibroblast cells deficient in Apaf-1 and caspase-9, and expressing c-Myc, are resistant to apoptotic stimuli that mimic conditions in developing tumors. Inactivation of Apaf-1 or caspase-9 substitutes for p53 loss in promoting the oncogenic transformation of Myc-expressing cells. These results imply a role for Apaf-1 and caspase-9 in controlling tumor development (Soengas, 1999).

The cytosolic protein APAF1, a human homolog of C. elegans CED-4, participates in the CASPASE 9 (CASP9)-dependent activation of CASP3 in the general apoptotic pathway. A null allele of the murine Apaf1 was generated by a gene trap. Homozygous mutants die at embryonic day 16.5. Their phenotype includes severe craniofacial malformations, brain overgrowth, persistence of the interdigital webs, and dramatic alterations of the lens and retina. Homozygous embryonic fibroblasts exhibit reduced response to various apoptotic stimuli. In situ immunodetection shows that the absence of Apaf1 protein prevents the activation of Casp3 in vivo. In agreement with the reported function of CED-4 in C. elegans, this phenotype can be correlated with a defect of apoptosis. Thesefindings suggest that Apaf1 is essential for Casp3 activation in embryonic brain and is a key regulator of developmental programmed cell death in mammals (Cecconi, 1998).

Caspase-9 is critical for cytochrome c (cyto-c)-dependent apoptosis and normal brain development. This apical protease in the cyto-c pathway for apoptosis resides inside mitochondria in several types of cells, including cardiomyocytes and many neurons. Caspase-9 is released from isolated mitochondria on treatment with Ca2+ or Bax, stimuli implicated in ischemic neuronal cell death that are known to induce cyto-c release from mitochondria. In neuronal cell culture models, apoptosis-inducing agents trigger translocation of caspase-9 from mitochondria to the nucleus, which is inhibitable by Bcl-2. Similarly, in an animal model of transient global cerebral ischemia, caspase-9 release from mitochondria and accumulation in nuclei is observed in hippocampal and other vulnerable neurons exhibiting early postischemic changes preceding apoptosis. Therefore, loss of mitochondrial barrier function during neuronal damage from ischemia or other insults may play an important role in making certain caspases available to participate in apoptosis (Krajewski, 1999).

Table of contents

Death caspase-1: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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