Search

Molluscum contagiosum virus proteins MC159 and MC160 and the equine herpesvirus 2 protein E8 share substantial homology to the death effector domain present in the adaptor molecule Fas-associated death domain protein (FADD) and the initiating death protease FADD-like interleukin-1beta-converting enzyme (FLICE) (caspase-8). FADD and FLICE participate in generating the death signal from both tumor necrosis factor receptor-1 (TNFR-1) and the CD-95 receptor. The flow of death signals from TNFR-1 occurs through the adaptor molecule tumor necrosis factor receptor-associated death domain protein (TRADD) to FADD to FLICE, whereas for CD-95 the receptor directly communicates with FADD and then FLICE. MC159 and E8 inhibited both TNFR-1- and CD-95-induced apoptosis as well as killing mediated by overexpression of the downstream adaptors TRADD and FADD. Neither viral molecule, however, inhibited FLICE-induced killing, consistent with an inhibitory action upstream of the active death protease. These data suggest the existence of a novel strategy employed by viruses to attenuate host immune killing mechanisms. Given that bovine herpesvirus 4 protein E1.1 and Kaposi's sarcoma associated-herpesvirus protein K13 also possess significant homology to the viral inhibitory molecules MC159, MC160, and E8, it may be that this class of proteins is used ubiquitously by viruses to evade host defense.

In the nematode Caenorhabditis elegans apoptosis is tightly regulated by a hierarchical set of genes. Two of these, ced-3 and ced-9, possess mammalian homologues encoding executional ICE proteases and inhibitory Bcl-2-related proteins, respectively. The function of a third key player, ced-4, is however completely unknown and no mammalian counterparts have been identified. Here we report that Ced-4 protein contains a structural region with similarity to the mammalian death effector domain which has previously been demonstrated to act as an important protein interaction motif in the signaling pathway of the mammalian surface receptor Fas (APO-1, CD95). Based on this finding and previously described genetic experiments, we propose that Ced-4, similar to the mammalian proteins FADD and FLICE, may possess a function as an adaptor protein in invertebrate apoptotic pathways.

Cancer affects one in three persons in the Developed World of whom half will die from their disease. Moreover, the half that survive suffer an unpleasant mixture of surgery and radio- and chemo-therapy, all of which can carry severe side effects and are of limited efficacy. Cancer is therefore both a very common disease and a frequent cause of death. Tumours are, however, clonal in origin: they arise from single mutant cells that progressively evolve an ensemble of mutations in critical genes that govern cell proliferation, survival, cell adhesion and cell mobility. Thus, if cancer affects one in three people, this means that the individual cancer cell only arises and propagates in one in three persons. As the human body comprises some ten thousand billion (1014) cells, that undergo a total of some 1016 cell divisions during the course of a human life, the cancer cell becomes extremely rare.

The widely expressed protein Fas is a member of the tumour necrosis factor receptor family which can trigger apoptosis1. However, Fas surface expression does not necessarily render cells susceptible to Fas ligand-induced death signals1,2, indicating that inhibitors of the apoptosis-signalling pathway must exist. Here we report the characterization of an inhibitor of apoptosis, designated FLIP (for FLICE-inhibitory protein), which is predominantly expressed in muscle and lymphoid tissues. The short form, FLIPS, contains two death effector domains and is structurally related to the viral FLIP inhibitors of apoptosis3, whereas the long form, FLIPL, contains in addition a caspase-like domain in which the active-centre cysteine residue is substituted by a tyrosine residue. FLIPS and FLIPL interact with the adaptor protein FADD4,5 and the protease FLICE6,7, and potently inhibit apoptosis induced by all known human death receptors1. FLIPL is expressed during the early stage of T-cell activation, but disappears when T cells become susceptible to Fas ligand-mediated apoptosis. High levels of FLIPL protein are also detectable in melanoma cell lines and malignant melanoma tumours. Thus FLIP may be implicated in tissue homeostasis as an important regulator of apoptosis.

Fas-associated death domain (DD) adaptor (FADD), a member of the DD superfamily, contains both a DD and a death effector domain (DED) that are important in mediating FAS ligand-induced apoptotic signaling. P45 is a unique member of the DD superfamily in that it has a domain with sequence and structural characteristics of both DD and DED. We show that p45 forms a complex with FADD and diminishes Fas-FADD mediated death signaling. The DED of FADD is required for the complex formation with p45. Following spinal cord injury, transgenic mice over-expressing p45 exhibit increased neuronal survival, decreased retraction of corticospinal tract fibers and improved functional recovery. Understanding p45-mediated cellular and molecular mechanisms may provide insights into facilitating nerve regeneration in humans. ISSN:1932-6203

PROGRAMMEDcell death (apoptosis) mediated by the cytokine receptor Fas is critical for the removal of autoreactive T cells1, the mechanism of immune privilege2,3, and for maintenance of immune-system homeostasis4. Signalling of programmed cell death involves the self-association of a conserved cytoplasmic region of Fas called the death domain5–7 and interaction with another death-domain-containing protein, FADD8 (also known as MORT1)9. Although death domains are found in several proteins10, their three-dimensional structure and the manner in which they interact is unknown. Here we describe the solution structure of the Fas death domain, as determined by NMR spectroscopy. The structure consists of six antiparallel, amphi-pathic α-helices arranged in a novel fold. From the structure and from site-directed mutagenesis, we have identified the region of the death domain involved in self-association and binding to the downstream signalling partner FADD.

Fas/APO-1 and p55 tumor necrosis factor (TNF) receptor (p55-R) activate cellular mechanisms that result in cell death. Upon activation of these receptors, Fas/APO-1 binds a protein called MORT1 (or FADD) and p55-R binds a protein called TRADD. MORT1 and TRADD can also bind to each other. We have cloned a novel protein, MACH, that binds to MORT1. This protein exists in multiple isoforms, some of which contain a region that has proteolytic activity and shows marked sequence homology to proteases of the ICE/CED-3 family. Cellular expression of the proteolytic MACH isoforms results in cell death. Expression of MACH isoforms that contain an incomplete ICE/CED-3 region provides effective protection against the cytotoxicity induced by Fas/APO-1 or p55-R triggering. These findings suggest that MACH is the most upstream enzymatic component in the Fas/APO-1- and p55-R-induced cell death signaling cascades.

Many diverse activities of tumor necrosis factor (TNF) are signaled through TNF receptor 1 (TNFR1). We have identified a novel 34 kDa protein, designated TRADD, that specifically interacts with an intracellular domain of TNFR1 known to be essential for mediating programmed cell death. Overexpression of TRADD leads to two major TNF-induced responses, apoptosis and activation of NF-kappa B. The C-terminal 118 amino acids of TRADD are sufficient to trigger both of these activities and likewise sufficient for interaction with the death domain of TNFR1. TRADD-mediated cell death can be suppressed by the crmA gene, which encodes a specific inhibitor of the interleukin-1 beta-converting enzyme. However, NF-kappa B activation by TRADD is not inhibited by crmA expression, demonstrating that the signaling pathways for TNF-induced cell death and NF-kappa B activation are distinct.

Besides inducing apoptosis, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) activates NF-κB. The apoptosis signaling pathway of TRAIL is well characterized involving TRAIL receptors, Fas-associated protein with death domain (FADD) and caspase-8. In contrast, the molecular mechanism of TRAIL signaling to NF-κB remains controversial. Here, we characterized the receptor-proximal mediators of NF-κB activation by TRAIL. Deletion of the DD of TRAIL receptors 1 and 2 revealed that it is essential in NF-κB signaling. Because FADD interacts with the TRAIL receptor DD, FADD was tested. RNAi-mediated knockdown of FADD or FADD deficiency in JURKAT T-cell leukemia cells decreased or disabled NF-κB signaling by TRAIL. In contrast, TRAIL-induced activation of NF-κB was maintained upon loss of receptor interacting protein 1 (RIP1) or knockdown of FLICE-like inhibitory protein (FLIP). Exogenous expression of FADD rescued TRAIL-induced NF-κB signaling. Loss-of-function mutations of FADD within the RHDLL motif of the death effector domain, which is required for TRAIL-induced apoptosis, abrogated FADD's ability to recruit caspase-8 and mediate NF-κB activation. Accordingly, deficiency of caspase-8 inhibited TRAIL-induced activation of NF-κB, which was rescued by wild-type caspase-8, but not by a catalytically inactive caspase-8 mutant. These data establish the mechanism of TRAIL-induced NF-κB activation involving the TRAIL receptor DD, FADD and caspase-8, but not RIP1 or FLIP. Our results show that signaling of TRAIL-induced apoptosis and NF-κB bifurcates downstream of caspase-8.

Fas death receptor-activated signaling pathways regulate apoptosis in a variety of cells, including cholangiocarcinoma and other cancer cells. Fas binding to Fas-associated death domain (FADD) activates FADD-caspase-8 binding to form the death-inducing signaling complex (DISC) to trigger apoptosis (Nature reviews 9:231-241, 2008, Cell 81:505-512, 1995). The Fas-Fas association exists primary as a dimer in the Fas-FADD complex and the Fas-FADD tetramer complex has the tendency to form higher oligomers (Nature 457:1019-1022, 2009). The importance of the oligomerized Fas-FADD complex in DISC formation has been confirmed (Nat Struct Mol Biol. 2010 Oct 10). This study sought to provide structural insight for the role of Fas binding to FADD and the oligomerization of Fas/FADD complex in activating FADD-procaspase-8 binding. In this study, we determined the conformational and motion changes of FADD by binding to Fas for the Fas-FADD complex in either dimer or tetramer behavior with molecular dynamics (MD) simulations. The conformational and motion changes in FADD by binding to Fas could directly affect FADD binding to procaspase-8. Results show Fas binding to FADD stabilized FADD conformation, including the increased stability of the critical residues in FADD death effector domain (DED) for FADD-procaspase-8 binding. Fas binding to FADD resulted in the decreased degree of both correlated and anti-correlated motion of the residues in FADD and caused the reversed correlated motion between FADD DED and FADD death domain. The exposure of procaspase-8 binding residues in FADD that allows FADD to interact with procaspase-8 was observed with Fas binding to FADD. We also observed the different degree of the conformational and motion changes of FADD in the Fas-FADD complex with the different degree of oligomerization. The increased conformational stability and the decreased degree of the correlated motion of the residues in FADD in Fas-FADD tetramer complex were observed compared to those in Fas-FADD dimer complex. This study provided structural evidence for the role of Fas binding to FADD and the oligomerization degree of Fas/FADD complex in DISC formation to signal apoptosis. The structural information should facilitate the identification of novel strategies and targets that regulate DISC formation and, thereby, modulate apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 19. doi:10.1158/1538-7445.AM2011-19

Huntington's disease is a neurodegenerative disorder characterized by a polyglutamine expansion at the N-terminus of the huntingtin protein (HTT) that leads to selective loss of neurons in the striatum by a hitherto unknown apoptotic mechanism. A molecular basis for the strong correlation between polyglutamine expansion of HTT and subsequent neurodegeneration has, until now, eluded scientists. The implication that caspase-8, a death effector domain (DED)-containing cysteine protease responsible for the initiation of receptor-mediated apoptosis, is required for this process raises the possibility that DEDs might facilitate the assembly of a death complex that engages a novel caspase-8-dependent cell death pathway.In an attempt to unravel the molecular basis of HTT-mediated cytotoxicity, several HTT-interacting proteins have been identified. One such protein, named HIP1 (HTT-interacting protein 1), contains a pseudo DED-like motif (pDED), reminiscent of the DED motif found in pro-caspase-8. Furthermore, HIP1 is pro-apoptotic and induces cell death in a pDED-dependent manner by activation of caspases.Although HIP1 and HTT colocalize in mammalian cells, the affinity of HTT for HIP1 decreases significantly as the polyglutamine tract in HTT expands. Consequently, a current model of Huntington's disease is that an expanded polyglutamine tract in HTT might result in the release of HIP1 protein and subsequent induction of a novel apoptotic pathway.To explore this possibility, Gervais and colleagues [1xRecruitment and activation of caspase-8 by the Huntingtin-interacting protein Hip-1 and a novel partner Hippi. Gervais, F.G et al. Nat. Cell Biol. 2002; 4: 95–105Crossref | PubMed | Scopus (208)See all References][1] employed a yeast two-hybrid screen using the pDED of Hip-1 as bait. This strategy yielded HIPPI (HIP1 protein interactor), a novel partner for HIP1 that contains a pDED domain strikingly similar to that present in HIP1. More importantly, Gervais et al. show that HIPPI is a molecular accomplice of HIP1, which results in recruitment and activation of procaspase-8 by the HIPPI–HIP1 complex. This HIPPI–HIP-1 interaction could therefore act as the initiating event in a non-receptor-mediated pathway for the activation of caspase-8 and subsequent cell death. The identification of this new apoptotic pathway that is dependent on the extent of polyglutamine expansion in HTT, goes some way to providing a molecular basis for the pathogenesis of Huntington's disease and could provide novel targets for inhibiting the selective neuronal loss associated with this disease.

The major apoptotic effector protein caspase-3 holds the power to destroy a huge number of proteins in a cell—over 34 thousand human proteins with a caspase-3 cleavage motif have been sequenced. Nevertheless, apoptosis proceeds in an ordered fashion; only specific substrates are cleaved at the proper time. On page 1051, Lee et al. describe a new function of the apoptosis regulator DEDD as a scaffolding protein that directs this orderly destruction. Figure DEDD (red) brings active caspase-3 (green) to its substrates. DEDD works by bringing together the important participants, much like scaffolding proteins that control signaling cascades. DEDD resides mainly in the cytosol, despite its previous identification based on its DNA-binding death effector domain (DED). Localization studies by Lee et al. revealed that, even in nonapoptotic epithelial cells, DEDD associated with the keratin intermediate filament network. Once apoptosis was induced, an apoptosis-related epitope within the DED of DEDD was exposed in a caspase-3–dependent manner. Activated DEDD returns the favor by bringing caspase-3 to its keratin substrate. Active caspase-3 localized almost entirely to keratin filaments, suggesting that keratin is its main substrate in epithelial cells. Later in apoptosis, DEDD, caspase-3, and fragments of keratin filaments formed inclusion bodies that eventually moved into apoptotic blebs. Ubiquitination of DEDD at apoptosis may regulate its scaffolding activity, as keratin and caspase-3 only associated with diubiquitinated DEDD. When DEDD could not be ubiquitinated, keratin filaments remained intact. Caspase-3 also remained largely inactive, indicating that DEDD further activates the protease and thereby regulates the destruction of substrates beyond keratin. The lag between the initial activation of caspase-3 by cytochrome c and later caspase-3 activation by DEDD may ensure that only small amounts of the protease are active until it reaches its main cytoskeletal target. ▪

We have characterized a phosphoprotein protein with a death effector domain that has a novel bifunctional role in programmed cell death. The 15-kDa phosphoprotein enriched in astrocytes (PEA-15) inhibits Fas-mediated apoptosis and increases tumor necrosis factor receptor-1 (TNF-R1)-mediated apoptosis in the same cell type in a ligand-dependent manner. Phosphorylation appears to play a role in its differential effects, since point mutations at one or both phosphorylation consensus sites within PEA-15 destroy its effect on Fas-mediated, but not TNF-R1-mediated, apoptosis. Furthermore, the differential effect is evident at the level of caspase-8 activity which is inhibited via Fas activation, but increased via TNF-R1 activation upon PEA-15 expression. These results show that PEA-15 provides a potential mechanism during development for distinguishing between diverse extracellular death-inducing signals that culminate either in apoptosis or in survival.

Tumor necrosis factor (TNF) can induce apoptosis and activate NF-kappa B through signaling cascades emanating from TNF receptor 1 (TNFR1). TRADD is a TNFR1-associated signal transducer that is involved in activating both pathways. Here we show that TRADD directly interacts with TRAF2 and FADD, signal transducers that activate NF-kappa B and induce apoptosis, respectively. A TRAF2 mutant lacking its N-terminal RING finger domain is a dominant-negative inhibitor of TNF-mediated NF-kappa B activation, but does not affect TNF-induced apoptosis. Conversely, a FADD mutant lacking its N-terminal 79 amino acids is a dominant-negative inhibitor of TNF-induced apoptosis, but does not inhibit NF-kappa B activation. Thus, these two TNFR1-TRADD signaling cascades appear to bifurcate at TRADD.

Much of our knowledge concerning the genetics that regulate cell death has come from the studies of cell death during the development of the nematode Caenorhabditis elegans. Of the 14 genes identified as components of nematode cell death pathways, two genes, ced-3 and ced-4, are required to promote cell death and a third, ced-9, blocks cell death. Recent studies show CED-4 to be an activator of CED-3 and CED-9 to be an inhibitor of CED-4. Two published sequence alignments suggest that CED-4 contains a death effector domain (DED), a protein sequence motif present in other death signaling proteins like Fadd and Flice; one study suggests a DED sequence similarity near the N-terminus while the other found sequence similarity near the C-terminus of CED-4. Using mutational analysis we have tested the functional significance of the conserved residues found within the putative DEDs of CED-4. Mutations in two conserved residues within the putative N-terminal DED of CED-4 affected its function, while mutations in the conserved residues within the putative C-terminal DED had no effect on CED-4 function. Our results do not support the presence of a DED in the C-terminus of CED-4 and suggest a potential role for the N-terminus in CED-4 function, possibly as a DED or as a CARD (caspase recruitment domain). We also found that CED-9 associated with all the CED-4 mutants and inhibited the activity of all the active-CED-4 mutants.

To identify potential biomarkers associated with Alzheimer’s disease (AD)-like neuropathologies in the murine brain, we conducted proteomic analyses of neocortices from TgCRND8 mice. Here we found that phosphoprotein enriched in astrocytes 15kDa (PEA-15) is expressed at higher levels in the neocortical proteomes from 6-month old TgCRND8 mice, as compared to non-transgenic mice. Immunostaining for PEA-15 revealed reactive astrocytes associated with the neocortical amyloid plaques in TgCRND8 mice and in post-mortem human AD brains. This is the first report of increased phosphoprotein enriched in astrocytes (PEA-15) expression in reactive astrocytes of an AD mouse model and human AD brains.

Proteases were, for a long time, mainly considered as protein degrading enzymes. However, in the last decade this view has changed dramatically, and the focus is now on proteases as signalling molecules. One of the best examples is apoptosis, the major mechanism used by eukaryotes to remove superfluous, damaged and potentially dangerous cells, in which a number of proteases have been found to play a central role. Of these the caspases have been considered to be the major players. However, more recently, other proteases have been increasingly suggested as being important in apoptosis, in particular the cysteine cathepsins. In this review the roles of caspases and cysteine cathepsins in apoptosis signalling are compared and discussed.

PEA-15 is a multifunctional protein that modulates signaling pathways which control cell proliferation and cell death. In particular, PEA-15 regulates the actions of the ERK MAP kinase cascade by binding to ERK and altering its subcellular localization. The three-dimensional structure of PEA-15 has been determined using NMR spectroscopy and its interaction with ERK defined by characterization of mutants that modulate ERK function. PEA-15 is composed of an N-terminal death effector domain (DED) and a C-terminal tail of irregular structure. NMR 'footprinting' and mutagenesis identified elements of both the DED and tail that are required for ERK binding. Comparison of the DED-binding surface for ERK2 with the death domain (DD)-binding surface of Drosophila Tube revealed an unexpected similarity between the interaction modes of the DD and DED motifs in these proteins. Despite a lack of functional or sequence similarity between PEA-15 and Tube, these proteins utilize a common surface of the structurally similar DD and DED to recognize functionally diverse targets.

PEA-15 is a small, death effector-domain (DED)–containing protein that was recently demonstrated to inhibit tumor necrosis factor-α–induced apoptosis and to reverse the inhibition of integrin activation due to H-Ras. This led us to investigate the involvement of PEA-15 in Ras signaling. Surprisingly, PEA-15 activates the extracellular signal receptor-activated kinase (ERK) mitogen-activated protein kinase pathway in a Ras-dependent manner. PEA-15 expression in Chinese hamster ovary cells resulted in an increased mitogen-activated protein kinase kinase and ERK activity. Furthermore, PEA-15 expression leads to an increase in Ras guanosine 5′-triphosphate loading. PEA-15 bypasses the anchorage dependence of ERK activation. Finally, the effects of PEA-15 on integrin signaling are separate from those on ERK activation. Heretofore, all known DEDs functioned in the regulation of apoptosis. In contrast, the DED of PEA-15 is essential for its capacity to activate ERK. The ability of PEA-15 to simultaneously inhibit apoptosis and potentiate Ras-to-Erk signaling may be of importance for oncogenic processes.

Background DJ-1 is a protein in association with Parkinson’s disease (PD) and cancers. DJ-1 has been reported to exhibit both cytoplasmic and nuclear distribution (Bonifati et al., Science, 2003; Nagakubo et al., BBRC, 1997). It functions in multiple pathways to affect cell survival. It suppresses the JNK signaling pathway in cytoplasma (Mo et al., Cell Death Differ, 2008) and interacts with Daxx and sequesters it within the nucleus, preventing the initiation of apoptotic signaling (Junn et al., PNAS, 2005). In contrast to its functions in cell survival, deletions or loss of function point mutations in DJ-1 are reported to be responsible for recessive early-onset Parkinson’s disease (PD) (Bonifati et al., Science, 2003). The most commonly studied PD-associated mutant, L166P, is reported to be unstable and to mislocalize to the mitochondria, leading to a loss of the cytoplasmic function of DJ-1 (Bonifati et al., Science, 2003). Although lines of evidence suggest that a high expression of DJ-1 enhances cell survival and loss of DJ-1 function is associated with PD, the detailed mechanisms are still not fully understood.