Explore the vast range of titles available.

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-FLIP S 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. The core FADD:Caspase-8 complex and its regulatory partners, such as the cell death inhibitor c-FLIP, coordinate cell fate. Here authors present the structure of full-length procaspase-8 in a complex with FADD and reveal how recruitment of c-FLIP S into this complex inhibits Caspase-8 activity.

Interferons (IFNs) are cytokines with powerful immunomodulatory and antiviral properties, but less is known about how they induce cell death. Here, we show that both type I (α/β) and type II (γ) IFNs induce precipitous receptor-interacting protein (RIP)1/RIP3 kinase-mediated necrosis when the adaptor protein Fas-associated death domain (FADD) is lost or disabled by phosphorylation, or when caspases (e.g., caspase 8) are inactivated. IFN-induced necrosis proceeds via progressive assembly of a RIP1–RIP3 “necrosome” complex that requires Jak1/STAT1-dependent transcription, but does not need the kinase activity of RIP1. Instead, IFNs transcriptionally activate the RNA-responsive protein kinase PKR, which then interacts with RIP1 to initiate necrosome formation and trigger necrosis. Although IFNs are powerful activators of necrosis when FADD is absent, these cytokines are likely not the dominant inducers of RIP kinase-driven embryonic lethality in FADD-deficient mice. We also identify phosphorylation on serine 191 as a mechanism that disables FADD and collaborates with caspase inactivation to allow IFN-activated necrosis. Collectively, these findings outline a mechanism of IFN-induced RIP kinase-dependent necrotic cell death and identify FADD and caspases as negative regulators of this process.

Colonizing enteric bacteria are shown to inhibit the antimicrobial process of host cell apoptosis through the action of NleB1, a type III secretion system effector with N- acetylglucosamine transferase activity, which can bind and modify eukaryotic death-domain-containing proteins. Virulence mechanism of bacterial effector NleB Previous studies identified a group of effectors from enteropathogenic Escherichia coli that can inhibit host nuclear factor-κB (NF-κB) signalling, yet only one of them, known as NleB, is required for bacterial virulence in vivo . Two papers published in this issue demonstrate the unique mechanism of action of NleB. It directly targets death receptor signalling complexes, binding to the death domains (DDs) of multiple DD-containing proteins including the TNF receptor, FAS, RIPK1, TRADD and FADD. NleB is shown to function as an N -acetylglucosamine (GlcNAc) transferase that modifies a conserved DD arginine, blocking the receptor-adapter interaction. These findings suggest that GlcNAc modification is essential for bacterial virulence and can regulate death receptor signalling. 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 1 , 2 , 3 . 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.

Several death-domain-containing proteins are directly inactivated by the enteropathogenic Escherichia coli type III secretion system effector NleB; NleB functions as an N -acetylglucosamine transferase that modifies a conserved death domain arginine residue, blocking the receptor–adapter interaction. Virulence mechanism of bacterial effector NleB Previous studies identified a group of effectors from enteropathogenic Escherichia coli that can inhibit host nuclear factor-κB (NF-κB) signalling, yet only one of them, known as NleB, is required for bacterial virulence in vivo . Two papers published in this issue demonstrate the unique mechanism of action of NleB. It directly targets death receptor signalling complexes, binding to the death domains (DDs) of multiple DD-containing proteins including the TNF receptor, FAS, RIPK1, TRADD and FADD. NleB is shown to function as an N -acetylglucosamine (GlcNAc) transferase that modifies a conserved DD arginine, blocking the receptor-adapter interaction. These findings suggest that GlcNAc modification is essential for bacterial virulence and can regulate death receptor signalling. The tumour necrosis factor (TNF) family is crucial for immune homeostasis, cell death and inflammation. These cytokines are recognized by members of the TNF receptor (TNFR) family of death receptors, including TNFR1 and TNFR2, and FAS and TNF-related apoptosis-inducing ligand (TRAIL) receptors 1 . Death receptor signalling requires death-domain-mediated homotypic/heterotypic interactions between the receptor and its downstream adaptors, including TNFR1-associated death domain protein (TRADD) and FAS-associated death domain protein (FADD) 2 . Here we discover that death domains in several proteins, including TRADD, FADD, RIPK1 and TNFR1, were directly inactivated by NleB, an enteropathogenic Escherichia coli (EPEC) type III secretion system effector known to inhibit host nuclear factor-κB (NF-κB) signalling 3 , 4 . NleB contained an unprecedented N -acetylglucosamine (GlcNAc) transferase activity that specifically modified a conserved arginine in these death domains (Arg 235 in the TRADD death domain). NleB GlcNAcylation (the addition of GlcNAc onto a protein side chain) of death domains blocked homotypic/heterotypic death domain interactions and assembly of the oligomeric TNFR1 complex, thereby disrupting TNF signalling in EPEC-infected cells, including NF-κB signalling, apoptosis and necroptosis. Type-III-delivered NleB also blocked FAS ligand and TRAIL-induced cell death by preventing formation of a FADD-mediated death-inducing signalling complex (DISC). The arginine GlcNAc transferase activity of NleB was required for bacterial colonization in the mouse model of EPEC infection. The mechanism of action of NleB represents a new model by which bacteria counteract host defences, and also a previously unappreciated post-translational modification.

The complexities of cell death The crystal structure of a complex between the cell surface receptor protein Fas and the Fas-associated death domain (FADD) protein - a central feature of the so-called death-inducing signalling complex of apoptosis-inducing cellular receptors - has been determined at 2.7 Å resolution. The structure reveals a previously unknown type of death domain interaction that allows four FADD and four Fas proteins to aggregate in the one complex. Surprisingly, a conformational change opens up the Fas death domain, which creates binding surfaces for FADD as well as Fas-Fas 'bridging' interactions. Only when a sufficient number of Fas molecules are in close proximity - as is the case when Fas ligand binds - can the open form of Fas be stabilized. This study presents the crystal structure of a Fas-FADD complex, a central feature of the so-called death inducing signalling complex. The structure reveals a new mode of death domain interactions that allows four FADD and four Fas proteins in one complex. The death inducing signalling complex (DISC) formed by Fas receptor, FADD (Fas-associated death domain protein) and caspase 8 is a pivotal trigger of apoptosis 1 , 2 , 3 . The Fas–FADD DISC represents a receptor platform, which once assembled initiates the induction of programmed cell death. A highly oligomeric network of homotypic protein interactions comprised of the death domains of Fas and FADD is at the centre of DISC formation 4 , 5 . Thus, characterizing the mechanistic basis for the Fas–FADD interaction is crucial for understanding DISC signalling but has remained unclear largely because of a lack of structural data. We have successfully formed and isolated the human Fas–FADD death domain complex and report the 2.7 Å crystal structure. The complex shows a tetrameric arrangement of four FADD death domains bound to four Fas death domains. We show that an opening of the Fas death domain exposes the FADD binding site and simultaneously generates a Fas–Fas bridge. The result is a regulatory Fas–FADD complex bridge governed by weak protein–protein interactions revealing a model where the complex itself functions as a mechanistic switch. This switch prevents accidental DISC assembly, yet allows for highly processive DISC formation and clustering upon a sufficient stimulus. In addition to depicting a previously unknown mode of death domain interactions, these results further uncover a mechanism for receptor signalling solely by oligomerization and clustering events.

The crystal structure of the complex formed by the death domains from Fas and FADD at physiological pH is now solved, revealing a 5:5 complex that is supported by electron microscopy data. Along with mutagenesis and mass spectrometry analyses, the work provide insight into Fas mutations that cause autoimmune lymphoproliferative syndrome. The death-inducing signaling complex (DISC) formed by the death receptor Fas, the adaptor protein FADD and caspase-8 mediates the extrinsic apoptotic program. Mutations in Fas that disrupt the DISC cause autoimmune lymphoproliferative syndrome (ALPS). Here we show that the Fas–FADD death domain (DD) complex forms an asymmetric oligomeric structure composed of 5–7 Fas DD and 5 FADD DD, whose interfaces harbor ALPS-associated mutations. Structure-based mutations disrupt the Fas–FADD interaction in vitro and in living cells; the severity of a mutation correlates with the number of occurrences of a particular interaction in the structure. The highly oligomeric structure explains the requirement for hexameric or membrane-bound FasL in Fas signaling. It also predicts strong dominant negative effects from Fas mutations, which are confirmed by signaling assays. The structure optimally positions the FADD death effector domain (DED) to interact with the caspase-8 DED for caspase recruitment and higher-order aggregation.

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-1β. 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.

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.

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.

NF- κ B-inducing kinase (NIK) is well-known for its role in promoting p100/NF- κ B2 processing into p52, a process defined as the alternative, or non-canonical, NF- κ B 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 LT β R, is required for TNF α -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/LT β R-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- κ B pathway.