The structure of FADD and its mode of interaction with procaspase-8

The structure of FADD has been solved in solution, revealing that the death effector domain (DED) and death domain (DD) are aligned with one another in an orthogonal, tail-to-tail fashion. Mutagenesis of FADD and functional reconstitution with its binding partners define the interaction with the intracellular domain of CD95 and the prodomain of procaspase-8 and reveal a self-association surface necessary to form a productive complex with an activated "death receptor." The identification of a procaspase-specific binding surface on the FADD DED suggests a preferential interaction with one, but not both, of the DEDs of procaspase-8 in a perpendicular arrangement. FADD self-association is mediated by a "hydrophobic patch" in the vicinity of F25 in the DED. The structure of FADD and its functional characterization, therefore, illustrate the architecture of key components in the death-inducing signaling complex.     

Evidence of complex formation between FADD and c-FLIP death effector domains for the death inducing signaling complex

Adaptor protein FADD forms the death inducing signaling complex (DISC) by recruiting the initiating caspases-8 and -10 through homotypic death effector domain (DED) interactions. Cellular FLICE-inhibitory protein (c-FLIP) is an inhibitor of death ligand-induced apoptosis downstream of death receptors, and FADD competes with procaspase-8/10 for recruitment for DISC. However, the mechanism of action of FADD and c-FLIP proteins remain poorly understood at the molecular level. In this study, we provide evidence indicating that the death effector domain (DED) of FADD interacts directly with the death effector domain of human c-FLIP. In addition, we use homology modeling to develop a molecular docking model of FADD and c-FLIP proteins. We also find that four structure-based mutants (E80A, L84A, K169A and Y171A) of c-FLIP DEDs disturb the interaction with FADD DED, and that these mutations lower the stability of the c-FLIP DED. [BMB Reports 2014; 47(9): 488-493]     

Homotypic FADD interactions through a conserved RXDLL motif are required for death receptor-induced apoptosis

Death receptors in the TNF receptor superfamily signal for apoptosis via the ordered recruitment of FADD and caspase-8 to a death-inducing signaling complex (DISC). However, the nature of the protein-protein interactions in the signaling complex is not well defined. Here we show that FADD self-associates through a conserved RXDLL motif in the death effector domain (DED). Despite exhibiting similar binding to both Fas and caspase-8 and preserved overall secondary structure, FADD RDXLL motif mutants cannot reconstitute FasL- or TRAIL-induced apoptosis and fail to recruit caspase-8 into the DISC of reconstituted FADD-deficient cells. Abolishing self-association can transform FADD into a dominant-negative mutant that interferes with Fas-induced apoptosis and formation of microscopically visible receptor oligomers. These findings suggest that lateral interactions among adapter molecules are required for death receptor apoptosis signaling and implicate self-association into oligomeric assemblies as a key function of death receptor adapter proteins in initiating apoptosis.     

Differential signaling to apoptotic and necrotic cell death by Fas-associated death domain protein FADD

Two general pathways for cell death have been defined, apoptosis and necrosis. Previous studies in Jurkat cells have demonstrated that the Fas-associated death domain (FADD) is required for Fas-mediated signaling to apoptosis and necrosis. Here we developed L929rTA cell lines that allow Tet-on inducible expression and FK506-binding protein (FKBP)-mediated dimerization of FADD, FADD-death effector domain (FADD-DED), or FADD-death domain (FADD-DD). We show that expression and dimerization of FADD leads to necrosis. However, pretreatment of the cells with the Hsp90 inhibitor geldanamycin, which leads to proteasome-mediated degradation of receptor interacting protein 1 (RIP1), reverts FKBP-FADD-induced necrosis to apoptosis. Expression and dimerization of FADD-DD mediates necrotic cell death. We found that FADD-DD is able to bind RIP1, another protein necessary for Fas-mediated necrosis. Expression and dimerization of FADD-DED initiates apoptosis. Remarkably, in the presence of caspase inhibitors, FADD-DED mediates necrotic cell death. Coimmunoprecipitation studies revealed that FADD-DED in the absence procaspase-8 C/A is also capable of recruiting RIP1. However, when procaspase-8 C/A and RIP1 are expressed simultaneously, FADD-DED preferentially recruits procaspase-8 C/A.     

The role of death effector domain-containing proteins in acute oxidative cell injury in hepatocytes

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.     

FADD self-association is required for stable interaction with an activated death receptor

Receptor-mediated programmed cell death proceeds through an activated receptor to which the death adaptor FADD and the initiator procaspases 8 and/or 10 are recruited following receptor stimulation. The adaptor FADD is responsible for both receptor binding and recruitment of the procaspases into the death-inducing signaling complex. Biochemical dissection of the FADD death effector domain and functional replacement with a coiled-coil motif demonstrates that there is an obligatory FADD self-association via the DED during assembly of the death-inducing signaling complex. Using engineered oligomerization motifs with defined stoichiometries, the requirement for FADD self-association through the DED can be separated from the caspase-recruitment function of the domain. Disruption of FADD self-association precludes formation of a competent signaling complex. On this basis, we propose an alternative architecture for the FADD signaling complex in which FADD acts as a molecular bridge to stitch together an array of activated death receptors.     

A death effector domain chain DISC model reveals a crucial role for caspase-8 chain assembly in mediating apoptotic cell death

Formation of the death-inducing signaling complex (DISC) is a critical step in death receptor-mediated apoptosis, yet the mechanisms underlying assembly of this key multiprotein complex remain unclear. Using quantitative mass spectrometry, we have delineated the stoichiometry of the native TRAIL DISC. While current models suggest that core DISC components are present at a ratio of 1:1, our data indicate that FADD is substoichiometric relative to TRAIL-Rs or DED-only proteins; strikingly, there is up to 9-fold more caspase-8 than FADD in the DISC. Using structural modeling, we propose an alternative DISC model in which procaspase-8 molecules interact sequentially, via their DED domains, to form a caspase-activating chain. Mutating key interacting residues in procaspase-8 DED2 abrogates DED chain formation in cells and disrupts TRAIL/CD95 DISC-mediated procaspase-8 activation in?a functional DISC reconstitution model. This provides direct experimental evidence for a DISC model in which DED chain assembly drives caspase-8 dimerization/activation, thereby triggering cell death.     

The death effector domain-associated factor plays distinct regulatory roles in the nucleus and cytoplasm

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.     

The three-dimensional solution structure and dynamic properties of the human FADD death domain

FADD (also known as MORT-1) is an essential adapter protein that couples the transmembrane receptors Fas (CD95) and tumor necrosis factor receptor-1 (TNF-R1) to intracellular cysteine proteases known as caspases, which propagate and execute the programmed cell death-inducing signal triggered by Fas ligand (FasL, CD95L) and TNF. FADD contains 208 amino acid residues, and comprises two functionally and structurally distinct domains: an N-terminal death effector domain (DED) that promotes activation of the downstream proteolytic cascade through binding of the DED domains of procaspase-8; and a C-terminal death domain (DD). FADD-DD provides the site of FADD recruitment to death receptor complexes at the plasma membrane by, for example, interaction with the Fas receptor cytoplasmic death domain (Fas-DD), or binding of the TNF-R1 adapter molecule TRADD. We have determined the three-dimensional solution structure and characterised the internal polypeptide dynamics of human FADD-DD using heteronuclear NMR spectroscopy of (15)N and (13)C,(15)N-labelled samples. The structure comprises six alpha-helices joined by short loops and displays overall similarity to the death domain of the Fas receptor. The analysis of the dynamic properties reveals no evidence of contiguous stretches of polypeptide chain with increased internal motion, except at the extreme chain termini. A pattern of increased rates of amide proton solvent exchange in the alpha3 helix correlates with a higher degree of solvent exposure for this secondary structure element. The properties of the FADD-DD structure are discussed with respect to previously reported mutagenesis data and emerging models for FasL-induced FADD recruitment to Fas and caspase-8 activation.     

Structural insight for the roles of fas death domain binding to fadd and oligomerization degree of the fas–fadd complex in the death‐inducing signaling complex formation: A computational study

Fas binding to Fas‐associated death domain (FADD) activates FADD–caspase‐8 binding to form death‐inducing signaling complex (DISC) that triggers apoptosis. The Fas–Fas association exists primarily as dimer in the Fas–FADD complex, and the Fas–FADD tetramer complexes have the tendency to form higher order oligomer. The importance of the oligomerized Fas–FADD complex in DISC formation has been confirmed. This study sought to provide structural insight for the roles of Fas death domain (Fas DD) binding to FADD and the oligomerization of Fas DD–FADD complex in activating FADD–procaspase‐8 binding. Results show Fas DD binding to FADD stabilized the FADD conformation, including the increased stability of the critical residues in FADD death effector domain (FADD DED) for FADD–procaspase‐8 binding. Fas DD binding to FADD resulted in the decreased degree of both correlated and anticorrelated motion of the residues in FADD and caused the reversed correlated motion between FADD DED and FADD death domain (FADD DD). The exposure of procaspase‐8 binding residues in FADD that allows FADD to interact with procaspase‐8 was observed with Fas DD binding to FADD. We also observed different degrees of conformational and motion changes of FADD in the Fas DD–FADD complex with different degrees of oligomerization. The increased conformational stability and the decreased degree of correlated motion of the residues in FADD in Fas DD–FADD tetramer complex were observed compared to those in Fas DD–FADD dimer complex. This study provides structural evidence for the roles of Fas DD binding to FADD and the oligomerization degree of Fas DD–FADD complex in DISC formation to signal apoptosis. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Direct binding of Fas-associated death domain (FADD) to the tumor necrosis factor-related apoptosis-inducing ligand receptor DR5 is regulated by the death effector domain of FADD

Members of the tumor necrosis factor superfamily of receptors induce apoptosis by recruiting adaptor molecules through death domain interactions. The central adaptor molecule for these receptors is the death domain-containing protein Fas-associated death domain (FADD). FADD binds a death domain on a receptor or additional adaptor and recruits caspases to the activated receptor. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signals apoptosis through two receptors, DR4 and DR5. Although there is much interest in TRAIL, the mechanism by which FADD is recruited to the TRAIL receptors is not clear. Using a reverse two-hybrid system we previously identified mutations in the death effector domain of FADD that prevented binding to Fas/CD95. Here we show that these mutations also prevent binding to DR5. FADD-deficient Jurkat cells stably expressing these FADD mutations did not transduce TRAIL or Fas/CD95 signaling. Second site compensating mutations that restore binding to and signaling through Fas/CD95 and DR5 were also in the death effector domain. We conclude that in contrast to current models where the death domain of FADD functions independently of the death effector domain, the death effector domain of FADD comes into direct contact with both TRAIL and Fas/CD95 receptors.     

Prolonged activation of ERK1,2 induces FADD-independent caspase 8 activation and cell death

Prolonged ERK/MAPK activation has been implicated in neuronal cell death in vitro and in vivo. We found that HEK293 cells, recently reported to express neuronal markers, are exquisitely sensitive to long term ERK stimulation. Activation of an inducible form of Raf-1 (Raf-1:ER) in HEK293 cells induced massive apoptosis characterized by DNA degradation, loss of plasma membrane integrity and PARP cleavage. Cell death required MEK activity and protein synthesis and occurred via the death receptor pathway independently of the mitochondrial pathway. Accordingly, prolonged ERK stimulation activated caspase 8 and strongly potentiated Fas signaling. The death receptor adaptator FADD was found to be rapidly induced upon ERK activation. However using RNA interference and ectopic expression, we demonstrated that neither FADD nor Fas were necessary for caspase 8 activation and cell death. These findings reveal that prolonged ERK/MAPK stimulation results in caspase 8 activation and cell death. This work was supported by grant from Association pour la Recherche sur le Cancer (CNRS6543/ARC). S. Cagnol is supported by a fellowship from the Ligue Nationale contre le Cancer.     

Stoichiometry of the CD95 death-inducing signaling complex: experimental and modeling evidence for a death effector domain chain model

The CD95 (Fas/APO-1) death-inducing signaling complex (DISC) is essential for the initiation of CD95-mediated apoptotic and nonapoptotic responses. The CD95 DISC comprises CD95, FADD, procaspase-8, procaspase-10, and c-FLIP proteins. Procaspase-8 and procaspase-10 are activated at?the DISC, leading to the formation of active caspases and apoptosis initiation. In this study we analyzed the?stoichiometry of the CD95 DISC. Using quantitative western blots, mass spectrometry, and mathematical modeling, we reveal that the amount of DED proteins procaspase-8/procaspase-10 and c-FLIP at the DISC exceeds that of FADD by several-fold. Furthermore, our findings imply that procaspase-8, procaspase-10, and c-FLIP could form DED chains at the DISC, enabling the formation of dimers and efficient activation of caspase-8. Taken together, our findings provide an enhanced understanding of caspase-8 activation and initiation of apoptosis at the DISC.     

P45 Forms a Complex with FADD and Promotes Neuronal Cell Survival Following Spinal Cord Injury

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.     

A docking model of key components of the DISC complex: death domain superfamily interactions redefined

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.     

Regulation of the Fas death pathway by FLICE-inhibitory protein in primary human B cells

The Fas/Fas ligand (L) system plays an important role in the maintenance of peripheral B cell tolerance and the prevention of misguided T cell help. CD40-derived signals are required to induce Fas expression on virgin B cells and to promote their susceptibility to Fas-mediated apoptosis. In the current study, we have analyzed the early biochemical events occurring upon Fas ligation in CD40L-activated primary human tonsillar B cells with respect to Fas-associated death domain protein (FADD), caspase-8/FADD-like IL-1beta-converting enzyme (FLICE), and c-FLICE inhibitory protein (FLIP). We report here that Fas-induced apoptosis in B cells does not require integrity of the mitochondrial Apaf-1 pathway and that caspase-8 is activated by association with the death-inducing signaling complex (DISC), i.e., upstream of the mitochondria. We show that both FADD and the zymogen form of caspase-8 are constitutively expressed at high levels in virgin B cells, whereas c-FLIP expression is marginal. In contrast, c-FLIP, but neither FADD nor procaspase-8, is strongly up-regulated upon ligation of CD40 or the B cell receptor on virgin B cells. Finally, we have found that c-FLIP is also recruited and cleaved at the level of the DISC in CD40L-activated virgin B cells. We propose that c-FLIP expression delays the onset of apoptosis in Fas-sensitive B cells. The transient protection afforded by c-FLIP could offer an ultimate safeguard mechanism against inappropriate cell death or allow recruitment of phagocytes to ensure efficient removal of apoptotic cells.     

Identification and Characterization of the Interaction Site between cFLIPL and Calmodulin

Overexpression of the cellular FLICE-like inhibitory protein (cFLIP) has been reported in a number of tumor types. As an inactive procaspase-8 homologue, cFLIP is recruited to the intracellular assembly known as the Death Inducing Signaling Complex (DISC) where it inhibits apoptosis, leading to cancer cell proliferation. Here we characterize the molecular details of the interaction between cFLIP_L and calmodulin, a ubiquitous calcium sensing protein. By expressing the individual domains of cFLIP_L, we demonstrate that the interaction with calmodulin is mediated by the N-terminal death effector domain (DED1) of cFLIP_L. Additionally, we mapped the interaction to a specific region of the C-terminus of DED1, referred to as DED1 R4. By designing DED1/DED2 chimeric constructs in which the homologous R4 regions of the two domains were swapped, calmodulin binding properties were transferred to DED2 and removed from DED1. Furthermore, we show that the isolated DED1 R4 peptide binds to calmodulin and solve the structure of the peptide-protein complex using NMR and computational refinement. Finally, we demonstrate an interaction between cFLIP_L and calmodulin in cancer cell lysates. In summary, our data implicate calmodulin as a potential player in DISC-mediated apoptosis and provide evidence for a specific interaction with the DED1 of cFLIP_L.     

The C-terminal tails of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and Fas receptors have opposing functions in Fas-associated death domain (FADD) recruitment and can regulate agonist-specific mechanisms of receptor activation

Members of the tumor necrosis factor (TNF) superfamily of receptors such as Fas/CD95 and the TNF-related apoptosis-inducing ligand (TRAIL) receptors DR4 and DR5 induce apoptosis by recruiting adaptor molecules and caspases. The central adaptor molecule for these receptors is a death domain-containing protein, FADD, which binds to the activated receptor via death domain-death domain interactions. Here, we show that in addition to the death domain, the C-terminal tails of DR4 and DR5 positively regulate FADD binding, caspase activation and apoptosis. In contrast, the corresponding region in the Fas receptor has the opposite effect and inhibits binding to the receptor death domain. Replacement of wild-type or mutant DR5 molecules into DR5-deficient BJAB cells indicates that some agonistic antibodies display an absolute requirement for the C-terminal tail for FADD binding and signaling while other antibodies can function in the absence of this mechanism. These data demonstrate that regions outside the death domains of DR4 and DR5 have opposite effects to that of Fas in regulating FADD recruitment and show that different death receptor agonists can use distinct molecular mechanisms to activate signaling from the same receptor.