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Donor lymphocyte infusions (DLI) following allogeneic stem cell transplantation are known to mediate graft-versus-leukemia effect (GVL). A major side effect of these immunotherapies is the development of graft-versus-host diseases (GVHD). One promising approach to prevent GVHD is to genetically modify donor T cells with a suicide mechanism that can be induced in the case of GVHD. Here we report on a retroviral vector containing the death effector domain (DED) of the human Fas-associated protein with death domain (FADD). The DED was fused to two copies of an FKBP506-binding protein and a truncated version of the human low-affinity receptor for nerve growth factor (LNGFR). Activation of the death signal pathway can be triggered upon the addition of chemical inducers of dimerization. This construct was functionally compared to an optimized HSV-TK vector in which a hypersensitive mutant of the herpes simplex virus thymidine kinase gene (TK39) was fused to a cytoplasmic truncated version of the cell surface antigen CD34. A direct comparison between both vectors in primary T lymphocytes showed that the number of T cells transduced with vectors containing the DED was significantly reduced within 24 h of drug administration whereas ganciclovir treatment of TK39-transduced T cells showed a delay in cell death of approximately 3-4 days. Our results indicate that constructs containing the DED may prove to be the most efficient mechanism to quickly eliminate alloreactive T cells.

The death effector domain (DED) occurs in proteins that regulate programmed cell death. Both pro- and anti-apoptotic proteins containing DEDs have been identified. For Fas and possibly other death receptors, homotypic DED interactions connect the Fas-associated death domain (FADD) protein to caspase-8 and caspase-10 to mediate formation of the death-inducing signal complex. This complex can be inhibited by other DED-containing proteins. Accumulating evidence now suggests that DED-containing proteins have additional roles in controlling pathways of cellular activation and proliferation. Thus, the DED defines a family of proteins that may be pivotal to cellular homeostasis by establishing a 'cell renewal set point' that coregulates proliferation and apoptosis in parallel.

Fas-associated death domain protein (FADD) is an adaptor protein bridging death receptors with initiator caspases. Thus, its function and localization are assumed to be cytoplasmic, although the localization of endogenous FADD has not been reported. Surprisingly, the data presented here demonstrate that FADD is mainly nuclear in several adherent cell lines. Its accumulation in the nucleus and export to the cytoplasm required the phosphorylation site Ser-194, which was also required for its interaction with the nucleocytoplasmic shuttling protein exportin-5. Within the nucleus, FADD interacted with the methyl-CpG binding domain protein 4 (MBD4), which excises thymine from GT mismatches in methylated regions of chromatin. The MBD4-interacting mismatch repair factor MLH1 was also found in a complex with FADD. The FADD–MBD4 interaction involved the death effector domain of FADD and a region of MBD4 adjacent to the glycosylase domain. The FADD-binding region of MBD4 was downstream of a frameshift mutation that occurs in a significant fraction of human colorectal carcinomas. Consistent with the idea that MBD4 can signal to an apoptotic effector, MBD4 regulated DNA damage-, Fas ligand-, and cell detachment-induced apoptosis. The nuclear localization of FADD and its interaction with a genome surveillance/DNA repair protein that can regulate apoptosis suggests a novel function of FADD distinct from direct participation in death receptor signaling complexes.

The adapter protein FADD consists of two protein interaction domains: a death domain and a death effector domain. The death domain binds to activated death receptors such as Fas, whereas the death effector domain binds to procaspase 8. An FADD mutant, which consists of only the death domain (FADD-DD), inhibits death receptor–induced apoptosis. FADD-DD can also activate a mechanistically distinct, cell type–specific apoptotic pathway that kills normal but not cancerous prostate epithelial cells. Here, we show that this apoptosis occurs through activation of caspases 9, 3, 6, and 7 and a serine protease. Simultaneous inhibition of caspases and serine proteases prevents FADD-DD–induced death. Inhibition of either pathway alone does not prevent cell death but does affect the morphology of the dying cells. Normal prostate epithelial cells require both the caspase and serine protease inhibitors to efficiently prevent apoptosis in response to TRAIL. In contrast, the serine protease inhibitor does not affect TRAIL-induced death in prostate tumor cells suggesting that the FADD-DD–dependent pathway can be activated by TRAIL. This apoptosis pathway is activated in a cell type–specific manner that is defective in cancer cells, suggesting that this pathway may be targeted during cancer development.

The adapter protein FADD consists of two protein interaction domains and is an essential component of the death inducing signaling complex (DISC) that is formed by activated death receptors of the tumor necrosis factor (TNF) receptor family. The FADD death domain binds to activated receptors such as Fas or other adapters such as TRADD, whereas the FADD death effector domain binds to procaspase 8. Each domain can interact with its target in the absence of the other domain, and this has led to the idea that the two domains function independently. FADD death domain interactions with Fas and TRADD are thought to occur on the same surface; however, the regulation of these interactions is poorly understood. We developed a modified reverse two-hybrid method that can identify mutations, which inhibit some protein-protein interactions without affecting other interactions. Using this method, we identified mutations in FADD that prevent binding to Fas but do not affect binding to TRADD. Surprisingly, these mutations were in the death effector domain rather than the death domain. To test whether the mutants function in mammalian cells, we expressed wild type or mutant FADD molecules in FADD-deficient cells. Wild type FADD rescued both Fas ligand- and TNF-dependent signaling, whereas the FADD death effector domain mutants rescued only TNF signaling. These data indicate that in contrast to current models, the death effector domain of FADD is involved in interaction with Fas.

Death effector domains (DEDs) are protein–protein interaction domains found in the death inducing signaling complex (DISC). Performing a structure-based alignment of all DED sequences we identified a region of high diversity in α-helix 3 and propose a classification of DEDs into class I DEDs typically containing a stretch of basic residues in the α-helix 3 region whereas DEDs of class II do not. Functional assays using mutants of Fas-associated death domain revealed that this basic region influences binding and recruitment of caspase-8 and cellular FLICE inhibitor protein to the DISC.

Over 500 researchers participated in a recent American Association for Cancer Research special conference, entitled “Apoptosis and Cancer: Basic Mechanisms and Therapeutic Opportunities in the Post-Genomic Era” (February 13–17, 2002) in sunny Hawaii (Hilton Waikoloa village, Kona, Hawaii). The meeting participants presented the most recent findings on the mechanisms regulating cell death in cancer. In the past decade, apoptosis research has undergone a quantum leap, metamorphosing from a descriptive, phenomenological discipline into a molecularly defined, highly complex signalling field. This transformation was highlighted in the conference's opening talk by meeting co-organizer, John Reed (The Burnham Institute, La Jolla, CA). Reed and colleagues used published protein functional information and bio-informatic mining of the available human genome databases to tabulate the number of human proteins predicted to be involved in regulating apoptosis. The list includes 11 catalytically active caspases, 26 CARD (caspase associated recruitment domain)-, 32 DD (death domain)-, 12 DED (death effector domain)-, 8 BIR (baculovirus inhibitor of apoptosis protein region)-, 24 BH (Bcl-2 homology)-, and 34 PAAD/PYD (pyrin/PAAD)-containing sequences.

We have analysed the genetic alteration of the entire coding region and all splice sites of caspase-8 and -10 genes in 99 gastric cancers by polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) and sequencing. We found LOH of the caspase-8 and -10 in nine (28%) of 32 and in four (15%) of 26 informative cases, respectively. Overall, three of 99 gastric cancers (3%) were found to have the caspase-10 mutations, which were identified in the coding regions of the death effector domain (codon 147) and the p17 large protease domain (codons 257 and 410), whereas no mutation was detected in caspase-8. In vitro expression studies, the M147T and Q257stop mutants severely impaired caspase-10-mediated apoptosis, whereas the V410I which was the same mutation detected in ALPS patient had a significant, albeit less severe, effect on apoptosis. The data presented here suggest that somatic alterations of the caspase-10 gene might contribute to the pathogenesis in a subset of gastric cancers through the loss of their apoptotic function.

FADD is a mammalian pro-apoptotic mediator consisting of the N-terminal death effector domain (DED) and the C-terminal death domain (DD). The N-terminal 88-residue fragment of murine FADD was defined as the stable structural unit of DED, as determined by proteolytic digestion and conformational analysis. This domain induced bacterial as well as mammalian cell death, whereas the full-length or DD of FADD did not. The Escherichia coli cells expressing FADD-DED showed elongated cell morphology and an increased level of nicked chromosomal DNA and mutation. The lethality of FADD-DED was abolished by co-expression of thioredoxin and superoxide dismutase or relieved by the addition of vitamin E as a reducing agent and under anaerobic growth conditions. The toxicity of FADD-DED was genetically suppressed by various oxidoreductases of E. coli. All these results suggest that the death effector domain of mammalian FADD induced bacterial cell death by enhancing cellular levels of reactive oxygen species (ROS).

Death receptors constitute a subgroup of the tumor necrosis factor (TNF) receptor superfamily, which are defined by the presence of a signaling domain with six α-helices in the cytoplasmic region that is termed the death domain (13). These receptors function to maintain homeostasis in the immune system by eliminating autoreactive cells, antigen-reactive T cells following an immune response, and virus-infected or malignant cells. In humans, six death receptors have been identified: Fas (also called CD95 and APO-1), TNF receptor 1 (TNFR1; also called TNFRSF1A and CD120a p55-R), DR3 (also called APO3, Wsl-1, TRAMP. and LARD), DR4 (also called TRAIL-R1 and Apo-2), DR5 (also called TRAIL-R2, KILLER, and TRICK2), and DR6 (17, 19). Binding of a death receptor to its ligand initiates a change in the receptor complex, resulting in signaling through a series of protein-protein interactions that culminate in apoptosis (4, 22). Fas binding to its ligand (FasL) leads to the recruitment of the adapter molecule FADD (Fas-associated death domain) through interactions of the death domains in Fas and FADD. FADD contains another region known as the death effector domain (DED). The FADD DED also possesses six α-helices in a folded region similar to the death domains, but it forms distinct contacts only with other DED-containing proteins, whereas death domain-containing proteins interact chiefly with other death domain-containing adapter proteins. Once FADD is recruited to Fas, its DED binds to the DEDs in the prodomain of caspase-8 or caspase-10 (1, 28). The complex containing Fas, FADD, and caspase-8 or caspase-10 is termed the death-inducing signaling complex (DISC). Recruitment of caspase-8 or caspase-10 into the DISC results in autocatalytic cleavage of the caspase into its active subunits and subsequent cleavage and activation of substrates, including other caspases, ultimately leading to apoptosis (15, 16). FADD also serves as an adapter molecule in other death receptor pathways (3, 5, 9, 10, 12, 24). Hence, death receptors may all work by a common mechanism involving recruitment of DED-containing caspases to form an active signaling complex. Apoptosis affords the host a defense mechanism to eliminate virus-infected cells. This must have proven sufficiently effective, because viruses in turn evolved mechanisms to interfere with host apoptosis pathways. A family of proteins known as the viral FLICE-inhibitory proteins (v-FLIPs) inhibit the signaling pathways in death receptor-induced apoptosis. These include the molluscum contagiosum virus (MCV) MC159 protein, the equine herpesvirus 2 (EHV-2) E8 protein, and the bovine herpesvirus E2 protein (2, 25). v-FLIPs block apoptosis induced through the Fas, TNFR1, DR3, DR4, and DR5 pathways. Apoptosis inhibition by the v-FLIPs stems from their two DEDs that can interact with the DEDs of FADD and caspase-8 (2, 11, 25, 27). The MCV MC159 protein is present in a complex with Fas when the receptor is activated (21). The EHV-2 E8 protein can be recruited to the DISC and may prevent activation of caspase-8 by blocking its recruitment to the DISC (25). v-FLIPs also inhibit formation of a cytoplasmic structure termed the death effector filament (18, 23). High-level expression of FADD or the prodomain of caspase-8 in cells results in their oligomerization as death effector filaments that recruit and activate caspases. Coexpression of v-FLIPs with FADD or the prodomain of caspase-8 blocks death effector filament formation and subsequent apoptosis. Despite these correlations, the precise inhibitory mechanism of v-FLIP, especially MC159, in the Fas signaling complex is unknown. The MC159 v-FLIP protein contains a six-amino-acid N-terminal sequence followed by a 74-amino-acid DED, a 14-amino-acid linker region, an 83-amino-acid DED, and a 64-amino-acid carboxy-terminal tail. An RXDL motif, conserved among other DED-containing proteins, is present at the carboxy end of each DED. MC159 DEDs are homologous to other DED-containing proteins, including FADD. Alignment of the MC159 DED sequences onto the nuclear magnetic resonance (NMR)-determined structure of the FADD DED suggests that each MC159 DED consists of six α-helices and contains a highly conserved hydrophobic patch on its surface (6). The role that each of these protein motifs might play in apoptosis inhibition has not yet been defined. Based on prior studies, v-FLIP is thought to prevent apoptosis by binding to FADD and caspase-8 (2, 21, 25, 26). In order to identify functionally important regions of MC159, we constructed a series of mutations in which charged amino acids were changed to alanines. Since charged amino acids are often on the surface of proteins, they are likely to participate in protein-protein interactions. Surprisingly, we found that the majority of the MC159 mutants that lost the ability to block apoptosis induced by Fas, TNF, and TNF-related apoptosis-inducing ligand (TRAIL) still bound FADD and caspase-8. The predicted hydrophobic patch 1 and α2 regions were important for FADD and caspase-8 binding. We also found that the conserved RXDL motif within the predicted α6 region is critical for protection from apoptosis and for inhibition of death effector filament formation.

Death receptor activation triggers recruitment of FADD, which via its death effector domain (DED) engages the DEDs of procaspase 8 and its inhibitor FLIP to form death-inducing signalling complexes (DISCs). The DEDs of FADD, FLIP and procaspase 8 interact with one another using two binding surfaces defined by α1/α4 and α2/α5 helices, respectively. Here we report that FLIP has preferential affinity for the α1/α4 surface of FADD, whereas procaspase 8 has preferential affinity for FADD's α2/α5 surface. These relative affinities contribute to FLIP being recruited to the DISC at comparable levels to procaspase 8 despite lower cellular expression. Additional studies, including assessment of DISC stoichiometry and functional assays, suggest that following death receptor recruitment, the FADD DED preferentially engages FLIP using its α1/α4 surface and procaspase 8 using its α2/α5 surface; these tripartite intermediates then interact via the α1/α4 surface of FLIP DED1 and the α2/α5 surface of procaspase 8 DED2.

Death ligands, a subset of the tumour necrosis factor (TNF) super family, play an important role in innate immunity, inflammatory disorders and cancer and have been the focus of intense study over the past two decades. Depending on the cellular context, the binding of these ligands to their cognate receptors (death receptors) can mediate different cell outcomes: gene activation and cell death. Whilst death receptor-mediated apoptosis has received most of the attention over the years, only recently TNF-induced necrosis has entered the spotlight. Although necrosis was long regarded as an unregulated form of cell death, death ligand-induced necrosis—also referred to as necroptosis—has recently been demonstrated to be dependent on the activity of the kinase activities of the receptor-interacting proteins 1 and 3 (RIP1 and RIP3) and, hence, to be a regulated process. This discovery revealed the opportunity of pharmacological interference with this form of cell death which appears to be involved in a number of pathological conditions. Signalling downstream of TNF is heavily dependent on degradative and non-degradative ubiquitination events. Here, we discuss the current understanding of how the necroptosis pathway is regulated and how the ubiquitin system is thought to control the balance between gene induction, apoptosis and necroptosis.

Homophilic interactions of death effector domains (DEDs) are crucial for the signaling pathways of death receptor-mediated apoptosis. The machinery that regulates proper oligomerization and autoactivation of procaspase-8 and/or procaspase-10 during T lymphocyte activation determines whether the cells will undergo caspase-mediated apoptosis or proliferation. We screened a yeast two-hybrid library by using the DEDs contained in the prodomains of procaspase-8 and procaspase-10 and isolated a DED-associated factor (DEDAF) that interacts with several DED-containing proteins but does not itself contain a DED. DEDAF is highly conserved between human and mouse (98% amino acid identity) and is homologous to a nuclear regulatory protein YAF-2. DEDAF is expressed at the highest levels in lymphoid tissues and placenta. DEDAF interacts with FADD, procaspase-8, and procaspase-10 in the cytosol as well as with the DED-containing DNA-binding protein (DEDD) in the nucleus. At the cell membrane, DEDAF augmented the formation of CD95-FADD-caspase-8 complexes and enhanced death receptor- as well as DED-mediated apoptosis. In the nucleus, DEDAF caused the DEDD protein to relocalize from subnuclear structures to a diffuse distribution in the nucleoplasm. Our data therefore suggest that DEDAF may be involved in the regulation of both cytoplasmic and nuclear events of apoptosis.

Activation of the transcription factor nuclear factor-kappa B (NF-kappa B) has been found to play an essential role in the inhibition of tumor necrosis factor (TNF)-mediated apoptosis. NF-kappa B regulates several antiapoptotic molecules including inhibitors of apoptosis, Bcl-2 family proteins (A1 and Bcl-X(L))(,) and IEX-IL. Here we report that the expression of a small death effector domain (DED)-containing protein, NDED (NF-kappa B-inducible DED-containing protein), depends on the activation of NF-kappa B. The inhibition of NF-kappa B by I kappa B alpha, a natural inhibitor of NF-kappa B, suppressed NDED mRNA expression induced by TNF. The restoration of NDED in NF-kappa B null cells inhibited TNF-induced apoptosis. Intriguingly, unlike the caspase-8 inhibitor cellular FADD-like interleukin-1 beta converting enzyme-inhibitory protein (c-FLIP), NDED suppressed TNF-mediated apoptosis by inhibiting TNF-induced caspase-8 enzymatic activity but not the processing of caspase-8. Furthermore, NDED could not inhibit etoposide-mediated apoptosis that is independent of caspase-8 activation. Our results provide the first demonstration that NF-kappa B transcriptionally induces the DED-containing protein to suppress TNF-mediated apoptosis by inhibiting caspase-8 activity, which offers new insight into the antiapoptotic mechanism of NF-kappa B.

Apoptosis is mediated by a highly regulated signal transduction cascade that eventually leads to precisely directed cell death. The death-inducing signaling complex (DISC), composed of Fas, FADD, and caspase-8, is an apical signaling complex that mediates receptor-induced apoptosis. We have docked the experimentally determined structures of the Fas and FADD death domains into a model of a partial DISC signaling complex. The arrangement of Fas and FADD was determined using the interaction modes of the two heterodimer crystal structures determined to date, Pelle/Tube and Apaf-1/procaspase-9. The proposed model reveals that both interactions can be accommodated in a single multimeric complex. Importantly, the model is consistent with reported site-directed mutagenesis data indicating residues throughout the domain are critical for function. These results imply that members of the death domain superfamily have the potential for multivalent interactions, offering novel possibilities for regulation of apoptotic signaling.

FLASH is a protein recently shown to interact with the death effector domain of caspase-8 and is likely to be a component of the death-inducing signaling complex in receptor-mediated apoptosis. Here we show that antisense oligonucleotide-induced inhibition of FLASH expression abolished TNF-alpha-induced activation of NF-kappaB in HEK293 cells, as determined by luciferase reporter gene expression driven by a NF-kappaB responsive promoter. Conversely, overexpression of FLASH dose-dependently activated NF-kappaB, an effect suppressed by dominant negative mutants of TRAF2, NIK, and IKKalpha, and partially by those of TRAF5 and TRAF6. TRAF2 was co-immunoprecipitated with FLASH from the cell extracts of HEK293 cells or HeLa cells stably expressing exogenous FLASH (HeLa/HA-FLASH). Furthermore, serial deletion mapping demonstrated that a domain spanning the residues 856-1191 of FLASH activated NF-kappaB as efficiently as the full-length and could directly bind to TRAF2 in vitro and in the transfected cells. Taken together, these results suggest that FLASH coordinates downstream NF-kappaB activity via a TRAF2-dependent pathway in the TNF-alpha signaling.

A caspase 8-deficient subline (JB6) of human Jurkat cells can be killed by the oligomerization of Fas-associated protein with death domain (FADD). This cell death process is not accompanied by caspase activation, but by necrotic morphological changes. Here, we show that the death effector domain of FADD is responsible for the FADD-mediated necrotic pathway. This process was accompanied by a loss of mitochondrial transmembrane potential (DeltaPsim), but not by the release of cytochrome c from mitochondria. Pyrrolidine dithiocarbamate, a metal chelator and antioxidant, efficiently inhibited the FADD-induced reduction of DeltaPsim and necrotic cell death. When human Jurkat, or its transformants, expressing mouse Fas were treated with Fas ligand or anti-mouse Fas antibodies, the cells died, showing characteristics of apoptosis. A broad caspase inhibitor (z-VAD-fmk) blocked the apoptotic morphological changes and the release of cytochrome c. However, the cells still died, and this cell death process was accompanied by a strong reduction in DeltaPsim, as well as necrotic morphological changes. The presence of z-VAD-fmk and pyrrolidine dithiocarbamate together blocked cell death, suggesting that both apoptotic and necrotic pathways can be activated through the Fas death receptor.

Tumor necrosis factor (TNF) induces a typical apoptotic cell death program in various cell lines by interacting with the p55 tumor necrosis factor receptor (TNF-R55). In contrast, triggering of the fibrosarcoma cell line L929sA gives rise to characteristic cellular changes resulting in necrosis. The intracellular domain of TNF-R55 can be subdivided into two parts: a membrane-proximal domain (amino acids 202–325) and a C-terminal death domain (DD) (amino acids 326–413), which has been shown to be necessary and sufficient for apoptosis. Structure/function analysis of TNF-R55-mediated necrosis in L929sA cells demonstrated that initiation of necrotic cell death, as defined by swelling of the cells, rapid membrane permeabilization, absence of nuclear condensation, absence of DNA hypoploidy, and generation of mitochondrial reactive oxygen intermediates, is also confined to the DD. The striking synergistic effect of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone on TNF-induced necrosis was also observed with receptors solely containing the DD. TNF-R55-mediated necrosis is not affected by the dominant negative deletion mutant of the Fas-associated death domain (FADD-(80–205)) that lacks the N-terminal death effector domain. Moreover, overexpression of FADD-(80–205) in L929sA is cytotoxic and insensitive to CrmA, while the cytotoxicity due to overexpression of the deletion mutant FADD-(1–111) lacking the DD is prevented by CrmA. These results demonstrate that the death domain of FADD can elicit an active necrotic cell death pathway.

Phospholipase D (PLD), a signal-transducing membrane-associated enzyme, is implicated in diverse processes including apoptosis, ERK activation, and glucose transport. Prior studies have identified specific PLD activators and repressors that directly regulate its enzymatic activity. Using two-hybrid screens, we have identified PEA-15 as a PLD interactor that unexpectedly functions to alter its level of expression. PEA-15 is a widely expressed death effector domain-containing phosphoprotein involved in signal transduction, apoptosis, ERK activation, and glucose transport. The PLD1-interacting site on PEA-15 consists of part of the death effector domain domain plus additional C-terminal flanking sequences, whereas the PEA-15-interacting site on PLD1 overlaps the previously identified RhoA-interacting site. PEA-15 did not affect basal or stimulated in vitro PLD1 enzymatic activation. However, co-expression of PEA-15 increased levels of PLD1 activity. This increased activation correlated with higher PLD1 protein expression levels, as marked by faster accumulation and longer persistence of PLD1 when PEA-15 was present. PEA-15 similarly increased protein expressions level of PLD2 and co-immunoprecipitated with it. These results suggest that PEA-15 may stabilize PLD or act as a PLD chaperone. The common involvement of PEA-15 and PLD in apoptosis, ERK activation, and glucose transport additionally suggests functional significance.

We report here the isolation and characterization of a novel tumor necrosis factor-alpha (TNF-alpha)-inducible gene, SCC-S2. Based on the nucleotide sequence, the SCC-S2 open reading frame contains a sequence in the amino terminus that shows a significant homology to death effector domain II of cell death regulatory protein, Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein (FLIP). Unlike FLIP, the SCC-S2 open reading frame contains only one death effector domain and lacks the carboxyl-terminal caspase-like homology domain, raising the possibility that SCC-S2 may be a novel member of the FLIP family. SCC-S2 mRNA expression is found in most normal tissues and malignant cells. The steady state level of SCC-S2 mRNA is significantly induced by TNF-alpha in different tumor cells (TNF-alpha at 20 ng/ml for 3 h: A549, approximately 2-9-fold; SKOV-3, approximately 3-fold; PCI-04A, approximately 3-6-fold). TNF-alpha treatment (100 ng/ml, 4 h) of HeLa cells transiently transfected with FLAG epitope-tagged SCC-S2 cDNA or expression vector alone led to an increase in the number of apoptotic cells as compared with the untreated counterpart. Interestingly, however, SCC-S2 transfectants revealed a significant decrease in the number of apoptotic cells as compared with the vector transfectants (p < 0.001). These data implicate a role of SCC-S2 as a negative mediator of apoptosis in certain cell types.

Apoptosis is a very general phenomenon, but only a few reports concern astrocytes. Indeed, astrocytes express receptors for tumor necrosis factor (TNF) α, a cytokine demonstrated on many cells and tissues to mediate apoptosis after recruitment of adaptor proteins containing a death effector domain (DED). PEA-15 is a DED-containing protein prominently expressed in the CNS and particularly abundant in astrocytes. This led us to investigate if PEA-15 expression could be involved in astrocytic protection against deleterious effects of TNF.In vitroassays evidence that PEA-15 may bind to DED-containing protein FADD and caspase-8 known to be apical adaptors of the TNF apoptotic signaling. After generation of PEA-15 null mutant mice, our results demonstrate that PEA-15 expression increases astrocyte survival after exposure to TNF.

Background The death effector domain (DED), which functions as a domain for a homophilic protein interaction, plays a role in death receptor-mediated apoptosis. Two tandemly repeated DEDs in the prodomain of caspase-8 (Casp8NC-DED) and those in MC159 (viral FLIP) have been shown to positively and negatively regulate apoptosis, respectively, by binding to caspase-8 and/or Fas- associated death domain (FADD). However, characteristics of each DED in Casp8NC-DED and those in MC159 have not been well examined. Results We analysed deletion and chimera mutants of DEDs derived from Casp8NC-DED and MC159, and found that MC159 and Casp8NC-DED require the combined effects of the two repeated DEDs to exert their binding and biological activities. The carboxy-terminal DED of Casp8NC-DED (Casp8C-DED) has the potential to induce apoptosis, and the amino-terminal DED of MC159 showed a dominant inhibitory effect on apoptosis when combined with Casp8C-DED. In addition, the two repeated DEDs in Casp8NC-DED and MC159 were shown to regulate the activities of caspase differently from the caspase recruitment domain (CARD) in the prodomains of caspase-2, -9 and Apaf-1. Conclusions Although each of the DEDs in Casp8NC-DED and MC159 has the potential to stimulate or inhibit apoptosis, the combination of the two-repeated DEDs is necessary for the DED-containing proteins to stimulate or inhibit apoptosis.

We have previously reported on the death effector domain containing E8 gene product from equine herpesvirus-2, designated FLICE inhibitory protein (v-FLIP), and on its cellular homologue, c-FLIP, which inhibit the activation of caspase-8 by death receptors. Here we report on the structure and function of the E10 gene product of equine herpesvirus-2, designated v-CARMEN, and on its cellular homologue, c-CARMEN, which contain a caspase-recruiting domain (CARD) motif. c-CARMEN is highly homologous to the viral protein in its N-terminal CARD motif but differs in its C-terminal extension. v-CARMEN and c-CARMEN interact directly in a CARD-dependent manner yet reveal different binding specificities toward members of the tumor necrosis factor receptor-associated factor (TRAF) family. v-CARMEN binds to TRAF6 and weakly to TRAF3 and, upon overexpression, potently induces the c-Jun N-terminal kinase (JNK), p38, and nuclear factor (NF)-kappaB transcriptional pathways. c-CARMEN or truncated versions thereof do not appear to induce JNK and NF-kappaB activation by themselves, nor do they affect the JNK and NF-kappaB activating potential of v-CARMEN. Thus, in contrast to the cellular homologue, v-CARMEN may have additional properties in its unique C terminus that allow for an autonomous activator effect on NF-kappaB and JNK. Through activation of NF-kappaB, v-CARMEN may regulate the expression of the cellular and viral genes important for viral replication.

▪ Abstract The immune system relies on cell death to maintain lymphoid homeostasis and avoid disease. Recent evidence has indicated that the caspase family of cysteine proteases is a central effector in apoptotic cell death and is absolutely responsible for many of the morphological features of apoptosis. Cell death, however, can occur through caspase-independent and caspase-dependent pathways. In the case of cells that are irreversibly neglected or damaged, death occurs even in the absence of caspase activity. In contrast, healthy cells require caspase activation to undergo cell death induced by surface receptors. This review summarizes the current understanding of these two pathways of cell death in the immune system.

Apoptosis is a physiological process by which multicellular organisms eliminate unwanted cells. Death factors such as Fas ligand induce apoptosis by triggering a series of intracellular protein-protein interactions mediated by defined motifs found in the signaling molecules. One of these motifs is the death effector domain (DED), a stretch of about 80 amino acids that is shared by adaptors, regulators, and executors of the death factor pathway. We have identified the human and rat complementary DNAs encoding a novel protein termed DEFT (Death EFfector domain-containing Testicular molecule). The N-terminus of DEFT shows a high degree of homology to the DEDs found in FADD (an adaptor molecule) as well as procaspase-8/FLICE and procaspase-10/Mch4 (executors of the death program). Northern blot hybridization experiments have shown that the DEFT messenger RNA (mRNA) is expressed in a variety of human and rat tissues, with particularly abundant expression in the testis. In situ hybridization analysis further in...

The CD95 signaling pathway comprises proteins that contain one or two death effector domains (DED), such as FADD/Mort1 or caspase-8. Here we describe a novel 37 kDa protein, DEDD, that contains an N-terminal DED. DEDD is highly conserved between human and mouse (98. 7% identity) and is ubiquitously expressed. Overexpression of DEDD in 293T cells induced weak apoptosis, mainly through its DED by which it interacts with FADD and caspase-8. Endogenous DEDD was found in the cytoplasm and translocated into the nucleus upon stimulation of CD95. Immunocytological studies revealed that overexpressed DEDD directly translocated into the nucleus, where it co-localizes in the nucleolus with UBF, a basal factor required for RNA polymerase I transcription. Consistent with its nuclear localization, DEDD contains two nuclear localization signals and the C-terminal part shares sequence homology with histones. Recombinant DEDD binds to both DNA and reconstituted mononucleosomes and inhibits transcription in a reconstituted in vitro system. The results suggest that DEDD is a final target of a chain of events by which the CD95-induced apoptotic signal is transferred into the nucleolus to shut off cellular biosynthetic activities.

REVIEWApoptosis is a cell suicide mechanism that enables metazoans to control cell number in tissues and to eliminate individual cells that threaten the animal's survival. Certain cells have unique sensors, termed death receptors, on their surface. Death receptors detect the presence of extracellular death signals and, in response, they rapidly ignite the cell's intrinsic apoptosis machinery.

The death-effector domain (DED) is a critical protein interaction domain that recruits caspases into complexes with members of the TNF-receptor superfamily. Apoptosis can also be induced by expressing certain DED-containing proteins without surface receptor cross-linking. Using Green Fluorescent Protein to examine DED-containing proteins in living cells, we show that these proteins cause apoptosis by forming novel cytoplasmic filaments that recruit and activate pro-caspase zymogens. Formation of these filaments, which we term death-effector filaments, was blocked by coexpression of viral antiapoptotic DED-containing proteins, but not by bcl-2 family proteins. Thus, formation of death-effector filaments allows a regulated intracellular assembly of apoptosis-signaling complexes that can initiate or amplify apoptotic stimuli independently of receptors at the plasma membrane.

When activated, membrane-bound receptors for Fas and tumour-necrosis factor initiate programmed cell death by recruiting the death domain of the adaptor protein FADD to the membrane. FADD then activates caspase 8 (also known as FLICE or MACH) through an interaction between the death-effector domains of FADD and caspase 8. This ultimately leads to the apoptotic response. Death-effector domains and homologous protein modules known as caspase-recruitment domains have been found in several proteins and are important regulators of caspase (FLICE) activity and of apoptosis. Here we describe the solution structure of a soluble, biologically active mutant of the FADD death-effector domain. The structure consists of six antiparallel, amphipathic alpha-helices and resembles the overall fold of the death domains of Fas and p75. Despite this structural similarity, mutations that inhibit protein-protein interactions involving the Fas death domain have no effect when introduced into the FADD death-effector domain. Instead, a hydrophobic region of the FADD death-effector domain that is not present in the death domains is vital for binding to FLICE and for apoptotic activity.

Activation of the cascade of proteolytic caspases has been identified as the final common pathway of apoptosis in diverse biological systems. We have isolated a gene, termed MRIT , that possesses overall sequence homology to FLICE (MACH), a large prodomain caspase that links the aggregated complex of the death domain receptors of the tumor necrosis factor receptor family to downstream caspases. However, unlike FLICE, the C-terminal domain of MRIT lacks the caspase catalytic consensus sequence QAC(R/Q)G. Nonetheless MRIT activates caspase-dependent death. Using yeast two-hybrid assays, we demonstrate that MRIT associates with caspases possessing large and small prodomains (FLICE, and CPP32/YAMA), as well as with the adaptor molecule FADD. In addition, MRIT simultaneously and independently interacts with BclX L and FLICE in mammalian cells. Thus, MRIT is a mammalian protein that interacts simultaneously with both caspases and a Bcl-2 family member.

We identified and cloned a novel human protein that contains FADD/Mort1 death effector domain homology regions, designated FLAME-1. FLAME-1, although most similar in structure to Mch4 and Mch5, does not possess caspase activity but can interact specifically with FADD, Mch4, and Mch5. Interestingly, FLAME-1 is recruited to the Fas receptor complex and can abrogate Fas/TNFR-induced apoptosis upon expression in FasL/tumor necrosis factor-sensitive MCF-7 cells, possibly by acting as a dominant-negative inhibitor. These findings identify a novel endogenous control point that regulates Fas/TNFR1-mediated apoptosis.

Viruses have evolved many distinct strategies to avoid the host's apoptotic response. Here we describe a new family of viral inhibitors (v-FLIPs) which interfere with apoptosis signalled through death receptors and which are present in several gamma-herpesviruses (including Kaposi's-sarcoma-associated human herpesvirus-8), as well as in the tumorigenic human molluscipoxvirus. v-FLIPs contain two death-effector domains which interact with the adaptor protein FADD, and this inhibits the recruitment and activation of the protease FLICE by the CD95 death receptor. Cells expressing v-FLIPs are protected against apoptosis induced by CD95 or by the related death receptors TRAMP and TRAIL-R. The herpesvirus saimiri FLIP is detected late during the lytic viral replication cycle, at a time when host cells are partially protected from CD95-ligand-mediated apoptosis. Protection of virus-infected cells against death-receptor-induced apoptosis may lead to higher virus production and contribute to the persistence and oncogenicity of several FLIP-encoding viruses.

The pivotal discovery that Fas-associated death domain protein (FADD) interleukin-1beta-converting enzyme (FLICE)/MACH was recruited to the CD95 signaling complex by virtue of its ability to bind the adapter molecule FADD established that this protease has a role in initiating the death pathway (Boldin, M. P., Goncharov, T. M. , Goltsev, Y. V., and Wallach, D. (1996) Cell 85, 803-815; Muzio, M., Chinnaiyan, A. M., Kischkel, K. C., O'Rourke, K., Shevchenko, A., Ni, J., Scaffidi, C., Bretz, J. D., Zhang, M., Gentz, R., Mann, M., Krammer, P. H., Peter, M. E., and Dixit, V. M. (1996) Cell 85, 817-827). In this report, we describe the cloning and characterization of a new member of the caspase family, a homologue of FLICE/MACH, 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. We show that 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.

To identify novel antiapoptotic proteins encoded by DNA viruses, we searched viral genomes for proteins that might interfere with Fas and TNFR1 apoptotic signaling pathways. We report here that equine herpesvirus type 2 E8 protein and molluscum contagiosum virus MC159 protein both show sequence similarity to the death effector domains (DEDs) of the Fas/TNFR1 signaling components FADD and caspase-8. Yeast two-hybrid analysis revealed that E8 protein interacted with the caspase-8 prodomain whereas MC159 protein interacted with FADD. Furthermore, expression of either E8 protein or MC159 protein protected cells from Fas- and TNFR1-induced apoptosis indicating that certain herpesviruses and poxviruses use DED-mediated interactions to interfere with apoptotic signaling pathways. These findings identify a novel control point exploited by viruses to regulate Fas- and TNFR1-mediated apoptosis.

Two members of the B-cell associated 31 (BAP31) family are found in humans; BAP29 and BAP31. These are ubiquitously expressed receptors residing in the endoplasmic reticulum. BAP31 functions in sorting of membrane proteins and in caspase-8 mediated apoptosis, while BAP29 appears to mainly corroborate with BAP31 in sorting. The N-terminal half of these proteins is membrane-bound while the C-terminal half is cytoplasmic. The latter include the so called variant of death effector domain (vDED), which shares weak sequence homology with DED domains. Here we present two structures of BAP31 vDED determined from a single and a twinned crystal, grown at pH 8.0 and pH 4.2, respectively. These structures show that BAP31 vDED forms a dimeric parallel coiled coil with no structural similarity to DED domains. Solution studies support this conclusion and strongly suggest that an additional α-helical domain is present in the C-terminal cytoplasmic region, probably forming a second coiled coil. The thermal stability of BAP31 vDED is quite modest at neutral pH, suggesting that it may assemble in a dynamic fashion in vivo. Surprisingly, BAP29 vDED is partially unfolded at pH 7, while a coiled coil is formed at pH 4.2 in vitro. It is however likely that folding of the domain is triggered by other factors than low pH in vivo. We found no evidence for direct interaction of the cytoplasmic domains of BAP29 and BAP31. Published version

AIM2 binds dsDNA and associates with ASC through their PYDs to form an inflammasome.The AIM2 PYD structure illustrates distinct charge distribution and a unique hydrophobic patch.The AIM2 PYD may bind the ASC PYD and the AIM2 HIN domain through overlapping surface.These findings provide insights into the mechanisms of AIM2 autoinhibition and inflammasome assembly. Absent in melanoma 2 (AIM2) is a cytosolic double-stranded (dsDNA) sensor essential for innate immune responses against DNA viruses and bacteria such as Francisella and Listeria. Upon dsDNA engagement, the AIM2 amino-terminal pyrin domain (PYD) is responsible for downstream signaling to the adapter protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) through homotypic PYD-PYD interactions and the assembly of an inflammasome. Toward a better understanding of the AIM2 signaling mechanism, we determined the crystal structure of the human AIM2 PYD. The structure reveals a death domain fold with a short α3 helix that is buttressed by a highly conserved lysine residue at the α2 helix, which may stabilize the α3 helix for potential interaction with partner domains. The surface of the AIM2 PYD exhibits distinct charge distribution with highly acidic α1-α2 helices and highly basic α5-α6 helices. A prominent solvent-exposed hydrophobic patch formed by residues Phe-27 and Phe-28 at the α2 helix resembles a similar surface involved in the death effector domain homotypic interactions. Docking studies suggest that the AIM2 PYD may bind the AIM2 hematopoietic interferon-inducible nuclear (HIN) domain or ASC PYD using overlapping surface near the α2 helix. This may ensure that AIM2 interacts with the downstream adapter ASC only upon release of the autoinhibition by the dsDNA ligand. Our work thus unveils novel structural features of the AIM2 PYD and provides insights into the potential mechanisms of the PYD-HIN and PYD-PYD interactions important for AIM2 autoinhibition and inflammasome assembly.

Emerging evidence suggests that an amplifiable protease cascade consisting of multiple aspartate specific cysteine proteases (ASCPs) is responsible for the apoptotic changes observed in mammalian cells undergoing programmed cell death. Here we describe the cloning of two novel ASCPs from human Jurkat T-lymphocytes. Like other ASCPs, the new proteases, named Mch4 and Mch5, are derived from single chain proenzymes. However, their putative active sites contain a QACQG pentapeptide instead of the QACRG present in ail known ASCPs. Also, their N termini contain FADD-like death effector domains, suggesting possible interaction with FADD. Expression of Mch4 in Escherichia coli produced an active protease that, like other ASCPs, was potently inhibited (Kj = 14 nM) by the tetrapeptide aldehyde DEVD-CHO. Interestingly, both Mch4 and the serine protease granzyme B cleave recombinant proCPP32 and proMch3 at a conserved IXXD-S sequence to produce the large and small subunits of the active proteases. Granzyme B also cleaves proMch4 at a homologous IXXD-A processing sequence to produce mature Mch4. These observations suggest that CPP32 and Mch3 are targets of mature Mch4 protease in apoptotic cells. The presence of the FADD-like domains in Mch4 and Mch5 suggests a role for these proteases in the Fas-apoptotic pathway. In addition, these proteases could participate in the granzyme B apoptotic pathways.

To identify CAP3 and CAP4, components of the CD95 (Fas/APO-1) death-inducing signaling complex, we utilized nano-electrospray tandem mass spectrometry, a recently developed technique to sequence femtomole quantities of polyacrylamide gel–separated proteins. Interestingly, CAP4 encodes a novel 55 kDa protein, designated FLICE, which has homology to both FADD and the ICE/CED-3 family of cysteine proteases. FLICE binds to the death effector domain of FADD and upon overexpression induces apoptosis that is blocked by the ICE family inhibitors, CrmA and z-VAD-fmk. CAP3 was identified as the FLICE prodomain which likely remains bound to the receptor after proteolytic activation. Taken together, this is unique biochemical evidence to link a death receptor physically to the proapoptotic proteases of the ICE/CED-3 family.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive cancer with a 5-year survival rate of ~50%. With the use of a custom cDNA-capture system coupled with massively parallel sequencing, we have now investigated transforming mechanisms for this malignancy. The cDNAs of cancer-related genes (n = 906) were purified from a human HNSCC cell line (T3M-1 Cl-10) and subjected to high-throughput resequencing, and the clinical relevance of non-synonymous mutations thus identified was evaluated with luciferase-based reporter assays. A CASP8 (procaspase-8) cDNA with a novel G-to-C point mutation that results in the substitution of alanine for glycine at codon 325 was identified, and the mutant protein, CASP8 (G325A), was found to activate nuclear factor-κB (NF-κB) signaling to an extent far greater than that achieved with the wild-type protein. Moreover, forced expression of wild-type CASP8 suppressed the growth of T3M-1 Cl-10 cells without notable effects on apoptosis. We further found that most CASP8 mutations previously detected in various epithelial tumors also increase the ability of the protein to activate NF-κB signaling. Such NF-κB activation was shown to be mediated through the COOH-terminal region of the second death effector domain of CASP8. Although CASP8 mutations associated with cancer have been thought to promote tumorigenesis as a result of attenuation of the proapoptotic function of the protein, our results now show that most such mutations, including the novel G325A identified here, separately confer a gain of function with regard to activation of NF-κB signaling, indicating another role of CASP8 in the transformation of human malignancies including HNSCC.

Using the cytoplasmic domain of Fas in the yeast two-hybrid system, we have identified a novel interacting protein, FADD, which binds Fas and Fas-FD5, a mutant of Fas possessing enhanced killing activity, but not the functionally inactive mutants Fas-LPR and Fas-FD8. FADD contains a death domain homologous to the death domains of Fas and TNFR-1. A point mutation in FADD, analogous to the Ipr mutation of Fas, abolishes its ability to bind Fas, suggesting a death domain to death domain interaction. Overexpression of FADD in MCF7 and BJAB cells induces apoptosis, which, like Fas-induced apoptosis, is blocked by CrmA, a specific inhibitor of the interieukin-lβ-converting enzyme. These findings suggest that FADD may play an important role in the proximal signal transduction of Fas.

Signaling for cell death by Fas/APO1 occurs via a distinct region in its intracellular domain. This region contains a conserved sequence motif, the death domain motif, that is also found in the intracellular domains of the p55 tumor necrosis factor receptor and the low affinity nerve growth factor receptor, as well as in the regulatory domain of the ankyrins. A novel protein that specifically binds to the death domain of Fas/APO1 but not to Fas/APO1 molecules with a loss of function point mutation occurring in lprcg mice was cloned by a two-hybrid screen of a HeLa cells' cDNA library. The cloned protein itself contains a death domain motif, and this region binds to the death domain of Fas/APO1, while the region upstream to the death domain prompts self-association of the protein. Induced expression of the protein results in ligand-independent triggering of cytotoxicity, suggesting that it is involved in cell death induction by Fas/APO1.

Fas-associated death domain (FADD) is a common adaptor molecule which plays an important role in transduction of death receptor mediated apoptosis. The FADD provides DED motif for binding to both procaspase-8 and cFLIP molecules which executes death receptor mediated apoptosis. Dysregulated expression of FADD and cFLIP may contribute to inhibition of apoptosis and promote cell survival in cancer. Moreover elevated intracellular level of cFLIP competitively excludes the binding of procaspase-8 to the death effector domain (DED) of FADD at the DISC to block the activation of death receptor signaling required for apoptosis. Increasing evidence shows that defects in FADD protein expression are associated with progression of malignancies and resistance to apoptosis. Therefore, improved expression and function of FADD may provide new paradigms for regulation of cell proliferation and survival in cancer. In the present study, we have examined the potential of FADD in induction of apoptosis by overexpression of FADD in HEK 293T cells and validated further its consequences on the expression of pro and anti-apoptotic proteins besides initiation of death receptor mediated signaling. We have found deficient expression of FADD and elevated expression of cFLIP(L) in HEK 293T cells. Our results demonstrate that over expression of FADD attenuates the expression of anti-apoptotic protein cFLIP and activates the cascade of extrinsic caspases to execution of apoptosis in HEK 293T cells.

Death effector domain (DED) proteins constitute a subfamily of the large death domain superfamily that is primarily involved in apoptosis pathways. DED structures have characteristic side chain–side chain interactions among polar residues on the protein surface, forming a network of hydrogen bonds and salt bridges. The polar interaction network is functionally important in promoting protein–protein interactions by maintaining optimal side chain orientations. We have refined the solution DED structure of the PED/PEA-15 protein, a representative member of DED subfamily, using traditional NMR restraints with the addition of residual dipolar coupling (RDC) restraints from two independent alignment media, and employed the explicit solvent refinement protocol. The newly refined DED structure of PED/PEA-15 possesses higher structural quality as indicated by WHAT IF Z-scores, with most significant improvement in the backbone conformation normality quality factor. This higher quality DED structure of PED/PEA-15 leads to the identification of a number of key polar side chain interactions, which are not typically observed in NMR protein structures. The elucidation of polar side chain interactions is a key step towards the understanding of protein–protein interactions involving the death domain superfamily. The NMR structures with extensive details of protein structural features are thereby termed high-definition (HD) NMR structures.

Tumor necrosis factor α (TNF) is a pleiotropic proinflammatory cytokine that plays a role in immunity and the control of cell proliferation, cell differentiation, and apoptosis. The pleiotropic nature of TNF is due to the formation of different signaling complexes upon the binding of TNF to its receptor, TNF receptor type 1 (TNFR1). TNF induces apoptosis in various mammalian cells when the cells are co-treated with a transcription inhibitor like actinomycin D (ActD). When TNFR1 is activated, it recruits an adaptor protein, TNF receptor-associated protein with death domain (TRADD), through its cytoplasmic death effector domain (DED). TRADD, in turn, recruits other signaling proteins, including TNF receptor-associated protein 2 (TRAF2) and receptor-associated protein kinase (RIPK) 1, to form a complex. Subsequently, this complex combines with FADD and procaspase-8, converts into a death-inducing signaling complex (DISC) to induce apoptosis. Cyclic AMP (cAMP) is a second messenger that regulates various cellular processes such as cell proliferation, gene expression, and apoptosis. cAMP analogues are reported to act as anti-apoptotic agents in various cell types, including hepatocytes. We found that a cAMP analogue, dibutyryl cAMP (db-cAMP), inhibits TNF + ActD-induced apoptosis in rat hepatocytes. The protein kinase A (PKA) inhibitor KT-5720 reverses this inhibitory effect of cAMP on apoptosis. Cytoprotection by cAMP involves down-regulation of various apoptotic signal regulators like TRADD and FADD and inhibition of caspase-8 and caspase-3 cleavage. We also found that cAMP exerts its affect at the proximal level of TNF signaling by inhibiting the formation of the DISC complex upon the binding of TNF to TNFR1. In conclusion, our study shows that cAMP prevents TNF + ActD-induced apoptosis in rat hepatocytes by inhibiting DISC complex formation.

Apoptosis is a mechanism that regulates hepatic tissue homeostasis and contributes to both acute and chronic injury in liver disease. The apoptotic signaling cascade involves activation of the death-inducing signaling complex (DISC) and subsequent recruitment of proteins containing death effector domains (DED), which regulate downstream effector molecules. Prominent among these are the Fas-associated death domain (FADD) and the cellular caspase 8-like inhibitory protein (cFLIP), and alterations in these proteins can lead to severe disruption of physiological processes, including acute liver failure or hepatocellular carcinoma. Their role in cell signaling events independent of the DISC remains undetermined. Oxidative stress can cause cell injury from direct effects on molecules or by activating intracellular signaling pathways including the mitogen-activated protein kinases (MAPKs). In this context, prolonged activation of the cJun N-terminal kinase (JNK)/AP-1/cJun signaling pathway promotes hepatocellular apoptosis, whereas activation of the extracellular signal-regulated kinase (Erk) exerts protection. We investigated the roles of FADD and cFLIP in acute oxidant stress induced by the superoxide generator menadione in hepatocytes. Menadione resulted in dose-dependent predominantly necrotic cell death. Hepatocytes expressing a truncated, dominant-negative FADD protein were partially protected, whereas cFLIP-deficient hepatocytes displayed increased cell death from menadione. In parallel, Erk phosphorylation was enhanced in hepatocytes expressing dnFADD and decreased in cFLIP-deficient hepatocytes. Hepatocyte injury was accompanied by increased release of proapoptotic factors and increased JNK/cJun activation. Thus, FADD and cFLIP contribute to the regulation of cell death from acute oxidant stress in hepatocytes involving MAPK signaling. This implies that DED-containing proteins are involved in the regulation of cellular survival beyond their role in cell death receptor–ligand-mediated apoptosis.

PED/PEA-15 is a small, non-catalytic, DED containing protein that is widely expressed in different tissues and highly conserved among mammals. PED/PEA-15 has been found to interact with several protein targets in various pathways, including FADD and procaspase-8 (apoptosis), ERK1/2 (cell cycle entry), and PLD1/2 (diabetes). In this research, we have studied the PED/PEA-15 in a complex with ERK2, a MAP kinase, using NMR spectroscopic techniques. MAP Kinase signaling pathways are involved in the regulation of many cellular functions, including cell proliferation, differentiation, apoptosis and survival. ERK1/2 are activated by a variety of external stimuli, including growth factors, hormones and neurotransmitters. Inactivated ERK2 is primarily found in the cytosol. Once the ERK/MAPK cascade is initiated, ERK2 is phosphorylated and stimulated, allowing it to redistribute in the cell nucleus and act as a transcription factor. Previous studies have shown that PED/PEA-15 complexes with ERK2 in the cytoplasm and prevents redistribution into the nucleus. Although the NMR structure and dynamics of PED/PEA-15 in the free form have been documented recently, no detailed structural and dynamic information for the ERK2-bound form is available. Here we report NMR chemical shift perturbation and backbone dynamic studies at the fast ps–ns timescale of PED/PEA-15, in its free form and in the complex with ERK2. These analyses characterize motions and conformational changes involved in ERK2 recognition and binding that orchestrate the reorganization of the DED and immobilization of the C-terminal tail. A new induced fit binding model for PED/PEA-15 is proposed.