Death domain assembly mechanism revealed by crystal structure of the oligomeric PIDDosome core complex

Proteins of the death domain (DD) superfamily mediate assembly of oligomeric signaling complexes for the activation of caspases and kinases via unknown mechanisms. Here we report the crystal structure of the PIDD DD and RAIDD DD complex, which forms the core of the caspase-2-activating complex PIDDosome. Although RAIDD DD and PIDD DD are monomers, they assemble into a complex that comprises seven RAIDD DDs and five PIDD DDs. Despite the use of an asymmetric assembly mechanism, all DDs in the complex are in quasi-equivalent environments. The structure provided eight unique asymmetric interfaces, which can be classified into three types. These three types of interactions together cover a majority of the DD surface. Mutagenesis on almost all interfaces leads to disruption of the assembly, resulting in defective caspase-2 activation. The three types of interactions may represent most, if not all, modes of interactions in the DD superfamily for assembling complexes of different stoichiometry.     

Structural analyses of death domains and their interactions

The death domain (DD), which is a versatle protein interaction module, is the prime mediator of the interactions necessary for apoptosis, innate immunity and the necrosis signaling pathway. Because DD mediated signaling events are associated with critical human diseases, studies in these areas are of great biological importance. Accordingly, many biochemical and structural studies of DD have been conducted in the past decade to investigate apoptotic and innate immune signaling. Evaluation of the molecular structure of DD and their interactions with partners have shown the underlying molecular basis for the assembly of DD mediated complexes and for the regulation of apoptosis and innate immunity. This review summarizes the structure and function of various DDs and DD:DD complexes involved in those signaling pathways.     

RIP death domain structural interactions implicated in TNF-mediated proliferation and survival

Death domain (DD)-containing proteins are involved in both apoptosis and survival/proliferation signaling induced by activated death receptors. Here, a phylogenetic and structural analysis was performed to highlight differences in DD domains and their key regulatory interaction sites. The phylogenetic analysis shows that receptor DDs are more conserved than DDs in adaptors. Adaptor DDs can be subdivided into those that activate or inhibit apoptosis. Modeling of six homotypic DD interactions involved in the TNF signaling pathway implicates that the DD of RIP (Receptor interacting protein kinase 1) is capable of interacting with the DD of TRADD (TNFR1-associated death domain protein) in two different, exclusive ways: one that subsequently recruits CRADD (apoptosis/inflammation) and another that recruits NFkappaB (survival/proliferation).     

Interaction patches of procaspase-1 caspase recruitment domains (CARDs) are differently involved in procaspase-1 activation and receptor-interacting protein 2 (RIP2)-dependent nuclear factor κB signaling

Protein interaction domains belonging to the death domain-fold superfamily are six-helix bundles that mediate the assembly of large protein complexes involved in apoptotic and inflammatory signaling. Typically, death domains (DDs), a subfamily of the death domain-fold superfamily, harbor six delineated interaction patches on their surfaces that mediate three distinct and conserved types of interaction designated as types I, II, and III. Here, we show that caspase recruitment domains (CARDs), another subfamily of the death domain-fold superfamily, multimerize by employing at least two of the three reported interaction types that were identified in DDs. On the one hand, the CARD of procaspase-1 binds the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) through a type I interaction that involves a patch surrounding residue Asp-27. On the other hand, the CARD of procaspase-1 auto-oligomerizes through a type III interaction involving a patch surrounding residue Arg-45. This oligomerization allows binding of receptor-interacting protein 2 (RIP2). In addition, we show that a 1:1 interaction between ASC and procaspase-1 is sufficient for procaspase-1 to gain proteolytic activity, whereas the formation of a higher order CARD complex involving ASC, procaspase-1, and RIP2 is required for effective procaspase-1-mediated NF-κB activation. These findings indicate that the CARD of procaspase-1 is differently involved in the formation of procaspase-1 activating platforms and procaspase-1-mediated, RIP2-dependent NF-κB activation.     

MyD88 oligomer size functions as a physical threshold to trigger IL1R Myddosome signaling

A recurring feature of innate immune receptor signaling is the self-assembly of signaling proteins into oligomeric complexes. The Myddosome is an oligomeric complex that is required to transmit inflammatory signals from TLR/IL1Rs and consists of MyD88 and IRAK family kinases. However, the molecular basis for how Myddosome proteins self-assemble and regulate intracellular signaling remains poorly understood. Here, we developed a novel assay to analyze the spatiotemporal dynamics of IL1R and Myddosome signaling in live cells. We found that MyD88 oligomerization is inducible and initially reversible. Moreover, the formation of larger, stable oligomers consisting of more than four MyD88s triggers the sequential recruitment of IRAK4 and IRAK1. Notably, genetic knockout of IRAK4 enhanced MyD88 oligomerization, indicating that IRAK4 controls MyD88 oligomer size and growth. MyD88 oligomer size thus functions as a physical threshold to trigger downstream signaling. These results provide a mechanistic basis for how protein oligomerization might function in cell signaling pathways.     

Solution structure of the isolated Pelle death domain

The interaction between the death domains (DDs) of Tube and the protein kinase Pelle is an important component of the Toll pathway. Published crystallographic data suggests that the Pelle-Tube DD interface is plastic and implies that in addition to the two predominant Pelle-Tube interfaces, a third interaction is possible. We present the NMR solution structure of the isolated death domain of Pelle and a study of the interaction between the DDs of Pelle and Tube. Our data suggests the solution structure of the isolated Pelle DD is similar to that of Pelle DD in complex with Tube. Additionally, they suggest that the plasticity observed in the crystal structure may not be relevant in the functioning death domain complex.     

Two human MYD88 variants, S34Y and R98C, interfere with MyD88-IRAK4-myddosome assembly

Innate immune receptors detect microbial pathogens and subsequently activate adaptive immune responses to combat pathogen invasion. MyD88 is a key adaptor molecule in both Toll-like receptor (TLR) and IL-1 receptor superfamily signaling pathways. This is illustrated by the fact that human individuals carrying rare, naturally occurring MYD88 point mutations suffer from reoccurring life-threatening infections. Here we analyzed the functional properties of six reported non-synonymous single nucleotide polymorphisms of MYD88 in an in vitro cellular system. Two variants found in the MyD88 death domain, S34Y and R98C, showed severely reduced NF-κB activation due to reduced homo-oligomerization and IRAK4 interaction. Structural modeling highlights Ser-34 and Arg-98 as residues important for the assembly of the Myddosome, a death domain (DD) post-receptor complex involving the DD of MyD88, IRAK4, and IRAK2 or IRAK1. Using S34Y and R98C as functional probes, our data show that MyD88 homo-oligomerization and IRAK4 interaction is modulated by the MyD88 TIR and IRAK4 kinase domain, demonstrating the functional importance of non-DD regions not observed in a recent Myddosome crystal structure. The differential interference of S34Y and R98C with some (IL-1 receptor, TLR2, TLR4, TLR5, and TLR7) but not all (TLR9) MyD88-dependent signaling pathways also suggests that receptor specificities exist at the level of the Myddosome. Given their detrimental effect on signaling, it is not surprising that our epidemiological analysis in several case-control studies confirms that S34Y and R98C are rare variants that may drastically contribute to susceptibility to infection in only few individuals.     

An Oligomeric Signaling Platform Formed by the Toll-like Receptor Signal Transducers MyD88 and IRAK-4

Toll-like receptors (TLRs) mediate responses to pathogen-associated molecules as part of the vertebrate innate immune response to infection. Receptor dimerization is coupled to downstream signal transduction by the recruitment of a post-receptor complex containing the adaptor protein MyD88 and the IRAK protein kinases. In this work, we show that the death domains of human MyD88 and IRAK-4 assemble into closed complexes having unusual stoichiometries of 7:4 and 8:4, the Myddosome. Formation of the Myddosome is likely to be a key event for TLR4 signaling in vivo as we show here that pathway activation requires that the receptors cluster into lipid rafts. Taken together, these findings indicate that TLR activation causes the formation of a highly oligomeric signaling platform analogous to the death-inducing signaling complex of the Fas receptor pathway.In vertebrates, the initial responses of innate immunity are mediated by a family of pattern recognition receptors, which are able to sense the presence of a variety of microbial products such as lipids and non-self nucleic acid (1). One important family of pattern recognition receptors is the Toll-like receptors (TLRs)⁴ that are expressed by many immune system cell types such as macrophages and dendritic cells. TLRs are class one transmembrane receptors that are activated by a process of stimulus-induced dimerization of their extracellular domains. This in turn causes the cytoplasmic Toll/interleukin-1 (IL-1) domains (TIRs) to dimerize, forming a scaffold for the recruitment of downstream signaling components (2). TLRs use five signaling adaptor proteins to couple receptor activation to downstream signal transduction (3). All of these adaptors have TIRs and engage with the activated TLRs by TIR-TIR interactions.One of the adaptor proteins, MyD88, is of particular importance because it is used by all but one of the TLRs as well as by the IL-1 and interferon-γ receptors. MyD88-deficient mice have profoundly impaired innate immune responses and are susceptible to a wide range of infectious diseases. The MyD88 sequence is tripartite and is comprised of a death domain (DD) at the N terminus, a short (40-amino-acid) intermediate domain (ID) of unknown structure, and a C-terminal TIR. Evidence from yeast two-hybrid experiments suggests that MyD88 can self-associate with contacts in both the DD and the TIR (4). The current view of post-receptor signal transduction is that two MyD88 TIR domains bind to the activated TLR, and this enables the recruitment of the protein kinases IRAK-4 and IRAK-1 (5). These kinases have DDs at their N termini, and both are recruited into a complex with MyD88 after signal initiation. It appears that IRAK-4 is recruited first, and this binding requires the ID of MyD88 (6, 7). Thus MyD88s, a splice variant that lacks the ID, down-regulates TLR signaling and cannot recruit IRAK-4 into the post-receptor complex. In contrast, IRAK-1 interacts with MyD88s presumably by DD-DD rather than DD-ID interactions. The next step in the signaling process is for IRAK-4 to phosphorylate IRAK-1, causing activation of the latter and hyper-autophosphorylation. IRAK-1 then dissociates from the complex and interacts with the ubiquitin-protein isopeptide ligase (E3) TRAF6 (8, 9).DDs together with the structurally related caspase recruitment domains (CARDs) and death effector domains (DEDs) form the death domain superfamily (10). There are 215 proteins encoded by the human genome that are predicted to have this fold, and they are widely used in cellular signaling including the TLR and apoptotic pathways. Structurally, DDs contain six antiparallel α-helices, and they are predominantly involved in protein-protein interactions with other DDs. Three modes of DD-DD interaction, types 1, 2, and 3 (10), have been characterized and are illustrated by the structures of the Drosophila Tube-Pelle heterodimer (11), the Procaspase-9 homodimer (12), and most remarkably, by the PIDDosome (13). In the latter case, PIDD, RAIDD, and Caspase-2 form a complex, which results in the proximity-induced activation of Caspase-2 protease activity, which in turn leads to cytochrome c release and apoptotic cell death. The DDs of PIDD and RAIDD interact to produce a complex having a stoichiometry of 5:7, and the subunits are arranged in three layers with five PIDDs, five RAIDDs, and then two RAIDDs. The structure is stabilized by 25 DD-DD contacts of which six are type 2, nine are type 1, and 10 are type 3.In this study, we report that like PIDD and RAIDD, the DDs of human MyD88 and IRAK-4 assemble into defined structures having stoichiometries of 7:4 and 8:4. We propose that the structure has two layers with a ring of seven or eight MyD88 subunits and a second layer of four IRAK-4 subunits. The formation of these higher order assemblies provides insight into the complex regulation and cross-talk observed in the TLR signaling pathways.     

Interleukin-1 (IL-1) receptor-associated kinase-dependent IL-1-induced signaling complexes phosphorylate TAK1 and TAB2 at the plasma membrane and activate TAK1 in the cytosol

Interleukin-1 (IL-1) receptor-associated kinase (IRAK) plays an important role in the sequential formation and activation of IL-1-induced signaling complexes. Previous studies showed that IRAK is recruited to the IL-1-receptor complex, where it is hyperphosphorylated. We now find that the phosphorylated IRAK in turn recruits TRAF6 to the receptor complex (complex I), which differs from the previous concept that IRAK interacts with TRAF6 after it leaves the receptor. IRAK then brings TRAF6 to TAK1, TAB1, and TAB2, which are preassociated on the membrane before stimulation to form the membrane-associated complex II. The formation of complex II leads to the phosphorylation of TAK1 and TAB2 on the membrane by an unknown kinase, followed by the dissociation of TRAF6-TAK1-TAB1-TAB2 (complex III) from IRAK and consequent translocation of complex III to the cytosol. The formation of complex III and its interaction with additional cytosolic factors lead to the activation of TAK1, resulting in NF-kappaB and JNK activation. Phosphorylated IRAK remains on the membrane and eventually is ubiquitinated and degraded. Taken together, the new data reveal that IRAK plays a critical role in mediating the association and dissociation of IL-1-induced signaling complexes, functioning as an organizer and transporter in IL-1-dependent signaling.     

Inhibition of both the extrinsic and intrinsic death pathways through nonhomotypic death-fold interactions

Death-fold domains constitute an evolutionarily conserved superfamily that mediates apoptotic signaling. These motifs, including CARD (caspase recruitment domain), DD (death domain), and DED (death effector domain), are believed to exert their effects solely through homotypic interactions. Herein we demonstrate that the CARD-containing protein ARC engages in nontraditional death-fold interactions to suppress both extrinsic and intrinsic death pathways. The extrinsic pathway is disrupted by heterotypic interactions between ARC's CARD and the DDs of Fas and FADD, which inhibit Fas-FADD binding and assembly of the death-inducing signaling complex (DISC). The intrinsic pathway is antagonized by ARC-Bax binding, involving ARC's CARD and the Bax C terminus. This inhibits Bax activation and translocation to the mitochondria. Knockdown of endogenous ARC facilitates DISC assembly and triggers spontaneous Bax activation and apoptosis. Conversely, physiological levels of ARC suppress these events. These studies establish a critical role for nonhomotypic death-fold interactions in the regulation of apoptosis.     

Identification of Critical Residues of the MyD88 Death Domain Involved in the Recruitment of Downstream Kinases

MyD88 couples the activation of the Toll-like receptors and interleukin-1 receptor superfamily with intracellular signaling pathways. Upon ligand binding, activated receptors recruit MyD88 via its Toll-interleukin-1 receptor domain. MyD88 then allows the recruitment of the interleukin-1 receptor-associated kinases (IRAKs). We performed a site-directed mutagenesis of MyD88 residues, conserved in death domains of the homologous FADD and Pelle proteins, and analyzed the effect of the mutations on MyD88 signaling. Our studies revealed that mutation of residues 52 (MyD88_E52A) and 58 (MyD88_Y58A) impaired recruitment of both IRAK1 and IRAK4, whereas mutation of residue 95 (MyD88_K95A) only affected IRAK4 recruitment. Since all MyD88 mutants were defective in signaling, recruitment of both IRAKs appeared necessary for activation of the pathway. Moreover, overexpression of a green fluorescent protein (GFP)-tagged mini-MyD88 protein (GFP-MyD88-(27–72)), comprising the Glu⁵² and Tyr⁵⁸ residues, interfered with recruitment of both IRAK1 and IRAK4 by MyD88 and suppressed NF-κB activation by the interleukin-1 receptor but not by the MyD88-independent TLR3. GFP-MyD88-(27–72) exerted its effect by titrating IRAK1 and suppressing IRAK1-dependent NF-κB activation. These experiments identify novel residues of MyD88 that are crucially involved in the recruitment of IRAK1 and IRAK4 and in downstream propagation of MyD88 signaling.MyD88 was first discovered during studies addressing the differentiation of mouse myeloid cells in response to growth-inhibitory stimuli (1). Subsequent investigations revealed that MyD88 possesses a modular organization (2), with an amino-terminal death domain (DD),³ found in proteins involved in cell death (3, 4), and a carboxyl-terminal Toll-interleukin-1 receptor (TIR) domain, present in the intracytoplasmic tail of receptors belonging to the Toll-like receptor (TLR)/interleukin-1 receptor (IL-1R) superfamily (5). MyD88 also has an intermediate domain (ID) that is crucial in TLR signaling due to its interaction with IRAK4 (6). The role of MyD88 as a signal transducer was first shown in the pathways triggered by the activation of IL-1R (7, 8) and TLR4 (9). Further studies showed that all TLRs, with the sole exception of TLR3, and the IL-1R family utilize the adaptor protein MyD88 to initiate their signaling pathway (10).By virtue of its modular organization, MyD88 critically bridges activated receptor complexes to downstream adaptors/effectors. Upon activation, MyD88 is recruited through its TIR domain by the homologous domain of the activated TLR/IL-1R (11, 12). MyD88, in turn, has been shown to interact with a family of downstream kinases, namely IRAK1 (13), IRAK2 (7), IRAK-M (15), and IRAK4 (16), through the interaction of its DD with the respective DDs present in the amino-terminal region of IRAKs (17). At this stage, this multimeric complex is competent to elicit the propagation of the signal downstream of the receptor(s). Although MyD88 recruits IRAK-1 via DD-DD interactions, its recruitment of IRAK-4 appears to be rather unusual. Burns et al. (6) first demonstrated that an alternatively spliced variant of MyD88 (MyD88s), lacking the ID domain, failed to interact with IRAK-4, suggesting that residues located in both the DD and ID of MyD88 are crucially involved in the recruitment of IRAK-4. Nevertheless, no information is available on the specific residues in the DD in MyD88 required for its interaction with either IRAK1 or IRAK4.The DD was initially defined as the region of homology between the cytoplasmic tails of the FAS/Apo1/CD95 and TNF receptors required for their induction of cytotoxic signaling (18, 19). In analogy with other DD-containing proteins, this domain in MyD88 is also involved in the formation of homomeric and heteromeric interactions. Herein, we have undertaken an alanine-scanning mutational analysis to identify amino acids that are required for downstream signaling and might participate in the homomeric and heteromeric interactions. Our studies revealed that MyD88_E52A and MyD88_Y58A mutants are strongly impaired in the recruitment of both IRAK1 and IRAK4, whereas the MyD88_K95A mutant is deficient in recruiting IRAK4. These findings identify residues within the DD of MyD88 crucially involved in the formation of higher order complexes containing IRAK1 and IRAK4 and required for the propagation of the TLR/IL1-R signaling pathways.     

Immune complex-mediated cell activation from systemic lupus erythematosus and rheumatoid arthritis patients elaborate different requirements for IRAK1/4 kinase activity across human cell types

IL-1R-associated kinases (IRAKs) are important mediators of MyD88-dependent signaling by the TLR/IL-1R superfamily and facilitate inflammatory responses. IRAK4 and IRAK1 function as active kinases and as scaffolds for protein-protein interactions. We report that although IRAK1/4 kinase activity is essential for human plasmacytoid dendritic cell (pDC) activation, it is dispensable in B, T, dendritic, and monocytic cells, which is in contrast with an essential active kinase role in comparable mouse cell types. An IRAK1/4 kinase inhibitor abrogated TLR7/9-induced IFN-α responses in both mouse and human pDCs, but other human immune cell populations activated via TLR7/9 or IL-1R were refractory to IRAK4 kinase inhibition. Gene ablation experiments using small interfering RNA demonstrated an essential scaffolding role for IRAK1 and IRAK4 in MyD88-dependent signaling. Finally, we demonstrate that autoimmune patient (systemic lupus erythematosus and rheumatoid arthritis) serum activates both pDC and B cells, but IRAK1/4 kinase inhibition affects only the pDC response, underscoring the differential IRAK1/4 functional requirements in human immune cells. These data reveal important species differences and elaborate cell type requirements for IRAK1/4 kinase activity.     

Regulation of interleukin-1- and lipopolysaccharide-induced NF-kappaB activation by alternative splicing of MyD88

MyD88 is an adaptor protein that is involved in interleukin-1 receptor (IL-1R)- and Toll-like receptor (TLR)-induced activation of NF-kappaB. It is composed of a C-terminal Toll/IL-1R homology (TIR) domain and an N-terminal death domain (DD), which mediate the interaction of MyD88 with the IL-1R/TLR and the IL-1R-associated kinase (IRAK), respectively. The interaction of MyD88 with IRAK triggers IRAK phosphorylation, which is essential for its activation and downstream signaling ability. Both domains of MyD88 are separated by a small intermediate domain (ID) of unknown function. Here, we report the identification of a splice variant of MyD88, termed MyD88(S), which encodes for a protein lacking the ID. MyD88(S) is mainly expressed in the spleen and can be induced in monocytes upon LPS treatment. Although MyD88(S) still binds the IL-1R and IRAK, it is defective in its ability to induce IRAK phosphorylation and NF-kappaB activation. In contrast, MyD88(S) behaves as a dominant-negative inhibitor of IL-1- and LPS-, but not TNF-induced, NF-kappaB activation. These results implicate the ID of MyD88 in the phosphorylation of IRAK. Moreover, the regulated expression and antagonistic activity of MyD88(S) suggest an important role for alternative splicing of MyD88 in the regulation of the cellular response to IL-1 and LPS.     

FADD negatively regulates lipopolysaccharide signaling by impairing interleukin-1 receptor-associated kinase 1-MyD88 interaction

Lipopolysaccharide (LPS) engages Toll-like receptor 4 (TLR4) on various cells to initiate inflammatory and angiogenic pathways. FADD is an adaptor protein involved in death receptor-mediated apoptosis. Here we report a role for FADD in regulation of TLR4 signals in endothelial cells. FADD specifically attenuates LPS-induced activation of c-Jun NH(2)-terminal kinase and phosphatidylinositol 3'-kinase in a death domain-dependent manner. In contrast, FADD-null cells show hyperactivation of these kinases. Examining physical associations of endogenous proteins, we show that FADD interacts with interleukin-1 receptor-associated kinase 1 (IRAK1) and MyD88. LPS stimulation increases IRAK1-FADD interaction and recruitment of the IRAK1-FADD complex to activated MyD88. IRAK1 is required for FADD-MyD88 interaction, as FADD does not associate with MyD88 in IRAK1-null cells. By shuttling FADD to MyD88, IRAK1 provides a mechanism for controlled and limited activation of the TLR4 signaling pathway. Functionally, enforced FADD expression inhibited LPS- but not vascular endothelial growth factor-induced endothelial cell sprouting, while FADD deficiency led to enhanced production of proinflammatory cytokines induced by stimulation of TLR4 and TLR2, but not TLR3. Reconstitution of FADD reversed the enhanced production of proinflammatory cytokines. Thus, FADD is a physiological negative regulator of IRAK1/MyD88-dependent responses in innate immune signaling.     

In vitro reconstitution of the interactions in the PIDDosome

Caspase-2 is critical for genotoxic stress induced apoptosis and is activated by formation of the PIDDosome, an oligomeric caspase-2 activating complex. The PIDDosome comprises three protein components, PIDD, RAIDD, and caspase-2. RAIDD contains both a death domain (DD) and a caspase recruitment domain (CARD). It acts as the bridge to recruit PIDD using the DD: DD interaction and to recruit caspase-2 via the CARD: CARD interaction. Here we report biochemical characterization and in vitro reconstitution of the core interactions in the PIDDosome. We show that RAIDD CARD and RAIDD DD interact with their binding partners, caspase-2 CARD and PIDD DD, respectively. However, full-length RAIDD fails to interact with either caspase-2 CARD or PIDD DD under a physiological buffer condition. We reveal that this lack of interaction of full-length RAIDD is not due to its tendency to aggregate under the physiological buffer condition, as decreasing full-length RAIDD aggregation using high salt or high pH is not able to promote complex formation. Instead, full-length RAIDD forms both binary and ternary complexes under a low salt condition. Successful in vitro reconstitution of the ternary complex provides a basis for further structural studies of the PIDDosome.     

PIDD mediates and stabilizes the interaction between RAIDD and Caspase-2 for the PIDDosome assembly

The PIDDosome, which is an oligomeric signaling complex composed of PIDD, RAIDD and caspase-2, can induce proximity-based dimerization and activation of caspase-2. In the PIDDosome assembly, the adaptor protein RAIDD interacts with PIDD and caspase-2 via CARD:CARD and DD:DD, respectively. To analyze the PIDDosome assembly, we purified all of the DD superfamily members and performed biochemical analyses. The results revealed that caspase-2 CARD is an insoluble protein that can be solubilized by its binding partner, RAIDD CARD, but not by full-length RAIDD; this indicates that full-length RAIDD in closed states cannot interact with caspase-2 CARD. Moreover, we found that caspase-2 CARD can be solubilized and interact with full-length RAIDD in the presence of PIDD DD, indicating that PIDD DD initially binds to RAIDD, after which caspase-2 can be recruited to RAIDD via a CARD:CARD interaction. Our study will be useful in determining the order of assembly of the PIDDosome. [BMB Reports 2013; 46(9): 471-476]     

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.     

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.