PEA-15 engages in allosteric interactions using a common scaffold in a phosphorylation-dependent manner.

Phosphoprotein enriched in astrocytes, 15 kDa (PEA-15) is a death-effector domain (DED) containing protein involved in regulating mitogen-activated protein kinase and apoptosis pathways. In this molecular dynamics study, we examined how phosphorylation of the PEA-15 C-terminal tail residues, Ser-104 and Ser-116, allosterically mediates conformational changes of the DED and alters the binding specificity from extracellular-regulated kinase (ERK) to Fas-associated death domain (FADD) protein. We delineated that the binding interfaces between the unphosphorylated PEA-15 and ERK2 and between the doubly phosphorylated PEA-15 and FADD are similarly composed of a scaffold that includes both the DED and the C-terminal tail residues of PEA-15. While the unphosphorylated serine residues do not directly interact with ERK2, the phosphorylated Ser-116 engages in strong electrostatic interactions with arginine residues on FADD DED. Upon PEA-15 binding, FADD repositions its death domain (DD) relative to the DED, an essential conformational change to allow the death-inducing signaling complex (DISC) assembly.

Reconstruction of the Fas-Based Death-Inducing Signaling Complex (DISC) Using a Protein-Protein Docking Meta-Approach.

The death-inducing signaling complex (DISC) is a fundamental multiprotein complex, which triggers the extrinsic apoptosis pathway through stimulation by death ligands. DISC consists of different death domain (DD) and death effector domain (DED) containing proteins such as the death receptor Fas (CD95) in complex with FADD, procaspase-8, and cFLIP. Despite many experimental and theoretical studies in this area, there is no global agreement neither on the DISC architecture nor on the mechanism of action of the involved species. In the current work, we have tried to reconstruct the DISC structure by identifying key protein interactions using a new protein-protein docking meta-approach. We combined the benefits of five of the most employed protein-protein docking engines, HADDOCK, ClusPro, HDOCK, GRAMM-X, and ZDOCK, in order to improve the accuracy of the predicted docking complexes. Free energy of binding and hot spot interacting residues were calculated and determined for each protein-protein interaction using molecular mechanics generalized Born surface area and alanine scanning techniques, respectively. In addition, a series of in-cellulo protein-fragment complementation assays were conducted to validate the protein-protein docking procedure. The results show that the DISC formation initiates by dimerization of adjacent FasDD trimers followed by recruitment of FADD through homotypic DD interactions with the oligomerized death receptor. Furthermore, the in-silico outcomes indicate that cFLIP cannot bind directly to FADD; instead, cFLIP recruitment to the DISC is a hierarchical and cooperative process where FADD initially recruits procaspase-8, which in turn recruits and heterodimerizes with cFLIP. Finally, a possible structure of the entire DISC is proposed based on the docking results.

Cryo-EM structural analysis of FADD:Caspase-8 complexes defines the catalytic dimer architecture for co-ordinated control of cell fate.

Regulated cell death is essential in development and cellular homeostasis. Multi-protein platforms, including the Death-Inducing Signaling Complex (DISC), co-ordinate cell fate via a core FADD:Caspase-8 complex and its regulatory partners, such as the cell death inhibitor c-FLIP. Here, using electron microscopy, we visualize full-length procaspase-8 in complex with FADD. Our structural analysis now reveals how the FADD-nucleated tandem death effector domain (tDED) helical filament is required to orientate the procaspase-8 catalytic domains, enabling their activation via anti-parallel dimerization. Strikingly, recruitment of c-FLIPS into this complex inhibits Caspase-8 activity by altering tDED triple helix architecture, resulting in steric hindrance of the canonical tDED Type I binding site. This prevents both Caspase-8 catalytic domain assembly and tDED helical filament elongation. Our findings reveal how the plasticity, composition and architecture of the core FADD:Caspase-8 complex critically defines life/death decisions not only via the DISC, but across multiple key signaling platforms including TNF complex II, the ripoptosome, and RIPK1/RIPK3 necrosome.

Fas/FasL of pacific cod mediated apoptosis.

Fas and Fas ligand (FasL) pathway plays important roles in virus defense and cell apoptosis. In our previous work, nervous necrosis virus (NNV) was discovered in Pacific cod (Gadus macrocephalus), and the Fas ligand (PcFasL) was up-regulated when NNV outbreak, however, signal transmission of Fas/FasL in fish are still unclear. In the present study, Pacific cod Fas (PcFas), PcFasL and Fas-associating protein with a novel death domain (PcFADD) were characterized. The predicted protein of PcFas, PcFasL and PcFADD includes 333 aa, 90 aa and 93 aa, separately. 3-D models of PcFas, PcFasL and PcFADD were well constructed based on reported templates, respectively, even though the sequence homology with other fish is very low. The transcript levels of PcFas increased gradually from 15 day-post hatching (dph) to 75dph. PcFas was significantly up-regulated when cod larvae had NNV symptoms at 24dph, 37dph, 46dph, 69dph, and 77dph. Subcellular localization revealed that PcFasL was located in the cytoplasm, while PcFas was mainly located in the cell membrane. Exogenous expressed PcFasL of 900 µg/mL could kill the Epithelioma papulosum cyprinid (EPC) cells by MTT test, but low concentration has no effect on the cells. qPCR analysis showed that overexpression of PcFas could significantly up-regulate the expression of genes related to Fas/FasL signaling pathway, including bcl-2, bax, and RIP3, while overexpression of PcFasL significantly up-regulate the expression of caspase-3, caspase-9, and MLKL. Overexpression of PcFas or PcFasL could induce EPC apoptosis significantly by flow cytometry, which was consistent with the results of caspase-3 mRNA level increasing. The results indicated that NNV could induce apoptosis through Fas/FasL signal pathway.

Cell-Penetrable Peptide-Conjugated FADD Induces Apoptosis and Regulates Inflammatory Signaling in Cancer Cells.

Dysregulated expression of Fas-associated death domain (FADD) is associated with the impediment of various cellular pathways, including apoptosis and inflammation. The adequate cytosolic expression of FADD is critical to the regulation of cancer cell proliferation. Importantly, cancer cells devise mechanisms to suppress FADD expression and, in turn, escape from apoptosis signaling. Formulating strategies, for direct delivery of FADD proteins into cancer cells in a controlled manner, may represent a promising therapeutic approach in cancer therapy. We chemically conjugated purified FADD protein with cell permeable TAT (transactivator of transcription) peptide, to deliver in cancer cells. TAT-conjugated FADD protein internalized through the caveolar pathway of endocytosis and retained in the cytosol to augment cell death. Inside cancer cells, TAT-FADD rapidly constituted DISC (death inducing signaling complex) assembly, which in turn, instigate apoptosis signaling. The apoptotic competency of TAT-FADD showed comparable outcomes with the conventional apoptosis inducers. Notably, TAT-FADD mitigates constitutive NF-κB activation and associated downstream anti-apoptotic genes Bcl2, cFLIPL, RIP1, and cIAP2, independent of pro-cancerous TNF-α priming. In cancer cells, TAT-FADD suppresses the canonical NLRP3 inflammasome priming and restricts the processing and secretion of proinflammatory IL-1ß. Our results demonstrate that TAT-mediated intracellular delivery of FADD protein can potentially recite apoptosis signaling with simultaneous regulation of anti-apoptotic and proinflammatory NF-κB signaling activation in cancer cells.

The volatile anesthetic sevoflurane reduces neutrophil apoptosis via Fas death domain-Fas-associated death domain interaction.

Sepsis remains a significant health care burden, with high morbidities and mortalities. Patients with sepsis often require general anesthesia for procedures and imaging studies. Knowing that anesthetic drugs can pose immunomodulatory effects, it would be critical to understand the impact of anesthetics on sepsis pathophysiology. The volatile anesthetic sevoflurane is a common general anesthetic derived from ether as a prototype. Using a murine sepsis model induced by cecal ligation and puncture surgery, we examined the impact of sevoflurane on sepsis outcome. Different from volatile anesthetic isoflurane, sevoflurane exposure significantly improved the outcome of septic mice. This was associated with less apoptosis in the spleen. Because splenic apoptosis was largely attributed to the apoptosis of neutrophils, we examined the effect of sevoflurane on FasL-induced neutrophil apoptosis. Sevoflurane exposure significantly attenuated apoptosis. Sevoflurane did not affect the binding of FasL to the extracellular domain of Fas receptor. Instead, in silico analysis suggested that sevoflurane would bind to the interphase between Fas death domain (DD) and Fas-associated DD (FADD). The effect of sevoflurane on Fas DD-FADD interaction was examined using fluorescence resonance energy transfer (FRET). Sevoflurane attenuated FRET efficiency, indicating that sevoflurane hindered the interaction between Fas DD and FADD. The predicted sevoflurane binding site is known to play a significant role in Fas DD-FADD interaction, supporting our in vitro and in vivo apoptosis results.-Koutsogiannaki, S., Hou, L., Babazada, H., Okuno, T., Blazon-Brown, N., Soriano, S. G., Yokomizo, T., Yuki, K. The volatile anesthetic sevoflurane reduces neutrophil apoptosis via Fas death domain-Fas-associated death domain interaction.

The first cloned sea cucumber FADD from Holothuria leucospilota: Molecular characterization, inducible expression and involvement of apoptosis.

In this study, a sea cucumber Fas-associated death domain (FADD) named HLFADD was first cloned from Holothuria leucospilota. The full-length cDNA of HLFADD is 2137 bp in size, containing a 116-bp 5'-untranslated region (UTR), a 1334-bp 3'-UTR and a 687-bp open reading frame (ORF) encoding a protein of 228 amino acids with a deduced molecular weight of 26.42 kDa. HLFADD protein contains a conserved death effector domain at its N-terminal and a conserved death domain at its C-terminal, structurally similar to its counterparts in vertebrates. The over-expressed HLFADD protein could induce apoptosis in HEK293 cells, suggesting a possible death receptor-mediated apoptosis pathway in echinoderms adapted with FADD. Moreover, HLFADD mRNA is ubiquitously expressed in all examined tissues, with the highest transcript level in the coelomocytes, followed by intestine. In vitro experiments performed in the H. leucospilota coelomocytes, the expression of HLFADD mRNA was significantly up-regulated by lipopolysaccharides (LPS) or polyriboinosinic-polyribocytidylic acid [poly (I:C)] challenge, suggesting that HLFADD might play important roles in the innate immune defense of sea cucumber against the invasion of bacteria and viruses.

S194-P-FADD as a marker of aggressiveness and poor prognosis in human T-cell lymphoblastic lymphoma.

T-cell lymphoblastic lymphoma is a haematological disease with an urgent need for reliable prognostic biomarkers that allow therapeutic stratification and dose adjustment. The scarcity of human samples is responsible for the delayed progress in the study and the clinical management of this disease, especially compared with T-cell acute lymphoblastic leukaemia, its leukemic counterpart. In the present work, we have determined by immunohistochemistry that S194-P-FADD protein is significantly reduced in a cohort of 22 samples from human T-cell lymphoblastic lymphoma. Notably, the extent of such reduction varies significantly among samples and has revealed determinant for the outcome of the tumour. We demonstrate that Fas-associated protein with death domain (FADD) phosphorylation status affects protein stability, subcellular localization and non-apoptotic functions, specifically cell proliferation. Phosphorylated FADD would be more stable and preferentially localized to the cell nucleus; there, it would favour cell proliferation. We show that patients with higher levels of S194-P-FADD exhibit more proliferative tumours and that they present worse clinical characteristics and a significant enrichment to an oncogenic signature. This supports that FADD phosphorylation may serve as a predictor for T-cell lymphoblastic lymphoma aggressiveness and clinical status. In summary, we propose FADD phosphorylation as a new biomarker with prognostic value in T-cell lymphoblastic lymphoma.

Molecular cloning and characterization of FADD from the orange-spotted grouper (Epinephelus coioides).

Fas-associated protein with death domain (FADD) is the key adaptor protein that transmits apoptotic signals mediated by the main death receptors. Besides being an essential instrument in cell death, FADD is also implicated in proliferation, cell cycle progression, tumor development, inflammation, innate immunity, and autophagy. In the present study, a FADD homologue (EcFADD) from the orange-spotted grouper (Epinephelus coioides) was cloned and its possible role in fish immunity was analyzed. The full length cDNA of EcFADD contains 808 base pairs (bp), including a 573 bp open reading frame that encodes a 190 amino acid protein with a predicted molecular mass of 21.81 kDa. Quantitative real-time polymerase chain reaction analysis indicated that EcFADD was distributed in all examined tissues. The expression of EcFADD in the spleen of E. coioides was differentially up-regulated when challenged with Singapore grouper iridovirus (SGIV) or polyinosine-polycytidylic acid(poly[I:C]). EcFADD was abundantly distributed in both the cytoplasm and nucleus in grouper spleen (GS) and fathead minnow (FHM) epithelial cells. Over-expression of EcFADD inhibited SGIV infection and replication and SGIV-induced apoptosis. To achieve antiviral and anti-apoptosis activities, FADD promoted the activation of interferon-stimulated response element (ISRE) and type I interferon (IFN) genes in the antiviral IFN signaling pathway and inhibited activation of apoptosis-related transcription factors p53. Our results not only characterize FADD but also reveal new immune functions and the molecular mechanisms by which FADD responds to virus infection and virus-induced apoptosis.

Cryo-EM Structure of Caspase-8 Tandem DED Filament Reveals Assembly and Regulation Mechanisms of the Death-Inducing Signaling Complex.

Caspase-8 activation can be triggered by death receptor-mediated formation of the death-inducing signaling complex (DISC) and by the inflammasome adaptor ASC. Caspase-8 assembles with FADD at the DISC and with ASC at the inflammasome through its tandem death effector domain (tDED), which is regulated by the tDED-containing cellular inhibitor cFLIP and the viral inhibitor MC159. Here we present the caspase-8 tDED filament structure determined by cryoelectron microscopy. Extensive assembly interfaces not predicted by the previously proposed linear DED chain model were uncovered, and were further confirmed by structure-based mutagenesis in filament formation in vitro and Fas-induced apoptosis and ASC-mediated caspase-8 recruitment in cells. Structurally, the two DEDs in caspase-8 use quasi-equivalent contacts to enable assembly. Using the tDED filament structure as a template, structural analyses reveal the interaction surfaces between FADD and caspase-8 and the distinct mechanisms of regulation by cFLIP and MC159 through comingling and capping, respectively.

Structural Insight for Roles of DR5 Death Domain Mutations on Oligomerization of DR5 Death Domain-FADD Complex in the Death-Inducing Signaling Complex Formation: A Computational Study.

Death receptor 5 (DR5)-induced apoptosis that prioritizes the death of tumor cells has been proposed as one of the promising cancer therapies. In this process, oligomerized DR5 death domain (DD) binding to Fas-associated death domain (FADD) leads to FADD activating caspase-8, which marks the formation of the death-inducing signaling complex (DISC) that initiates apoptosis. DR5 DD mutations found in cancer cells have been suggested to play an important pathological role, the mechanism through which those mutants prevent the DR5-activated DISC formation is not clear yet. This study sought to provide structural and molecular insight for the roles of four selected DR5 DD mutations (E355K, E367K, K415N, and L363F) in the oligomerization of DR5 DD-FADD complex during the DISC formation. Results from the molecular dynamics simulations show that the simulated mutants induce conformational, dynamical motions and interactions changes in the DR5 DD-FADD tetramer complex, including changes in a protein's backbone flexibility, less exposure of FADD DED's caspase-8 binding site, reduced H-bonding and hydrophobic contacts at the DR5 DD-FADD DD binding, altered distribution of the electrostatic potentials and correlated motions of residues, and reduced binding affinity of DR5 DD binding to FADD. This study provides structural and molecular insight for the influence of DR5 DD mutations on oligomerization of DR5 DD-FADD complex, which is expected to foster understanding of the DR5 DD mutants' resistance mechanism against DR5-activated DISC formation.

Structure Calculation and Reconstruction of Discrete-State Dynamics from Residual Dipolar Couplings.

Residual dipolar couplings (RDCs) acquired by nuclear magnetic resonance (NMR) spectroscopy are an indispensable source of information in investigation of molecular structures and dynamics. Here, we present a comprehensive strategy for structure calculation and reconstruction of discrete-state dynamics from RDC data that is based on the singular value decomposition (SVD) method of order tensor estimation. In addition to structure determination, we provide a mechanism of producing an ensemble of conformations for the dynamical regions of a protein from RDC data. The developed methodology has been tested on simulated RDC data with ±1 Hz of error from an 83 residue α protein (PDB ID 1A1Z ) and a 213 residue α/ß protein DGCR8 (PDB ID 2YT4 ). In nearly all instances, our method reproduced the structure of the protein including the conformational ensemble to within less than 2 Å. On the basis of our investigations, arc motions with more than 30° of rotation are identified as internal dynamics and are reconstructed with sufficient accuracy. Furthermore, states with relative occupancies above 20% are consistently recognized and reconstructed successfully. Arc motions with a magnitude of 15° or relative occupancy of less than 10% are consistently unrecognizable as dynamical regions within the context of ±1 Hz of error.

Identification of the Calmodulin-Binding Domains of Fas Death Receptor.

The extrinsic apoptotic pathway is initiated by binding of a Fas ligand to the ectodomain of the surface death receptor Fas protein. Subsequently, the intracellular death domain of Fas (FasDD) and that of the Fas-associated protein (FADD) interact to form the core of the death-inducing signaling complex (DISC), a crucial step for activation of caspases that induce cell death. Previous studies have shown that calmodulin (CaM) is recruited into the DISC in cholangiocarcinoma cells and specifically interacts with FasDD to regulate the apoptotic/survival signaling pathway. Inhibition of CaM activity in DISC stimulates apoptosis significantly. We have recently shown that CaM forms a ternary complex with FasDD (2:1 CaM:FasDD). However, the molecular mechanism by which CaM binds to two distinct FasDD motifs is not fully understood. Here, we employed mass spectrometry, nuclear magnetic resonance (NMR), biophysical, and biochemical methods to identify the binding regions of FasDD and provide a molecular basis for the role of CaM in Fas-mediated apoptosis. Proteolytic digestion and mass spectrometry data revealed that peptides spanning residues 209-239 (Fas-Pep1) and 251-288 (Fas-Pep2) constitute the two CaM-binding regions of FasDD. To determine the molecular mechanism of interaction, we have characterized the binding of recombinant/synthetic Fas-Pep1 and Fas-Pep2 peptides with CaM. Our data show that both peptides engage the N- and C-terminal lobes of CaM simultaneously. Binding of Fas-Pep1 to CaM is entropically driven while that of Fas-Pep2 to CaM is enthalpically driven, indicating that a combination of electrostatic and hydrophobic forces contribute to the stabilization of the FasDD-CaM complex. Our data suggest that because Fas-Pep1 and Fas-Pep2 are involved in extensive intermolecular contacts with the death domain of FADD, binding of CaM to these regions may hinder its ability to bind to FADD, thus greatly inhibiting the initiation of apoptotic signaling pathway.

Structural Characterizations of the Fas Receptor and the Fas-Associated Protein with Death Domain Interactions.

The Fas receptor is a representative death receptor, and the Fas-associated protein with death domain (FADD) is a crucial adapter protein needed to support the Fas receptor's activity. The Fas-FADD interactions constitute an important signaling pathway that ultimately induces apoptosis or programmed cell death in biological systems. The interactions responsible for this cell-death process are governed by the binding process of the Fas ligand to the Fas, followed by the caspase cascade activation. Using a computational approach, the present communication explores certain essential structural aspects of the Fas-FADD death domains and their interfacial interactions.

DED or alive: assembly and regulation of the death effector domain complexes.

Death effector domains (DEDs) are protein-protein interaction domains initially identified in proteins such as FADD, FLIP and caspase-8 involved in regulating apoptosis. Subsequently, these proteins have been shown to have important roles in regulating other forms of cell death, including necroptosis, and in regulating other important cellular processes, including autophagy and inflammation. Moreover, these proteins also have prominent roles in innate and adaptive immunity and during embryonic development. In this article, we review the various roles of DED-containing proteins and discuss recent developments in our understanding of DED complex formation and regulation. We also briefly discuss opportunities to therapeutically target DED complex formation in diseases such as cancer.

NIK promotes tissue destruction independently of the alternative NF-κB pathway through TNFR1/RIP1-induced apoptosis.

NF-κB-inducing kinase (NIK) is well-known for its role in promoting p100/NF-κB2 processing into p52, a process defined as the alternative, or non-canonical, NF-κB pathway. Here we reveal an unexpected new role of NIK in TNFR1-mediated RIP1-dependent apoptosis, a consequence of TNFR1 activation observed in c-IAP1/2-depleted conditions. We show that NIK stabilization, obtained by activation of the non-death TNFRs Fn14 or LTßR, is required for TNFα-mediated apoptosis. These apoptotic stimuli trigger the depletion of c-IAP1/2, the phosphorylation of RIP1 and the RIP1 kinase-dependent assembly of the RIP1/FADD/caspase-8 complex. In the absence of NIK, the phosphorylation of RIP1 and the formation of RIP1/FADD/caspase-8 complex are compromised while c-IAP1/2 depletion is unaffected. In vitro kinase assays revealed that recombinant RIP1 is a bona fide substrate of NIK. In vivo, we demonstrated the requirement of NIK pro-death function, but not the processing of its substrate p100 into p52, in a mouse model of TNFR1/LTßR-induced thymus involution. In addition, we also highlight a role for NIK in hepatocyte apoptosis in a mouse model of virus-induced TNFR1/RIP1-dependent liver damage. We conclude that NIK not only contributes to lymphoid organogenesis, inflammation and cell survival but also to TNFR1/RIP1-dependent cell death independently of the alternative NF-κB pathway.

The response of Greek key proteins to changes in connectivity depends on the nature of their secondary structure.

What governs the balance between connectivity and topology in regulating the mechanism of protein folding? We use circular permutation to vary the order of the helices in the all-α Greek key protein FADD (Fas-associated death domain) to investigate this question. Unlike all-ß Greek key proteins, where changes in the order of secondary structure cause a shift in the folding nucleus, the position of the nucleus in FADD is unchanged, even when permutation reduces the complexity significantly. We suggest that this is because local helical contacts are so dominant that permutation has little effect on the entropic cost of forming the folding nucleus whereas, in all-ß Greek key proteins, all interactions in the nucleus are long range. Thus, the type of secondary structure modulates the sensitivity of proteins to changes in connectivity.

Structural study of the RIPoptosome core reveals a helical assembly for kinase recruitment.

Receptor interaction protein kinase 1 (RIP1) is a molecular cell-fate switch. RIP1, together with Fas-associated protein with death domain (FADD) and caspase-8, forms the RIPoptosome that activates apoptosis. RIP1 also associates with RIP3 to form the necrosome that triggers necroptosis. The RIPoptosome assembles through interactions between the death domains (DDs) of RIP1 and FADD and between death effector domains (DEDs) of FADD and caspase-8. In this study, we analyzed the overall structure of the RIP1 DD/FADD DD complex, the core of the RIPoptosome, by negative-stain electron microscopy and modeling. The results show that RIP1 DD and FADD DD form a stable complex in vitro similar to the previously described Fas DD/FADD DD complex, suggesting that the RIPoptosome and the Fas death-inducing signaling complex share a common assembly mechanism. Both complexes adopt a helical conformation that requires type I, II, and III interactions between the death domains.

Characterization of the ripoptosome and its components: implications for anti-inflammatory and cancer therapy.

Most intracellular signaling cascades rely on the formation of multiprotein signaling complexes assembled in large protein signaling platforms. Especially in cell death signaling, there is a large variety of these complexes, including the apoptosome, the necrosome, or the death-inducing signaling complex (DISC), to name only a few. During the last years, a number of cellular conditions were identified that lead to the formation of another signaling platform, the so-called ripoptosome. Diverse stimuli such as genotoxic stress, death receptor or Toll-like-receptor (TLR) ligation, or degradation of cellular inhibitor of apoptosis proteins (cIAPs) are able to induce ripoptosome formation. The ripoptosome is tightly regulated by cIAPs that control intracellular RIP1 assembly and the association with other cell death-regulating proteins, most likely by ubiquitin linkage. The suppression of cIAP activity results in accumulation of RIP1 platforms that ultimately triggers necroptosis by activation of RIP3-MLKL-dependent necrosis signaling pathways. The ripoptosome is a 2-MDa protein complex, which consists of the core components caspase-8, FADD, different cFLIP isoforms, and RIP1. It represents one of the rheostats in cell death signaling, as it can activate apoptotic and necroptotic cell death responses. The specific formation and activation of the ripoptosome in cancer but not in primary cells suggests that this complex is a potential novel target for cancer or anti-inflammatory therapy, as suggested by the potential proinflammatory effects of necroptosis. Therefore, the better understanding and characterization of this signaling platform is of enormous importance for the development of novel cancer therapeutics. In this chapter, we describe several methods for purification and investigation of the ripoptosome in human cells. We also describe methods for monitoring apoptotic as well as necroptotic cell death.

A type III effector antagonizes death receptor signalling during bacterial gut infection.

Successful infection by enteric bacterial pathogens depends on the ability of the bacteria to colonize the gut, replicate in host tissues and disseminate to other hosts. Pathogens such as Salmonella, Shigella and enteropathogenic and enterohaemorrhagic (EPEC and EHEC, respectively) Escherichia coli use a type III secretion system (T3SS) to deliver virulence effector proteins into host cells during infection that promote colonization and interfere with antimicrobial host responses. Here we report that the T3SS effector NleB1 from EPEC binds to host cell death-domain-containing proteins and thereby inhibits death receptor signalling. Protein interaction studies identified FADD, TRADD and RIPK1 as binding partners of NleB1. NleB1 expressed ectopically or injected by the bacterial T3SS prevented Fas ligand or TNF-induced formation of the canonical death-inducing signalling complex (DISC) and proteolytic activation of caspase-8, an essential step in death-receptor-induced apoptosis. This inhibition depended on the N-acetylglucosamine transferase activity of NleB1, which specifically modified Arg 117 in the death domain of FADD. The importance of the death receptor apoptotic pathway to host defence was demonstrated using mice deficient in the FAS signalling pathway, which showed delayed clearance of the EPEC-like mouse pathogen Citrobacter rodentium and reversion to virulence of an nleB mutant. The activity of NleB suggests that EPEC and other attaching and effacing pathogens antagonize death-receptor-induced apoptosis of infected cells, thereby blocking a major antimicrobial host response.