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Death receptors are a subset of the tumor necrosis factor receptor (TNFR) family of proteins and share a characteristic cytoplasmic motif called the “death domain.” In addition to mediating cell death, these receptors regulate cell proliferation, inflammatory responses, and tumor progression. Receptor occupancy triggers the assembly of several cytoplasmic molecules into distinct complexes, each initiating separate signaling events leading to different biological responses. Post-translational modifications involving ubiquitin, a peptide of 76 amino acids, regulate events at nearly all stages of signaling. All ubiquitin chains function as docking platforms for molecules with specific recognition motifs that either propagate the signal or target the protein for proteasomal degradation. Moreover, enzymes with ubiquitin thioesterase activity (deubiquitinating enzymes, or DUBs) reverse modifications by removing the ubiquitin chains, allowing ubiquitin editing at the molecular level. Ubiquitin protein ligases (E3s), DUBs, and signaling molecules with ubiquitin recognition motifs control TNFR1 mediated cell death and activation of NF-κB and JNK. Here, we discuss the current understanding of how these proteins regulate TNFR1 signaling.

Originally described in insect viruses, cellular proteins with Baculoviral IAP repeat (BIR) motifs have been thought to function primarily as inhibitors of apoptosis. The subsequent finding that a subset of IAPs that contain a RING domain have ubiquitin protein ligase (E3) activity implied the presence of other functions. It is now known that IAPs are involved in mitotic chromosome segregation, cellular morphogenesis, copper homeostasis, and intracellular signaling. Here, we review the current understanding of the roles of IAPs in apoptotic and nonapoptotic processes and explore the notion that the latter represents the primary physiologic activities of IAPs.

Summary Background The proinflammatory cytokine tumor necrosis factor-α (TNF-α) elicits cellular responses by signaling through a receptor complex that includes the essential adaptor molecule RIP. One important consequence of signaling is activation of the transcription factor NF-κB, and failure to downregulate TNF-induced NF-κB transcriptional activity results in chronic inflammation and death. Internalization of the receptor complex plays an important regulatory role in TNF signaling. Results We report that CARP-2, a RING domain-containing ubiquitin protein ligase (E3), is a negative regulator of TNF-induced NF-κB activation. By virtue of its phospholipid-binding FYVE domain, CARP-2 localized to endocytic vesicles, where it interacted with internalized TNF-receptor complex, resulting in RIP ubiquitination and degradation. Knockdown of CARP-2 stabilized TNFR1-associated polyubiquitinated RIP levels after TNF simulation and enhanced activation of NF-κB. Conclusions CARP-2 acts at the level of endocytic vesicles to limit the intensity of TNF-induced NF-κB activation by the regulated elimination of a necessary signaling component within the receptor complex.

We have isolated a novel interferon (IFN)-retinoid regulated cell death regulatory protein genes associated with retinoid-IFN-induced mortality (GRIM)-19 earlier. To understand its mechanism of action, we have employed a yeast-two-hybrid screen and identified serine protease HtrA2 as its binding partner. GRIM-19 physically interacts with HtrA2 and augments cell death in an IFN/all-trans retinoic acid (RA)-dependent manner. In the presence of GRIM-19, the HtrA2-driven destruction of the antiapoptotic protein X-linked inhibitor of apoptosis (XIAP) is augmented. These interactions were disrupted by an human herpes virus-8 (HHV-8)-coded oncoprotein, vIRF1, and conferred resistance to IFN/RA-induced cell death. These data show a critical role of HtrA2 in a cytokine-induced cell death response for the first time and its inhibition by a viral protein.

The transcription factor NF-kappaB is sequestered in the cytoplasm in a complex with IkappaB. Almost all NF-kappaB activation pathways converge on IkappaB kinase (IKK), which phosphorylates IkappaB resulting in Lys 48-linked polyubiquitination of IkappaB and its degradation. This allows migration of NF-kappaB to the nucleus where it regulates gene expression. IKK has two catalytic subunits, IKKalpha and IKKbeta, and a regulatory subunit, IKKgamma or NEMO. NEMO is essential for NF-kappaB activation, and NEMO dysfunction in humans is the cause of incontinentia pigmenti and hypohidrotic ectodermal dysplasia and immunodeficiency (HED-ID). The recruitment of IKK to occupied cytokine receptors, and its subsequent activation, are dependent on the attachment of Lys 63-linked polyubiquitin chains to signalling intermediates such as receptor-interacting protein (RIP). Here, we show that NEMO binds to Lys 63- but not Lys 48-linked polyubiquitin, and that single point mutations in NEMO that prevent binding to Lys 63-linked polyubiquitin also abrogates the binding of NEMO to RIP in tumour necrosis factor (TNF)-alpha-stimulated cells, the recruitment of IKK to TNF receptor (TNF-R) 1, and the activation of IKK and NF-kappaB. RIP is also destabilized in the absence of NEMO binding and undergoes proteasomal degradation in TNF-alpha-treated cells. These results provide a mechanism for NEMO's critical role in IKK activation, and a key to understanding the link between cytokine-receptor proximal signalling and IKK and NF-kappaB activation.

Caspases are responsible for the execution of programmed cell death (apoptosis) and must undergo proteolytic activation, in response to apoptotic stimuli, to function. The mechanism of initiator caspase activation has been generalized by the induced proximity model, which is thought to drive dimerization-mediated activation of caspases. The initiator caspase, caspase-9, exists predominantly as a monomer in solution. To examine the induced proximity model, we engineered a constitutively dimeric caspase-9 by relieving steric hindrance at the dimer interface. Crystal structure of the engineered caspase-9 closely resembles that of the wild-type (WT) caspase-9, including all relevant structural details and the asymmetric nature of two monomers. Compared to the WT caspase-9, this engineered dimer exhibits a higher level of catalytic activity in vitro and induces more efficient cell death when expressed. However, the catalytic activity of the dimeric caspase-9 is only a small fraction of that for the Apaf-1-activated caspase-9. Furthermore, in contrast to the WT caspase-9, the activity of the dimeric caspase-9 can no longer be significantly enhanced in an Apaf-1-dependent manner. These findings suggest that dimerization of caspase-9 may be qualitatively different from its activation by Apaf-1, and in conjunction with other evidence, posit an induced conformation model for the activation of initiator caspases., Evidence from an engineered caspase-9 dimer suggests that, contrary to the prevailing hypothesis, dimerization may not be the critical step in caspase-9 activation.

Glioblastoma multiforme, the most common brain tumor, typically exhibits markedly increased angiogenesis, which is crucial for tumor growth and invasion. Antiangiogenic strategies based on disruption of the tumor microvasculature have proven effective for the treatment of experimental brain tumors. Here, we have overexpressed human caspase-9 by stable transfection in the SNB19 glioblastoma cell line, which normally expresses low levels of caspase-9. Our studies revealed that overexpression of caspase-9 coupled with radiation has a synergistic effect on the inhibition of glioma invasion as demonstrated by Matrigel assay (> 65%). Furthermore, sense caspase stable clones cocultured with fetal rat brain aggregates along with radiation showed complete inhibition as compared to the parental and vector controls. During in vitro angiogenesis, SNB19 cells cocultured with human microvascular endothelial cells (HMEC) showed vascular network formation after 48-72 h. In contrast, these capillary-like structures were inhibited when HMEC cells were cocultured with sense caspase stable SNB19 cells. This effect was further enhanced by radiation (5 Gy). Signaling mechanisms revealed that apoptosis is induced by cleavage of caspase-9 by radiation, loss of mitochondrial membrane potential and activation of caspase-3. These results demonstrate that activation of caspase-9 disrupts glioma cell invasion and angiogenesis in vitro. Hence, overexpression of proapoptotic molecules such as caspase-9 may be an important determinant of the therapeutic effect of radiation in cancer therapy.

Caspases mediate essential key proteolytic events in inflammatory cascades and the apoptotic cell death pathway. Human caspases functionally segregate into two distinct subfamilies: those involved in cytokine maturation (caspase-1, -4 and -5) and those involved in cellular apoptosis (caspase-2, -3, -6, -7, -8, -9 and -10)1,2. Although caspase-12 is phylogenetically related to the cytokine maturation caspases, in mice it has been proposed as a mediator of apoptosis induced by endoplasmic reticulum stress including amyloid-β cytotoxicity, suggesting that it might contribute to the pathogenesis of Alzheimer's disease3. Here we show that a single nucleotide polymorphism in caspase-12 in humans results in the synthesis of either a truncated protein (Csp12-S) or a full-length caspase proenzyme (Csp12-L). The read-through single nucleotide polymorphism encoding Csp12-L is confined to populations of African descent and confers hypo-responsiveness to lipopolysaccharide-stimulated cytokine production in ex vivo whole blood, but has no significant effect on apoptotic sensitivity. In a preliminary study, we find that the frequency of the Csp12-L allele is increased in African American individuals with severe sepsis. Thus, Csp12-L attenuates the inflammatory and innate immune response to endotoxins and in doing so may constitute a risk factor for developing sepsis.

Smac/Diablo and HtrA2/Omi are inhibitors of apoptosis (IAP)-binding proteins released from the mitochondria of human cells during apoptosis and regulate apoptosis by liberating caspases from IAP inhibition. Here we describe the identification of a proteolytically processed isoform of the polypeptide chain-releasing factor GSPT1/eRF3 protein, which functions in translation, as a new IAP-binding protein. In common with other IAP-binding proteins, the processed GSPT1 protein harbors a conserved N-terminal IAP-binding motif (AKPF). Additionally, processed GSPT1 interacts biochemically with IAPs and could promote caspase activation, IAP ubiquitination and apoptosis. The IAP-binding motif of the processed GSPT1 is absolutely required for these activities. Our findings are consistent with a model whereby processing of GSPT1 into the IAP-binding isoform could potentiate apoptosis by liberating caspases from IAP inhibition, or target IAPs and the processed GSPT1 for proteasome-mediated degradation.

Apoptosis control in adult testis is crucial to achieve normal spermatogenesis. In this study c-FLIP, an apoptosis-modulating protein, was investigated. In Western blot and immunohistochemical analyses, the 55 KDa c-FLIP long isoform (c-FLIP(L)) was found to be expressed strongly in spermatocytes and spermatids, at low levels in spermatogonia and at almost undetectable levels in Sertoli cells. This expression pattern was confirmed by Northern blot analyses. Further experiments carried out on GC-1spg germ cell line revealed that reducing c-FLIP(L) expression increases Fas-dependent apoptosis. Conversely, restoring c-FLIP(L) expression reduces this response to control levels. Caspase-10 expression was found to match c-FLIP(L) expression pattern; further, caspase-10 activation upon anti-Fas treatment inversely correlated with c-FLIP(L) expression. Finally, TUNEL staining of seminiferous tubules incubated with anti-Fas antibody showed that apoptosis occurs mostly in basally located germ cells, indicating that such cells, expressing low levels of c-FLIP(L), are sensitive to Fas-mediated apoptosis. These data indicate for the first time that c-FLIP(L) might control germ cell apoptosis and caspase activity in the adult testis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo2L induces apoptosis in a wide variety of cancer and transformed cells. Activation of BID, a "BH3-domain-only" Bcl-2 family member, triggers the oligomerization of proapoptotic family members Bak or Bax, resulting in the release of mitochondrial proteins to cytosol. In this study, we have shown the importance of Bax and Bak in TRAIL-induced apoptosis by studying in murine embryonic fibroblasts (MEFs) from Bax(-/-) and Bak(-/-) animals. TRAIL induced cytochrome c release and apoptosis in wild-type, Bid(-/-), Bax(-/-), or Bak(-/-) MEFs, but not in Bax(-/-) Bak(-/-) double knockout (DKO) MEFs. Bid, which functions upstream of cytochrome c release, was cleaved in all of the knockout cells except in Bid(-/-) MEFs. The release of cytochrome c was correlated with caspase-9 activity. TRAIL increased caspase-3 activity in all of the cells except in DKO cells. TRAIL-induced drop in mitochondrial membrane potential was not observed in DKO MEFs. Unlike cytochrome c release, TRAIL-induced Smac/DIABLO release was blocked in Bid(-/-), Bax(-/-), Bak(-/-), or DKO MEFs, suggesting the differential regulation of these mitochondrial proteins during apoptosis. The apoptotic events downstream of mitochondria were intact in DKO MEFs, because microinjection of cytochrome c, or ectopic expression of mature Smac/DIABLO or pretreatment of Smac N7 peptide completely restored TRAIL sensitivity. In conclusion, the data suggest that Bax and Bak differentially regulate the release of cytochrome c and Smac/DIABLO from mitochondria, and Smac/DIABLO can be used to sensitize cells that are deficient in Bax and Bak genes, or resistant to TRAIL.

Smac (or DIABLO) is a recently identified, novel proapoptotic molecule, which is released from mitochondria into the cytosol during apoptosis. Smac functions by eliminating the caspase-inhibitory properties of the inhibitors of apoptosis proteins (IAP), particularly XIAP. In this study, we stably transfected both full-length (FL) and mature (MT) Smac genes into the K562 and CEM leukaemic cell lines. Both FL and MT Smac transfectants increased the sensitivity of leukaemic cells to UV light-induced apoptosis and the activation of caspase-9 and caspase-3. Purified cytosol from the mature Smac transfectants, or the addition of human recombinant Smac protein or N-7 peptide into nontransfected cytosol, showed an increased sensitivity to cytochrome c-induced activation of caspase-3. The mature Smac enhanced the susceptibility of both K562 and CEM cells to TRAIL-induced apoptosis. Overexpression of the mature Smac protein also inhibited proliferation, as detected by reduced colony formation and Ki-67 expression in leukaemic cells. Cell cycle analysis revealed that Smac transfectants displayed significant G0/G1 arrest and reduction in 5-bromo-2'-deoxyuridine (BrdU) incorporation. Smac sensitized human acute myeloid leukaemia blasts to cytochrome c-induced activation of caspase-3. However, Smac failed to overcome Apaf-1-deficiency-mediated resistance to cytochrome c in primary leukaemic blasts. In summary, this study reveals that Smac/DIABLO exhibits a potential role in increasing apoptosis and suppressing proliferation in human leukaemic cells. Importantly, it also indicates that it is crucial to evaluate the levels of Apaf-1 and XIAP proteins in patient samples before using Smac peptide therapy in the treatment of human leukaemia.

Omi/HtrA2 is a mammalian serine protease with high homology to bacterial HtrA chaperones. Omi/HtrA2 is localized in mitochondria and is released to the cytoplasm in response to apoptotic stimuli. Omi/HtrA2 induces cell death in a caspase-dependent manner by interacting with the inhibitor of apoptosis protein as well as in a caspase-independent manner that relies on its protease activity. We describe the identification and characterization of a novel compound as a specific inhibitor of the proteolytic activity of Omi/HtrA2. This compound (ucf-101) was isolated in a high throughput screening of a combinatorial library using bacterially made Omi-(134-458) protease and fluorescein-casein as a generic substrate. ucf-101 showed specific activity against Omi/HtrA2 and very little activity against various other serine proteases. This compound has a natural fluorescence that was used to monitor its ability to enter mammalian cells. ucf-101, when tested in caspase-9 (-/-) null fibroblasts, was found to inhibit Omi/HtrA2-induced cell death.

SCL/Tal-1 is a helix-loop-helix (HLH) transcription factor required for blood cell development, whose abnormal expression is responsible for induction of T-cell acute lymphoblastic leukemia. We show here that SCL/Tal-1 is a key target of caspases in developing erythroblasts. SCL/Tal-1 degradation occurred rapidly after caspase activation and preceded the cleavage of the major erythroid transcription factor GATA-1. Expression of a caspase-resistant SCL/Tal-1 in erythroid progenitors was able to prevent amplification of caspase activation, GATA-1 degradation and impaired erythropoiesis induced by growth factor deprivation or death receptor triggering. The potent proerythropoietic activity of uncleavable SCL/Tal-1 was clearly evident in the absence of erythropoietin, a condition that did not allow survival of normal erythroid cells or expansion of erythroblasts expressing caspase-resistant GATA-1. In the absence of erythropoietin, cells expressing caspase-resistant SCL/Tal-1 maintain high levels of Bcl-X(L), which inhibits amplification of the caspase cascade and mediates protection from apoptosis. Thus, SCL/TAL-1 is a survival factor for erythroid cells, whereas caspase-mediated cleavage of SCL/Tal-1 results in amplification of caspase activation, GATA-1 degradation and impaired erythropoiesis.

HtrA2/Omi, a mitochondrial serine protease in mammals, is important in programmed cell death. However, the underlining mechanism of HtrA2/Omi-mediated apoptosis remains unclear. Analogous to the bacterial homolog HtrA (DegP), the mature HtrA2 protein contains a central serine protease domain and a C-terminal PDZ domain. The 2.0 A crystal structure of HtrA2/Omi reveals the formation of a pyramid-shaped homotrimer mediated exclusively by the serine protease domains. The peptide-binding pocket of the PDZ domain is buried in the intimate interface between the PDZ and the protease domains. Mutational analysis reveals that the monomeric HtrA2/Omi mutants are unable to induce cell death and are deficient in protease activity. The PDZ domain modulates HtrA2/Omi-mediated cell death activity by regulating its serine protease activity. These structural and biochemical observations provide an important framework for deciphering the mechanisms of HtrA2/Omi-mediated apoptosis.

The PYRIN and CARD domains are members of the six-helix bundle death domain-fold superfamily that mediates assembly of large signaling complexes in the apoptotic and inflammatory signaling pathways. Here we show that the PYRIN-CARD protein ASC functions as a caspase-1-activating adaptor. ASC interacted specifically with procaspase-1 via CARD-CARD interactions and induced its oligomerization. Consistent with these results ectopic expression of full-length ASC, but not its isolated CARD or PYRIN domain, with procaspase-1 induced activation of procaspase-1 and processing of pro-interleukin-1beta in transfected cells. Substitution of the PYRIN domain of ASC with an inducible FKBP12 oligomerization domain produced a molecule that can induce caspase-1 activation in response to stimulation with the oligomerization drug AP20187, suggesting that the PYRIN domain functions as an oligomerization domain, whereas the CARD domain functions as the effector domain in the caspase-1 activation pathway. Furthermore stable expression of an isolated CARD of ASC in THP-1 cells diminished interleukin-1beta generation in response to pro-inflammatory cytokines. These results indicate that ASC is involved in the caspase-1 signaling pathway by mediating the assembly of a caspase-1-inflammasome signaling complex in response to pro-inflammatory cytokine stimulation.

Death effector domain-containing proteins are involved in important cellular processes such as death-receptor induced apoptosis, NF-kappaB activation and ERK activation. Here we report the identification of a novel nuclear DED-containing protein, FLAME-3. FLAME-3 shares significant sequence (46.6% identical) and structural homology to another DED-containing protein, DEDD. FLAME-3 interacts with DEDD and c-FLIP (FLAME-1) but not with the other DED-containing proteins FADD, caspase-8 or caspase-10. FLAME-3 translocates to, and sequesters c-FLIP in the nucleus upon overexpression in human cell lines. Using the yeast two-hybrid system to identify DEDD-interacting proteins, the TFIIIC102 subunit of human transcription factor TFIIIC was identified as a DEDD- and FLAME-3-specific interacting protein. Co-expression of either DEDD or FLAME-3 with hTFIIIC102 in MCF-7 cells induces the translocation from the cytoplasm and sequestration of hTFIIIC102 in the nucleus, indicating that DEDD and FLAME-3 form strong heterocomplexes with hTFIIIC102 and might be important regulators of the activity of the hTFIIIC transcriptional complex. Consistent with this, overexpression of DEDD or FLAME-3 in 293 cells inhibited the expression of a luciferase-reporter gene under the control of the NF-kappaB promoter. Our data provide the first direct evidence for the involvement of DED-containing proteins in the regulation of components of the general transcription machinery in the nucleus.

Caspase-2 is one of the earliest identified caspases, but the mechanism of caspase-2-induced apoptosis remains unknown. We show here that caspase-2 engages the mitochondria-dependent apoptotic pathway by inducing the release of cytochrome c (Cyt c) and other mitochondrial apoptogenic factors into the cell cytoplasm. In support of these observations we found that Bcl-2 and Bcl-xL can block caspase-2- and CRADD (caspase and RIP adaptor with death domain)-induced cell death. Unlike caspase-8, which can process all known caspase zymogens directly, caspase-2 is completely inactive toward other caspase zymogens. However, like caspase-8, physiological levels of purified caspase-2 can cleave cytosolic Bid protein, which in turn can trigger the release of Cyt c from isolated mitochondria. Interestingly, caspase-2 can also induce directly the release of Cyt c, AIF (apoptosis-inducing factor), and Smac (second mitochondria-derived activator of caspases protein) from isolated mitochondria independent of Bid or other cytosolic factors. The caspase-2-released Cyt c is sufficient to activate the Apaf-caspase-9 apoptosome in vitro. In combination, our data suggest that caspase-2 is a direct effector of the mitochondrial apoptotic pathway.

Caspase-associated recruitment domains (CARD) are protein-protein interaction modules found extensively in proteins that play important roles in apoptosis, NFkappaB activation, and cytokine regulation. In this study we identified a novel human protein, CARD-8, which contains a C-terminal CARD domain with high similarity to the CARD domain of caspase-1/ICE. We demonstrate that CARD-8 interacts physically with caspase-1 and negatively regulates caspase-1-dependent IL-1beta generation in the THP-1 monocytic cell line. CARD-8 binds also to ICEBERG and pseudo-ICE, two other recently identified proteins, which bind to the CARD domain of caspase-1 and negatively regulate its activity. Reverse transcriptase-PCR analysis revealed that CARD-8 is expressed mainly in monocytes, placenta, lymph nodes, and spleen. This pattern of expression is consistent with caspase-1 expression in the same cells and tissues. CARD-8 was also found to negatively regulate NF-kappaB activation by TNF-alpha stimulation and by ectopically expressed RICK, suggesting that this protein may control cell survival. Consistent with these results, stable expression of CARD-8 in U937 or THP-1 cells sensitizes the cells to differentiation-induced apoptosis. Overexpression of CARD-8 can also induce apoptosis in transfected cells. The results suggest that CARD-8 represents a new signaling molecule involved in the regulation of caspase-1 and NF-kappaB activation.

A20 is a potent anti-inflammatory protein that inhibits NF-κB, and A20 dysfunction is associated with autoimmunity and B-cell lymphoma. A20 harbors a deubiquitination enzyme domain and can employ multiple mechanisms to antagonize ubiquitination upstream of NEMO, a regulatory subunit of the IκB kinase complex (IKK). However, direct evidence of IKK inhibition by A20 is lacking, and the inhibitory mechanism remains poorly understood. Here we show that A20 can directly impair IKK activation without deubiquitination or impairment of ubiquitination enzymes. We find that polyubiquitin binding by A20, which is largely dependent on A20’s seventh zinc finger motif (ZnF7), induces specific binding to NEMO. Remarkably, this ubiquitin-induced recruitment of A20 to NEMO is sufficient to block IKK phosphorylation by its upstream kinase TAK1. Our results suggest a non-catalytic mechanism of IKK inhibition by A20 and a means by which polyubiquitin chains can specify a signaling outcome.

Caspase-14 is a recent addition to the caspase family of aspartate proteases involved in apoptotic processes. Human caspase-14 appears to be only weakly processed during apoptosis, and it does not cleave classical caspase substrates. Post partum, caspase-14 is prominently expressed by human keratinocytes and reportedly participates in terminal differentiation of complex epithelia. Here we provide evidence challenging the view that caspase-14 expression or processing is linked exclusively to terminal keratinocyte differentiation. We demonstrate that caspase-14 expression extended to multiple cell lines derived from simple epithelia of the breast, prostate, and stomach. In keratinocytes and breast epithelial cells, caspase-14 expression was upregulated in high-density cultures and during forced suspension culture. These effects were primarily due to transcriptional activation as indicated by reporter gene assays using a 2 kb caspase-14 promoter fragment. Importantly, caspase-14 was not cleaved during forced suspension culture of either cell type although this treatment induced caspase-dependent apoptosis (anoikis). Forced expression of caspase-14 in immortalized human keratinocytes had no effect on cell death in forced suspension nor was the transfected caspase-14 processed in this setting. In contrast to postconfluent and forced suspension culture, terminal differentiation of keratinocytes induced in vitro by Ca2+ treatment was not associated with increased caspase-14 expression or promoter activity. Our results indicate that (1) caspase-14 is expressed not only in complex but also simple epithelia; (2) cells derived from complex and simple epithelia upregulate caspase-14 expression in conditions of high cell density or lack of matrix interaction and; (3) in both cell types this phenomenon is due to transcriptional regulation.

The human leukemia cell lines K562, CEM, CEM/VLB100, human leukemic blasts, and the bladder cancer J82 cell line have different sensitivities to UV light–induced apoptosis. It is reported that resistance to UV light–induced apoptosis occurs at a point in the apoptotic pathway upstream of caspase-3 but downstream of mitochondrial cytochrome c release. It is demonstrated that the block is due to deficiency of Apaf-1, a critical member of the apoptosome. Sensitivity to apoptosis was independent of caspase-9b or XIAP (inhibitors of apoptosis proteins) expression or levels of procaspase-9. Transfection of Apaf-1 conferred sensitivity to apoptosis in resistant cells. Apaf-1 deficiency may constitute a significant mode of resistance to apoptosis in human leukemia.

The caspase recruitment domain (CARD) is a protein-binding module that mediates the assembly of CARD-containing proteins into apoptosis and NF-kappaB signaling complexes. We report here that CARD protein 11 (CARD11) and CARD protein 14 (CARD14) are novel CARD-containing proteins that belong to the membrane-associated guanylate kinase (MAGUK) family, a class of proteins that functions as molecular scaffolds for the assembly of multiprotein complexes at specialized regions of the plasma membrane. CARD11 and CARD14 have homologous structures consisting of an N-terminal CARD domain, a central coiled-coil domain, and a C-terminal tripartite domain comprised of a PDZ domain, an Src homology 3 domain, and a GUK domain with homology to guanylate kinase. The CARD domains of both CARD11 and CARD14 associate specifically with the CARD domain of BCL10, a signaling protein that activates NF-kappaB through the IkappaB kinase complex in response to upstream stimuli. When expressed in cells, CARD11 and CARD14 activate NF-kappaB and induce the phosphorylation of BCL10. These findings suggest that CARD11 and CARD14 are novel MAGUK family members that function as upstream activators of BCL10 and NF-kappaB signaling.

The Tpl-2 proto-oncoprotein promotes cellular proliferation when overexpressed in a variety of tumor cell lines. Here, we present evidence that when overexpressed in immortalized non-transformed cells, Tpl-2 induces apoptosis by promoting the activation of caspase-3 via a caspase-9-dependent mechanism, and that apoptosis is enhanced when Tpl-2 is co-expressed with the newly identified ankyrin repeat protein Tvl-1. The activation of caspase-3 by caspase-9 is known to depend on the assembly of a multimolecular complex that includes Apaf-1 and caspase-9. Data presented here show that co-expression of Tpl-2 with Tvl-1 promotes the assembly of a complex that involves several proteins that bind Apaf-1 including Tvl-1, itself, Tpl-2 and phosphorylated procaspase-9. More important, procaspase-3, which under normal growth conditions is not associated with the complex, binds Tvl-1 conditionally in response to Tpl-2–generated apoptotic signals. The conditional association of procaspase-3 with Tvl-1 promotes the in vivo proteolytic maturation of procaspase-3 by caspase-9, a process casually linked to apoptosis. © 2001 Wiley-Liss, Inc.

We report here the identification and functional characterization of two new human caspase recruitment domain (CARD) molecules, termed Pseudo-interleukin-1beta converting enzyme (ICE) and ICEBERG. Both proteins share a high degree of homology, reaching 92% and 53% identity, respectively, to the prodomain of caspase-1/ICE. Interestingly, both Pseudo-ICE and ICEBERG are mapped to chromosome 11q22 that bears caspases-1, -4- and -5 genes, all involved in cytokine production rather than in apoptosis. We demonstrate that Pseudo-ICE and ICEBERG interact physically with caspase-1 and block, in a monocytic cell line, the interferon-gamma and lipopolysaccharide-induced secretion of interleukin-1beta which is a well-known consequence of caspase-1 activation. Moreover, Pseudo-ICE, but not ICEBERG, interacts with the CARD-containing kinase RICK/RIP2/CARDIAK and activates NF-kappaB. Our data suggest that Pseudo-ICE and ICEBERG are intracellular regulators of caspase-1 activation and could play a role in the regulation of IL-1beta secretion and NF-kappaB activation during the pro-inflammatory cytokine response.

Publisher Summary This chapter provides an overview on the structure, organization and the mechanism of activation of caspases, with focus particularly on the assay of caspase-9. The human caspase family consists of 11 members and divided in three subfamilies is based on phylogenetic analysis. Caspases, synthesized as inactive zymogens, with molecular sizes ranging from 30 to 55 kDa are based on functional analysis of caspases, and are broadly classified in three groups—namely, cytokine activators, upstream apoptosis initiator caspases, and downstream apoptosis executioner caspases. Caspases exist as latent zymogens in normal cells with extremely low intrinsic protease activity. In caspases with short prodomains, there is no significant autoactivation observed in mammalian cells. These caspases are known as “downstream caspases” because they depend on active initiator caspases for their activation by proteolytic cleavage. Another mechanism of caspase activation is through aspartate-specific serine proteases.

Although the type 1 insulin-like growth factor receptor (IGF-IR) is a potent inhibitor of apoptosis, its C-terminus sequence sends contradictory signals, including a clearly proapoptotic signal. We have synthesized a peptide, peptide 2, having the sequence of the IGF-IR from residue 1282 to residue 1298 (C-terminus of the β subunit). To favor its uptake into cells, we linked it to a stearic acid moiety at its NH-terminus. Peptide 2 is taken up by the cells, where it inhibits DNA synthesis and causes apoptosis, while a scrambled peptide (with stearic acid) and peptide 2 without stearic acid are completely ineffective. Peptide 2 is more effective when cells are in anchorage-independent conditions than when they grow in monolayer cultures. Accordingly, we find that peptide 2 can inhibit the growth of a human prostatic cell line in nude mice. The proapoptotic effect of peptide 2 is inhibited by the expression of Bcl-2 or by a dominant negative mutant of caspase 9. These and other data indicate that peptide 2 does not seem to be competing directly with the IGF-IR for common substrates, but that its proapoptotic effect is related to its ability to activate the caspase cascade. J. Cell. Physiol. 181:124–135, 1999. © 1999 Wiley-Liss, Inc.

Caspase-9-mediated apoptosis (programmed cell death) plays a central role in the development and homeostasis of all multicellular organisms. Mature caspase-9 is derived from its procaspase precursor as a result of recruitment by the activating factor Apaf-1. The crystal structures of the caspase-recruitment domain of Apaf-1 by itself and in complex with the prodomain of procaspase-9 have been determined at 1.6 and 2.5 A resolution, respectively. These structures and other evidence reveal that each molecule of Apaf-1 interacts with a molecule of procaspase-9 through two highly charged and complementary surfaces formed by non-conserved residues; these surfaces determine recognition specificity through networks of intermolecular hydrogen bonds and van der Waals interactions. Mutation of the important interface residues in procaspase-9 or Apaf-1 prevents or reduces activation of procaspase-9 in a cell-free system. Wild-type, but not mutant, prodomains of caspase-9 completely inhibit catalytic processing of procaspase-9. Furthermore, analysis of homologues from Caenorhabditis elegans indicates that recruitment of CED-3 by CED-4 is probably mediated by the same set of conserved structural motifs, with a corresponding change in the specificity-determining residues.

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.

Molecules that regulate NF-kappaB activation play critical roles in apoptosis and inflammation. We describe the cloning of the cellular homolog of the equine herpesvirus-2 protein E10 and show that both proteins regulate apoptosis and NF-kappaB activation. These proteins were found to contain N-terminal caspase-recruitment domains (CARDs) and novel C-terminal domains (CTDs) and were therefore named CLAPs (CARD-like apoptotic proteins). The cellular and viral CLAPs induce apoptosis downstream of caspase-8 by activating the Apaf-1-caspase-9 pathway and activate NF-kappaB by acting upstream of the NF-kappaB-inducing kinase, NIK, and the IkB kinase, IKKalpha. Deletion of either the CARD or the CTD domain inhibits both activities. The CARD domain was found to be important for homo- and heterodimerization of CLAPs. Substitution of the CARD domain with an inducible FKBP12 oligomerization domain produced a molecule that can induce NF-kappaB activation, suggesting that the CARD domain functions as an oligomerization domain, whereas the CTD domain functions as the effector domain in the NF-kappaB activation pathway. Expression of the CARD domain of human CLAP abrogates tumor necrosis factor-alpha-induced NF-kappaB activation, suggesting that cellular CLAP plays an essential role in this pathway of NF-kappaB activation.

The production of red blood cells follows the sequential formation of proerythroblasts and basophilic, polychromatophilic and orthochromatic erythroblasts, and is promoted by the hormone erythropoietin (Epo) in response to tissue hypoxia. However, little is known about the negative regulation of this process. Death receptors are a family of surface molecules that trigger caspase activation and apoptosis in a variety of cell types. Here we show that immature erythroid cells express several death receptors whose ligands are produced by mature erythroblasts. Exposure of erythroid progenitors to mature erythroblasts or death-receptor ligands resulted in caspase-mediated degradation of the transcription factor GATA-1, which is associated with impaired erythroblast development. Expression of a caspase-resistant GATA-1 mutant, but not of the wild-type gene, completely restored erythroid expansion and differentiation following the triggering of death receptors, indicating that there is regulatory feedback between mature and immature erythroblasts through caspase-mediated cleavage of GATA-1. Similarly, erythropoiesis blockade following Epo deprivation was largely prevented by the expression of caspase-inhibitory proteins or caspase-resistant GATA-1 in erythroid progenitors. Caspase-mediated cleavage of GATA-1 may therefore represent an important negative control mechanism in erythropoiesis.

Caspases play a major role in the transduction of the apoptotic signal and execution of apoptosis in mammalian cells. Ectopic overexpression of the short prodomain caspases-3 and -6 precursors in mammalian cells does not induce apoptosis. This is due to their inability to undergo autocatalytic processing/activation and suggests that they depend on the long prodomain caspases for activation. To investigate directly the apoptotic activity of these two caspases in vivo, we engineered constitutively active recombinant caspases-3 and -6 precursors. This was achieved by making contiguous precursor caspases-3 and -6 molecules, which have their small subunits preceding their large subunits. Unlike their wild type counterparts, these recombinant molecules were capable of autocatalytic processing in an in vitro translation reaction, suggesting that they are catalytically active. They were also capable of autoprocessing and inducing apoptosis in vivo independent of the upstream caspases. Furthermore, their autocatalytic and apoptotic activities were inhibited by the pancaspase inhibitor z-VAD-fluoromethylketone, but not by CrmA or Bcl-2, thus directly demonstrating that the targets of inhibition of apoptosis by CrmA and Bcl-2 are upstream of caspases-3 and -6. Since caspases-3 and -6 are the most downstream executioners of apoptosis, the constitutively active versions of these caspases could be used at very low concentrations in gene therapy model systems to induce apoptosis in target tissues or tumors.

Inhibitor of apoptosis (IAP) gene products play an evolutionarily conserved role in regulating programmed cell death in diverse species ranging from insects to humans. Human XIAP, cIAP1 and cIAP2 are direct inhibitors of at least two members of the caspase family of cell death proteases: caspase-3 and caspase-7. Here we compared the mechanism by which IAPs interfere with activation of caspase-3 and other effector caspases in cytosolic extracts where caspase activation was initiated by caspase-8, a proximal protease activated by ligation of TNF-family receptors, or by cytochrome c, which is released from mitochondria into the cytosol during apoptosis. These studies demonstrate that XIAP, cIAP1 and cIAP2 can prevent the proteolytic processing of pro-caspases -3, -6 and -7 by blocking the cytochrome c-induced activation of pro-caspase-9. In contrast, these IAP family proteins did not prevent caspase-8-induced proteolytic activation of pro-caspase-3; however, they subsequently inhibited active caspase-3 directly, thus blocking downstream apoptotic events such as further activation of caspases. These findings demonstrate that IAPs can suppress different apoptotic pathways by inhibiting distinct caspases and identify pro-caspase-9 as a new target for IAP-mediated inhibition of apoptosis.

Employing the degenerate primer-dependent polymerase chain reaction approach used recently to clone human Mch2, we have identified and cloned the insect Spodoptera frugiperda target of the baculovirus antiapoptotic protein p35. This protein named Sf caspase-1 belongs to the family of caspases and is highly related to human Mch3 and CPP32 in sequence and specific activity. The proenzyme of Sf caspase-1 is 299 amino acids in length and can undergo autocatalytic processing in Escherichia coli to an active enzyme heterocomplex. Autoprocessing occurs at Asp-28, Asp-184, and Asp-195 to generate the large p19/p18 and small p12 subunits. Sf caspase-1 is able to induce apoptosis in Sf9 cells and is capable of cleaving p35 to similar sized fragments as observed with extracts from p35 null mutant baculovirus-infected Sf9 cells. Sf caspase-1 activity is potently inhibited by p35, suggesting that it is an important target of this antiapoptotic protein. Finally, the Sf9 nuclear immunophilin FKBP46 was identified as a death-associated substrate for Sf caspase-1.

The Fas/APO-1-receptor associated cysteine protease Mch5 (MACH/FLICE) is believed to be the enzyme responsible for activating a protease cascade after Fas-receptor ligation, leading to cell death. The Fas-apoptotic pathway is potently inhibited by the cowpox serpin CrmA, suggesting that Mch5 could be the target of this serpin. Bacterial expression of proMch5 generated a mature enzyme composed of two subunits, which are derived from the precursor proenzyme by processing at Asp-227, Asp-233, Asp-391, and Asp-401. We demonstrate that recombinant Mch5 is able to process/activate all known ICE/Ced-3-like cysteine proteases and is potently inhibited by CrmA. This contrasts with the observation that Mch4, the second FADD-related cysteine protease that is also able to process/activate all known ICE/Ced-3-like cysteine proteases, is poorly inhibited by CrmA. These data suggest that Mch5 is the most upstream protease that receives the activation signal from the Fas-receptor to initiate the apoptotic protease cascade that leads to activation of ICE-like proteases (TX, ICE, and ICE-relIII), Ced-3-like proteases (CPP32, Mch2, Mch3, Mch4, and Mch6), and the ICH-1 protease. On the other hand, Mch4 could be a second upstream protease that is responsible for activation of the same protease cascade in CrmA-insensitive apoptotic pathways.

We report here the purification of the third protein factor, Apaf-3, 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. 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 attenuated 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.