Crystal structure of MC159 reveals molecular mechanism of DISC assembly and FLIP inhibition

The death-inducing signaling complex (DISC) comprising Fas, Fas-associated death domain (FADD), and caspase-8/10 is assembled via homotypic associations between death domains (DDs) of Fas and FADD and between death effector domains (DEDs) of FADD and caspase-8/10. Caspase-8/10 and FLICE/caspase-8 inhibitory proteins (FLIPs) that inhibit caspase activation at the DISC level contain tandem DEDs. Here, we report the crystal structure of a viral FLIP, MC159, at 1.2 Angstroms resolution. It reveals a noncanonical fold of DED1, a dumbbell-shaped structure with rigidly associated DEDs and a different mode of interaction in the DD superfamily. Whereas the conserved hydrophobic patch of DED1 interacts with DED2, the corresponding region of DED2 mediates caspase-8 recruitment and contributes to DISC assembly. In contrast, MC159 cooperatively assembles with Fas and FADD via an extensive surface that encompasses the conserved charge triad. This interaction apparently competes with FADD self-association and disrupts higher-order oligomerization required for caspase activation in the DISC.     

Mutational analyses of c-FLIPR, the only murine short FLIP isoform, reveal requirements for DISC recruitment

Cellular FLICE-inhibitory protein (c-FLIP) proteins are known as potent inhibitors of death receptor-mediated apoptosis by interfering with caspase-8 activation at the death-inducing signaling complex (DISC). Among the three human isoforms, c-FLIP(long), c-FLIP(short) and c-FLIP(R), the latter isoform is poorly characterized. We report here the characterization of murine c-FLIP(R) and show that it is the only short c-FLIP isoform expressed in mice. By generating several mutants, we demonstrate that both death effector domains (DEDs) are required for DISC binding and the antiapoptotic function of c-FLIP(R). Surprisingly, the C-terminal tail is important for both protein stability and DISC recruitment. Three-dimensional modeling of c-FLIP(R) revealed a substantial similarity of the overall structures and potential interaction motifs with the viral FLIP MC159. We found, however, that c-FLIP(R) uses different structural motifs for its DISC recruitment. Whereas MC159 interferes with interaction and self-oligomerization of the DISC component FADD by its extensive hydrophilic surface, a narrow hydrophobic patch of c-FLIP(R) on the surface of DED2 is crucial for DISC association. Thus, despite the presence of similar tandem DEDs, viral and cellular FLIPs inhibit apoptosis by remarkably divergent mechanisms.     

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]     

Crystal structure of a viral FLIP: insights into FLIP-mediated inhibition of death receptor signaling

Death receptor signaling is initiated by the assembly of the death-inducing signaling complex, which culminates in the activation of the initiator caspase, either caspase-8 or caspase-10. A family of viral and cellular proteins, known as FLIP, plays an essential role in the regulation of death receptor signaling. Viral FLIP (v-FLIP) and short cellular FLIP (c-FLIPS) inhibit apoptosis by interfering with death receptor signaling. The structure and mechanisms of v-FLIP and c-FLIPS remain largely unknown. Here we report a high resolution crystal structure of MC159, a v-FLIP derived from the molluscum contagiosum virus, which is a member of the human poxvirus family. Unexpectedly, the two tandem death effector domains (DEDs) of MC159 rigidly associate with each other through a hydrophobic interface. Structure-based sequence analysis suggests that this interface is conserved in the tandem DEDs from other v-FLIP, c-FLIPS, and caspase-8 and -10. Strikingly, the overall packing arrangement between the two DEDs of MC159 resembles that between the caspase recruitment domains of Apaf-1 and caspase-9. In addition, each DED of MC159 contains a highly conserved binding motif on the surface, to which loss-of-function mutations in MC159 map. These observations, in conjunction with published evidence, reveal significant insights into the function of v-FLIP and suggest a mechanism by which v-FLIP and c-FLIPS inhibit death receptor signaling.     

Revival of apoptotic cells that display early-stage dynamic membrane blebbing

The critical point at which apoptosis becomes irreversible and how cells attain an anti-apoptotic state remain unknown. Here, we report that apoptotic cells undergoing early-stage dynamic membrane blebbing revive. We examined this phenomenon in cell lines that stably express 2DED2DD, a modified FADD produced by fusing the tandem death effector domains (DEDs) and tandem death domains (DDs). Induction of apoptosis caused rapid blebbing. Eight hours later, most cells shrunk while some detached from the flask. Twenty-four hours later, when activated caspase 3 decreased, more than half the cells revived and appeared normal, probably due to the induction of unidentified anti-apoptotic proteins.     

Structure and chromosome localization of the human CASP8 gene

The human CASP8 gene, whose product is also known as caspase 8 and FLICE, encodes an interleukin-1β converting enzyme (ICE)-related cysteine protease that is activated by the engagement of several different death receptors. Caspase 8 is immediately recruited to the Fas receptor once it oligomerizes, and its protease activity is crucial for the apoptotic response generated by the resulting death-inducing signaling complex (DISC). We report here that the CASP8 gene contains at least 11 exons spanning ∼30 kb on human chromosome band 2q33–34. This region of human chromosome 2 was previously reported as the location of the CASP10 gene, whose product is closely related to caspase 8. Chromosome 2 band q33–34 is also involved in tumorigenesis, with loss of heterogeneity (LOH) being reported in a number of tumors. We also report EcoRI and HindIII polymorphisms that may prove to be useful in disease analysis. Both caspases 8 and 10 contain long pro-domains with duplicated death effector domains (DEDs), as well as their corresponding cysteine protease catalytic domains. Thus, it appears that CASP8 and CASP10 have evolved by tandem gene duplication, much like the CASP1, CASP4 and CASP5 gene cluster on human chromosome 11q22.2–22.3.     

Characterization of the {alpha}-helix region in domain 3 of the haemolytic lectin CEL-III: implications for self-oligomerization and haemolytic processes

CEL-III is a haemolytic lectin, which has two beta-trefoil domains (domains 1 and 2) and a beta-sheet-rich domain (domain 3). In domain 3 (residues 284-432), there is a hydrophobic region containing two alpha-helices (H8 and H9, residues 317-357) and a loop between them, in which alternate hydrophobic residues, especially Val residues, are present. To elucidate the role of the alpha-helix region in the haemolytic process, peptides corresponding to different parts of this region were synthesized and characterized. The peptides containing the sequence that corresponded to the loop and second alpha-helix (H9) showed the strongest antibacterial activity for Staphylococcus aureus and Bacillus subtilis through a marked permeabilization of the bacterial cell membrane. The recombinant glutathione S-transferase (GST)-fusion proteins containing domain 3 or the alpha-helix region peptide formed self-oligomers, whereas mutations in the alternate Val residues in the alpha-helix region lead to decreased oligomerization ability of the fusion proteins. These results suggest that the alpha-helix region, particularly its alternate Val residues are important for oligomerization of CEL-III in target cell membranes, which is also required for a subsequent haemolytic action.     

Binding of FADD and Caspase-8 to Molluscum Contagiosum Virus MC159 v-FLIP Is Not Sufficient for Its Antiapoptotic Function

Molluscum contagiosum virus (MCV), a member of the human poxvirus family, encodes the MC159 protein that inhibits Fas-, tumor necrosis factor (TNF)-, and TNF-related apoptosis-inducing ligant (TRAIL)-induced apoptosis. We used site-directed mutagenesis to change charged or hydrophobic amino acid residues to alanines to identify regions of MC159 that are critical for protection from apoptosis and for protein-protein interactions. Surprisingly, while MC159 is thought to block apoptosis by binding to Fas-associated death domain (FADD) or caspase-8, several mutants that lost apoptosis blocking activity still bound to both FADD and caspase-8. Mutations in the predicted hydrophobic patch 1 and alpha2 regions of both death effector domains (DEDs) within MC159 resulted in loss of the ability to bind to FADD or caspase-8 and to block apoptosis. Amino acid substitutions in the RXDL motif located in the alpha6 region of either DED resulted in loss of protection from apoptosis induced by Fas, TNF, and TRAIL and abolished the ability of MC159 to block death effector filament formation. Thus, charged or hydrophobic amino acids in three regions of the MC159 DEDs (hydrophobic patch 1, alpha2, and alpha6) are critical for the protein's ability to interact with cellular proteins and to block apoptosis.     

The Death Effector Domains of Caspase-8 Induce Terminal Differentiation

The differentiation and senescence programs of metazoans play key roles in regulating normal development and preventing aberrant cell proliferation, such as cancer. These programs are intimately associated with both the mitotic and apoptotic pathways. Caspase-8 is an apical apoptotic initiator that has recently been appreciated to coordinate non-apoptotic roles in the cell. Most of these functions are attributed to the catalytic domain, however, the amino-terminal death effector domains (DED)s, which belong to the death domain superfamily of proteins, can also play key roles during development. Here we describe a novel role for caspase-8 DEDs in regulating cell differentiation and senescence. Caspase-8 DEDs accumulate during terminal differentiation and senescence of epithelial, endothelial and myeloid cells; genetic deletion or shRNA suppression of caspase-8 disrupts cell differentiation, while re-expression of DEDs rescues this phenotype. Among caspase-8 deficient neuroblastoma cells, DED expression attenuated tumor growth in vivo and proliferation in vitro via disruption of mitosis and cytokinesis, resulting in upregulation of p53 and induction of differentiation markers. These events occur independent of caspase-8 catalytic activity, but require a critical lysine (K156) in a microtubule-binding motif in the second DED domain. The results demonstrate a new function for the DEDs of caspase-8, and describe an unexpected mechanism that contributes to cell differentiation and senescence.     

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.     

Cloning and characterization of Disc1, the mouse ortholog of DISC1 (Disrupted-in-Schizophrenia 1)

We cloned the mouse ortholog of DISC1 (Disrupted-in-Schizophrenia 1), a candidate gene for schizophrenia. Disc1 is 3163 nucleotides long and has 60% identity with the human DISC1. Disc1 encodes 851 amino acids and has 56% identity with the human protein. Disc1 maps to the DISC1 syntenic region in the mouse, and genomic structure is conserved. A Disc1 splice variant deletes a portion of Disc1 beginning at amino acids orthologous to the human truncation. Bioinformatic analysis and cross-species comparisons revealed sequence conservation distributed across the genes and conservation of leucine zipper and coiled-coil domains in both orthologs. In situ hybridization in adult mouse brain revealed a restricted expression pattern, with highest levels in the dentate gyrus of the hippocampus and lower expression in CA1-CA3 of the hippocampus, cerebellum, cerebral cortex, and olfactory bulbs. Identification of Disc1 will facilitate the study of DISC1's function and creation of mouse models of DISC1 disruption.     

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.     

Protein kinase C alpha and zeta differentially regulate death-inducing signaling complex formation in cigarette smoke extract-induced apoptosis

Cigarette smoke, a major risk factor in emphysema, causes cell death by incompletely understood mechanisms. Death-inducing signaling complex (DISC) formation is an initial event in Fas-mediated apoptosis. We demonstrate that cigarette smoke extract (CSE) induces DISC formation in human lung fibroblasts (MRC-5) and promotes DISC trafficking from the Golgi complex to membrane lipid rafts. We demonstrate a novel role of protein kinase C (PKC) in the regulation of DISC formation and trafficking. The PKC isoforms, PKCalpha, zeta, epsilon, and eta, were activated by CSE exposure. Overexpression of wild-type PKCalpha inhibited, while PKCzeta promoted, CSE-induced cell death. Dominant-negative (dn)PKCzeta protected against CSE-induced cell death by suppressing DISC formation and caspase-3 activation, while dnPKCalpha enhanced cell death by promoting these events. DISC formation was augmented by wortmannin, an inhibitor of PI3K. CSE-induced Akt phosphorylation was reduced by dnPKCalpha, but it was increased by dnPKCzeta. Expression of PKCalpha in vivo inhibited DISC formation, caspase-3/8 activation, lung injury, and cell death after prolonged cigarette smoke exposure, whereas expression of PKCzeta promoted caspase-3 activation. In conclusion, CSE-induced DISC formation is differentially regulated by PKCalpha and PKCzeta via the PI3K/Akt pathway. These results suggest that modulation of PKC may have therapeutic potential in the prevention of smoke-related lung injury.     

The PYRIN domain: a member of the death domain-fold superfamily

PYRIN domains were identified recently as putative protein-protein interaction domains at the N-termini of several proteins thought to function in apoptotic and inflammatory signaling pathways. The approximately 95 residue PYRIN domains have no statistically significant sequence homology to proteins with known three-dimensional structure. Using secondary structure prediction and potential-based fold recognition methods, however, the PYRIN domain is predicted to be a member of the six-helix bundle death domain-fold superfamily that includes death domains (DDs), death effector domains (DEDs), and caspase recruitment domains (CARDs). Members of the death domain-fold superfamily are well established mediators of protein-protein interactions found in many proteins involved in apoptosis and inflammation, indicating further that the PYRIN domains serve a similar function. An homology model of the PYRIN domain of CARD7/DEFCAP/NAC/NALP1, a member of the Apaf-1/Ced-4 family of proteins, was constructed using the three-dimensional structures of the FADD and p75 neurotrophin receptor DDs, and of the Apaf-1 and caspase-9 CARDs, as templates. Validation of the model using a variety of computational techniques indicates that the fold prediction is consistent with the sequence. Comparison of a circular dichroism spectrum of the PYRIN domain of CARD7/DEFCAP/NAC/NALP1 with spectra of several proteins known to adopt the death domain-fold provides experimental support for the structure prediction.     

Activation of the c-Jun N-terminal kinase/stress-activated protein kinase pathway by overexpression of caspase-8 and its homologs.

Caspase-8 is the most proximal caspase in the caspase cascade and possesses a prodomain consisting of two homologous death effector domains (DEDs). We have discovered that caspase-8 and its homologs can physically interact with tumor necrosis factor receptor-associated factor family members and activate the c-Jun N-terminal kinase (JNK, or stress-activated protein kinase) pathway. This ability resides in the DED-containing prodomain of these proteins and is independent of their role as cell death proteases. A point mutant in the first DED of caspase-8 can block JNK activation induced by several death domain receptors. Inhibition of JNK activation blocks apoptosis mediated by caspase-10, Mach-related inducer of toxicity/cFLIP, and Fas/CD95, thereby suggesting a cooperative role of this pathway in the mediation of caspase-induced apoptosis.     

Structural analysis of the phototactic transducer protein HtrII linker region from Natronomonas pharaonis

Halobacterial pharaonis phoborhodopsin [ppR, also called Natronomonas pharaonis sensory rhodopsin II (NpSRII)] is a phototaxis protein which transmits a light signal to the cytoplasm through its transducer protein (pHtrII). pHtrII, a two-transmembrane protein that interacts with ppR, belongs to the group of methyl-accepting chemotaxis proteins (MCPs). Several mutation studies have indicated that the linker region connecting the transmembrane and methylation regions is necessary for signal transduction. However, the three-dimensional (3D) structure of an MCP linker region has yet to be reported, and hence, details concerning the signal transduction mechanism remain unknown. Here the structure of the pHtrII linker region was investigated biochemically and biophysically. Following limited proteolysis, only one trypsin resistant fragment in the pHtrII linker region was identified. This fragment forms a homodimer with a Kd value of 115 microM. The 3D structure of this fragment was determined by solution NMR, and only one alpha-helix was found between two HAMP domains of the linker region. This alpha-helix was significantly stabilized within transmembrane protein pHtrII as revealed by CW-EPR. The presence of Af1503 HAMP domain-like structures in the linker region was supported by CD, NMR, and ELDOR data. The alpha-helix determined here presumably works as a mechanical joint between two HAMP domains in the linker region to transfer the photoactivated conformational change downstream.     

A New C-Terminal Cleavage Product of Procaspase-8, p30, Defines an Alternative Pathway of Procaspase-8 Activation

Caspase-8 is the main initiator caspase in death receptor-induced apoptosis. Procaspase-8 is activated at the death-inducing signaling complex (DISC). Previous studies suggested a two-step model of procaspase-8 activation. The first cleavage step occurs between the protease domains p18 and p10. The second cleavage step takes place between the prodomain and the large protease subunit (p18). Subsequently, the active caspase-8 heterotetramer p18₂-p10₂ is released into the cytosol, starting the apoptotic signaling cascade. In this report, we have further analyzed procaspase-8 processing upon death receptor stimulation directly at the DISC and in the cytosol. We have found an alternative sequence of cleavage events for procaspase-8. We have demonstrated that the first cleavage can also occur between the prodomain and the large protease subunit (p18). The resulting cleavage product, p30, contains both the large protease subunit (p18) and the small protease subunit (p10). p30 is further processed to p10 and p18 by active caspases. Furthermore, we show that p30 can sensitize cells toward death receptor-induced apoptosis. Taken together, our data suggest an alternative mechanism of procaspase-8 activation at the DISC.Apoptosis can be triggered by a number of factors, including UV or γ-irradiation, chemotherapeutic drugs, and signaling from death receptors (11, 12). CD95 (APO-1/Fas) is a member of the death receptor family, a subfamily of the tumor necrosis factor receptor (TNF-R) superfamily (1, 30). Eight members of the death receptor subfamily have been characterized so far: TNF-R1 (DR1, CD120a, p55, p60), CD95 (DR2, APO-1, Fas), DR3 (APO-3, LARD, TRAMP, WSL1), TRAIL-R1 (APO-2, DR4), TRAIL-R2 (DR5, KILLER, TRICK2), DR6, EDA-R, and NGF-R (13). Cross-linking of CD95 by its natural ligand, CD95L (CD178) (29), or by agonistic antibodies induces apoptosis in sensitive cells (31, 36). The death-inducing signaling complex (DISC) is formed within seconds after CD95 stimulation (9). The DISC consists of oligomerized, probably trimerized CD95 receptors, the adaptor molecule FADD, two isoforms of procaspase-8 (procaspase-8a and -8b), procaspase-10, and c-FLIP_L/S/R (6, 19, 21, 25, 27). The interactions between molecules at the DISC are based on homotypic contacts. The death domain of the receptor interacts with the death domain of FADD, while the death effector domain (DED) of FADD interacts with the N-terminal tandem DEDs of procaspase-8 and -10 and c-FLIP_L/S/R.Two isoforms of procaspase-8 (procaspase-8a and procaspase-8b) were reported to be bound to the DISC (24). Both isoforms possess two tandem DEDs, as well as the catalytic subunits p18 and p10 (see Fig. ​Fig.1A).1A). Procaspase-8a contains an additional 2-kDa (15-amino-acid [aa]) fragment, which results from the translation of exon 9. This small fragment is located between the second DED and the large catalytic subunit, resulting in different lengths of procaspase-8a and -8b (p55 and p53 kDa), respectively.Open in a separate windowFIG. 1.A new 30-kDa protein is detected by the anti-caspase-8 MAb C15. (A) Scheme of procaspase-8 and its cleavage products. The binding sites of the anti-caspase-8 MAbs C5 and C15 are indicated. (B) The B-lymphoblastoid cell lines SKW6.4, Raji, and BJAB and the T-cell lines CEM, Jurkat 16, and caspase-8-deficient Jurkat (clone JI9.2) were stimulated with LZ-CD95L for the indicated times, followed by caspase-8 immunoprecipitation (C8-IP) using the anti-caspase-8 MAb C15 directed against the p18 subunit of procaspase-8. Western blotting of immunoprecipitates was performed using the anti-caspase-8 MAb C15 (**, Ig heavy chain; *, unspecific band). (C) SKW6.4 cells were stimulated with LZ-CD95L for different times, and procaspase-8 processing in total cellular lysates was analyzed by Western blotting using the anti-caspase-8 MAb C15. (D) B-lymphoblastoid BJAB cells were stimulated with LZ-TRAIL for different times, and procaspase-8 processing was analyzed as described for panel C. (E) Primary human T cells (day 6) were stimulated with LZ-CD95L, and procaspase-8 processing was analyzed as described for panel C (*, unspecific band).Activation of procaspase-8 is believed to follow an “induced-proximity” model in which high local concentrations and a favorable mutual orientation of procaspase-8 molecules at the DISC lead to their autoproteolytic processing (2, 3, 20). There is strong evidence from several in vitro studies that autoproteolytic activation of procaspase-8 occurs after oligomerization at the receptor complex (20). Furthermore, it has been shown that homodimers of procaspase-8 have proteolytic activity and that proteolytic processing of procaspase-8 occurs between precursor homodimers (3).Procaspase-8a/b (p55/p53) processing at the DISC has been described to involve two sequential cleavage steps (see Fig. ​Fig.1A).1A). This process is referred to as the “two-step model” (3, 17). The first cleavage step occurs between the two protease domains, and the second cleavage step takes place between the prodomain and the large protease subunit (see Fig. ​Fig.1A)1A) (15). During the first cleavage step, the cleavage at Asp³⁷⁴ generates the two subunits p43/p41 and p12. Both cleavage products remain bound to the DISC: p43/p41 by DED interactions and p12 by interactions with the large protease domain of p43/p41. The second cleavage step takes place at Asp²¹⁶ and Asp³⁸⁴, producing the active enzyme subunits p18, p10, and the prodomain p26/p24. As a result of procaspase-8 processing, the active caspase-8 heterotetramer p18₂-p10₂ is formed at the DISC. This heterotetramer is subsequently released into the cytosol, starting the apoptotic signaling cascade (14).Recent studies have shown that processing of procaspase-8 at the DISC is more complicated and can involve additional steps like the generation of a prolonged prodomain of procaspase-8, termed CAP3 (p27), that is quickly converted to p26 (see Fig. ​Fig.1A)1A) (7).In addition to its central role in death receptor-induced apoptosis, caspase-8 was reported to be required for proliferation of lymphocytes (12, 23). Recently caspase-8 was shown to be an important factor for NF-κB activation following T-cell receptor stimulation (28). The mechanism underlying the dual role of caspase-8 activity and its regulation is largely unknown.In the present study, we show that upon death receptor stimulation, p30 is formed by cleavage at Asp²¹⁰, a yet-unknown cleavage product of procaspase-8, which comprises the C terminus of procaspase-8. p30 turned out to be a key intermediate product in the course of procaspase-8 processing. Furthermore, we suggest that the p30-mediated activation of procaspase-8 plays an important role in the amplification of the death signal. Taken together, our findings provide a new mechanism of procaspase-8 activation and extend the current two-step cleavage model by an alternative activation pathway.     

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.     

Substantial Conformational Change Mediated by Charge-Triad Residues of the Death Effector Domain in Protein-Protein Interactions

Protein conformational changes are commonly associated with the formation of protein complexes. The non-catalytic death effector domains (DEDs) mediate protein-protein interactions in a variety of cellular processes, including apoptosis, proliferation and migration, and glucose metabolism. Here, using NMR residual dipolar coupling (RDC) data, we report a conformational change in the DED of the phosphoprotein enriched in astrocytes, 15 kDa (PEA-15) protein in the complex with a mitogen-activated protein (MAP) kinase, extracellular regulated kinase 2 (ERK2), which is essential in regulating ERK2 cellular distribution and function in cell proliferation and migration. The most significant conformational change in PEA-15 happens at helices α2, α3, and α4, which also possess the highest flexibility among the six-helix bundle of the DED. This crucial conformational change is modulated by the D/E-RxDL charge-triad motif, one of the prominent structural features of DEDs, together with a number of other electrostatic and hydrogen bonding interactions on the protein surface. Charge-triad motif promotes the optimal orientation of key residues and expands the binding interface to accommodate protein-protein interactions. However, the charge-triad residues are not directly involved in the binding interface between PEA-15 and ERK2.     

The TRAF3-binding site of human molluscipox virus FLIP molecule MC159 is critical for its capacity to inhibit Fas-induced apoptosis

Members of the viral Flice/caspase-8 inhibitory protein (v-FLIP) family prevent induction of apoptosis by death receptors through inhibition of the processing and activation of procaspase-8 and -10 at the level of the receptor-associated death-inducing signaling complex (DISC). Here, we have addressed the molecular function of the v-FLIP member MC159 of the human molluscum contagiosum virus. MC159 FLIP powerfully inhibited both caspase-dependent and caspase-independent cell death induced by Fas. The C-terminal region of MC159 bound TNF receptor-associated factor (TRAF)3, was necessary for optimal TRAF2 binding, and mediated the recruitment of both TRAFs into the Fas DISC. TRAF-binding-deficient mutants of MC159 showed impaired inhibition of FasL-induced caspase-8 processing and Fas internalization, and had reduced antiapoptotic activity. Our findings provide evidence that a MC159/TRAF2/TRAF3 complex regulates a new aspect of Fas signaling, and identify MC159 FLIP as a molecule that targets multiple features of Fas-induced cell death.