ABSTRACT
TNF Receptor Associated Factor 2 (TRAF2) is a trimeric protein that belongs to the TNF receptor associated factor family (TRAFs). The TRAF2 oligomeric state is crucial for receptor binding and for its interaction with other proteins involved in the TNFR signaling. The monomer-trimer equilibrium of a C- terminal domain truncated form of TRAF2 (TRAF2-C), plays also a relevant role in binding the membrane, causing inward vesiculation. In this study, we have investigated the conformational dynamics of TRAF2-C through circular dichroism, fluorescence, and dynamic light scattering, performing temperature-dependent measurements. The data indicate that the protein retains its oligomeric state and most of its secondary structure, while displaying a significative increase in the heterogeneity of the tyrosines signal, increasing the temperature from ≈15 to ≈35 °C. The peculiar crowding of tyrosine residues (12 out of 18) at the three subunit interfaces and the strong dependence on the trimer concentration indicate that such conformational changes mainly involve the contact areas between each pair of monomers, affecting the oligomeric state. Molecular dynamic simulations in this temperature range suggest that the interfaces heterogeneity is an intrinsic property of the trimer that arises from the continuous, asymmetric approaching and distancing of its subunits. Such dynamics affect the results of molecular docking on the external protein surface using receptor peptides, indicating that the TRAF2-receptor interaction in the solution might not involve three subunits at the same time, as suggested by the static analysis obtainable from the crystal structure. These findings shed new light on the role that the TRAF2 oligomeric state might have in regulating the protein binding activity in vivo.
Subject(s)
Protein Subunits/chemistry , TNF Receptor-Associated Factor 2/chemistry , Tyrosine/chemistry , Binding Sites , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Inhibitor of Apoptosis Proteins/chemistry , Inhibitor of Apoptosis Proteins/genetics , Inhibitor of Apoptosis Proteins/metabolism , Molecular Docking Simulation , Molecular Dynamics Simulation , Nuclear Pore Complex Proteins/chemistry , Nuclear Pore Complex Proteins/genetics , Nuclear Pore Complex Proteins/metabolism , Proprotein Convertases/chemistry , Proprotein Convertases/genetics , Proprotein Convertases/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Multimerization , Protein Structure, Tertiary , Protein Subunits/genetics , Protein Subunits/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Serine Endopeptidases/chemistry , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , TNF Receptor-Associated Death Domain Protein/chemistry , TNF Receptor-Associated Death Domain Protein/genetics , TNF Receptor-Associated Death Domain Protein/metabolism , TNF Receptor-Associated Factor 2/genetics , TNF Receptor-Associated Factor 2/metabolism , Thermodynamics , Tyrosine/metabolism , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolismABSTRACT
The p75 neurotrophin receptor (p75NTR) is a critical mediator of neuronal death and tissue remodeling and has been implicated in various neurodegenerative diseases and cancers. The death domain (DD) of p75NTR is an intracellular signaling hub and has been shown to interact with diverse adaptor proteins. In breast cancer cells, binding of the adaptor protein TRADD to p75NTR depends on nerve growth factor and promotes cell survival. However, the structural mechanism and functional significance of TRADD recruitment in neuronal p75NTR signaling remain poorly understood. Here we report an NMR structure of the p75NTR-DD and TRADD-DD complex and reveal the mechanism of specific recognition of the TRADD-DD by the p75NTR-DD mainly through electrostatic interactions. Furthermore, we identified spatiotemporal overlap of p75NTR and TRADD expression in developing cerebellar granule neurons (CGNs) at early postnatal stages and discover the physiological relevance of the interaction between TRADD and p75NTR in the regulation of canonical NF-κB signaling and cell survival in CGNs. Our results provide a new structural framework for understanding how the recruitment of TRADD to p75NTR through DD interactions creates a membrane-proximal platform, which can be efficiently regulated by various neurotrophic factors through extracellular domains of p75NTR, to propagate downstream signaling in developing neurons.
Subject(s)
NF-kappa B/metabolism , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/metabolism , Neurons/metabolism , Receptors, Nerve Growth Factor/chemistry , Receptors, Nerve Growth Factor/metabolism , TNF Receptor-Associated Death Domain Protein/metabolism , Animals , Death Domain , Female , Humans , Male , Mice, Inbred C57BL , Mice, Knockout , Protein Conformation , Protein Interaction Domains and Motifs , Receptor, Nerve Growth Factor/metabolism , Signal Transduction , TNF Receptor-Associated Death Domain Protein/chemistryABSTRACT
Tumor necrosis factor receptor 1 (TNFR1) associated death domain protein (TRADD) is a pivotal adaptor in TNF signaling pathway and up-regulates MAVS/IFN signaling pathway in human and mammal. However, the role of TRADD in teleost fish remains obscure. To reveal the function of teleost TRADD in the innate immune response, the TRADD homologue (bcTRADD) of black carp (Mylopharyngodon piceus) has been cloned and the function of bcTRADD is investigated in this study, which shares similar functional domain to its mammalian counterpart. bcTRADD mRNA expression level increased in response to different stimuli, including LPS, poly (I:C) and virus infection in host cells. bcTRADD activated the transcriptional activity of NF-κB promoter in the reporter assay; however, showed hardly any effect on the transcriptional activity of IFN promoter. It was interesting that black carp mitochondria antiviral signaling protein (bcMAVS)-activated IFN promoter transcription were dramatically depressed by bcTRADD and the C-terminal death domain of bcTRADD was indispensable for its regulation of bcMAVS. Accordingly, the plaque assay result showed that EPC cells co-expressing bcMAVS and bcTRADD presented much attenuated antiviral activity than EPC cells expressing bcMAVS alone. Knockdown of bcTRADD slightly promoted the antiviral ability of the host cells against SVCV. The current data support the conclusion that bcTRADD suppresses MAVS-mediated antiviral signaling, which is different to its mammalian counterpart.
Subject(s)
Carps/genetics , Carps/immunology , Fish Diseases/immunology , Gene Expression Regulation/immunology , Immunity, Innate/genetics , TNF Receptor-Associated Death Domain Protein/genetics , TNF Receptor-Associated Death Domain Protein/immunology , Amino Acid Sequence , Animals , Cell Line , Fish Proteins/chemistry , Fish Proteins/genetics , Fish Proteins/immunology , Gene Expression Profiling/veterinary , HEK293 Cells , Humans , Lipopolysaccharides/pharmacology , Phylogeny , Poly I-C/pharmacology , Rhabdoviridae/physiology , Rhabdoviridae Infections/immunology , Rhabdoviridae Infections/veterinary , Sequence Alignment/veterinary , TNF Receptor-Associated Death Domain Protein/chemistryABSTRACT
Cell death in human diseases is often a consequence of disrupted cellular homeostasis. If cell death is prevented without restoring cellular homeostasis, it may lead to a persistent dysfunctional and pathological state. Although mechanisms of cell death have been thoroughly investigated1-3, it remains unclear how homeostasis can be restored after inhibition of cell death. Here we identify TRADD4-6, an adaptor protein, as a direct regulator of both cellular homeostasis and apoptosis. TRADD modulates cellular homeostasis by inhibiting K63-linked ubiquitination of beclin 1 mediated by TRAF2, cIAP1 and cIAP2, thereby reducing autophagy. TRADD deficiency inhibits RIPK1-dependent extrinsic apoptosis and proteasomal stress-induced intrinsic apoptosis. We also show that the small molecules ICCB-19 and Apt-1 bind to a pocket on the N-terminal TRAF2-binding domain of TRADD (TRADD-N), which interacts with the C-terminal domain (TRADD-C) and TRAF2 to modulate the ubiquitination of RIPK1 and beclin 1. Inhibition of TRADD by ICCB-19 or Apt-1 blocks apoptosis and restores cellular homeostasis by activating autophagy in cells with accumulated mutant tau, α-synuclein, or huntingtin. Treatment with Apt-1 restored proteostasis and inhibited cell death in a mouse model of proteinopathy induced by mutant tau(P301S). We conclude that pharmacological targeting of TRADD may represent a promising strategy for inhibiting cell death and restoring homeostasis to treat human diseases.
Subject(s)
Apoptosis/drug effects , Homeostasis/drug effects , TNF Receptor-Associated Death Domain Protein/antagonists & inhibitors , TNF Receptor-Associated Death Domain Protein/metabolism , Animals , Autophagy/drug effects , Baculoviral IAP Repeat-Containing 3 Protein/metabolism , Beclin-1/chemistry , Beclin-1/metabolism , Bortezomib/antagonists & inhibitors , Bortezomib/pharmacology , Cell Line , Humans , Huntingtin Protein/metabolism , Inhibitor of Apoptosis Proteins/metabolism , Male , Mice , Models, Molecular , Neurofibrillary Tangles/metabolism , Proteome/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/chemistry , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , TNF Receptor-Associated Death Domain Protein/chemistry , TNF Receptor-Associated Death Domain Protein/deficiency , TNF Receptor-Associated Factor 2/metabolism , Ubiquitination , alpha-Synuclein/metabolism , tau Proteins/metabolismABSTRACT
In the present study, we characterized tumor necrosis factor receptor-associated factor 2/7 (lcTRAF2/7) and TNFR1-associated death domain protein (lcTRADD) in Larimichthys crocea (L. crocea) and examined their expression profiles in tissues of Vibrio-challenged and unchallenged fish. The coding sequences of lcTRAF2, lcTRAF7, and lcTRADD were 1488, 2454, and 744 nucleotides, and they encoded proteins of 495, 344, and 248 amino acids, respectively. The results of phylogenetic analysis revealed that lcTRAF2, lcTRAF7, and lcTRADD were closest to Oplegnathus fasciatus (85%), Xiphophorus maculatus (97%), and Acanthochromis polyacanthus (65%), respectively. Multiple sequence alignment showed that lcTRAF2 and lcTRAF7 were highly conserved with other vertebrate TRAFs in their functional domains; however, lcTRADD was poorly conserved. The results of quantitative real-time polymerase chain reaction analysis indicated that lcTRAF2, lcTRAF7, and lcTRADD were constitutively expressed in the spleen, liver, kidney, heart, brain, gill, bladder, skin, fin, eye, and muscle. After challenging fish with Vibrio parahaemolyticus, the mRNA expression levels of lcTRAF2, lcTRAF7, and lcTRADD were upregulated in liver, spleen, and kidney. Immunofluorescence staining revealed that lcTRAF2 and lcTRADD were cytoplasmic in localization, whereas lcTRAF7 targeted both the cytoplasm and nucleus. In addition, the NF-κB protein level was upregulated after lipopolysaccharide stimulation in lcTRAF2, lcTRAF7, or lcTRADD overexpressing cells. Taken collectively, these results have improved our understanding of the functions of TRAF2, TRAF7, and TRADD in pathogenic infections in teleosts.
Subject(s)
Fish Diseases/immunology , Fish Proteins/genetics , Fish Proteins/immunology , Gene Expression Regulation/immunology , Immunity, Innate/genetics , Perciformes/genetics , Perciformes/immunology , Amino Acid Sequence , Animals , Fish Proteins/chemistry , Gene Expression Profiling/veterinary , Phylogeny , Sequence Alignment/veterinary , TNF Receptor-Associated Death Domain Protein/chemistry , TNF Receptor-Associated Death Domain Protein/genetics , TNF Receptor-Associated Death Domain Protein/immunology , Tumor Necrosis Factor Receptor-Associated Peptides and Proteins/chemistry , Tumor Necrosis Factor Receptor-Associated Peptides and Proteins/genetics , Tumor Necrosis Factor Receptor-Associated Peptides and Proteins/immunology , Vibrio Infections/immunology , Vibrio Infections/veterinary , Vibrio parahaemolyticus/physiologyABSTRACT
Enteropathogenic E. coli NleB and related type III effectors catalyze arginine GlcNAcylation of death domain (DD) proteins to block host defense, but the underlying mechanism is unknown. Here we solve crystal structures of NleB alone and in complex with FADD-DD, UDP, and Mn2+ as well as NleB-GlcNAcylated DDs of TRADD and RIPK1. NleB adopts a GT-A fold with a unique helix-pair insertion to hold FADD-DD; the interface contacts explain the selectivity of NleB for certain DDs. The acceptor arginine is fixed into a cleft, in which Glu253 serves as a base to activate the guanidinium. Analyses of the enzyme-substrate complex and the product structures reveal an inverting sugar-transfer reaction and a detailed catalytic mechanism. These structural insights are validated by mutagenesis analyses of NleB-mediated GlcNAcylation in vitro and its function in mouse infection. Our study builds a structural framework for understanding of NleB-catalyzed arginine GlcNAcylation of host death domain.
Subject(s)
Enteropathogenic Escherichia coli/genetics , Escherichia coli Proteins/chemistry , Host-Pathogen Interactions/genetics , Protein Conformation , Virulence Factors/chemistry , Animals , Apoptosis/genetics , Arginine/chemistry , Arginine/genetics , Coenzyme A Ligases/chemistry , Coenzyme A Ligases/genetics , Crystallography, X-Ray , Death Domain/genetics , Enteropathogenic Escherichia coli/pathogenicity , Escherichia coli Proteins/genetics , Guanidine/chemistry , Humans , Manganese/chemistry , Mice , Mutagenesis , TNF Receptor-Associated Death Domain Protein/chemistry , TNF Receptor-Associated Death Domain Protein/genetics , Virulence Factors/geneticsABSTRACT
Strains of Salmonella utilize two distinct type three secretion systems to deliver effector proteins directly into host cells. The Salmonella effectors SseK1 and SseK3 are arginine glycosyltransferases that modify mammalian death domain containing proteins with N-acetyl glucosamine (GlcNAc) when overexpressed ectopically or as recombinant protein fusions. Here, we combined Arg-GlcNAc glycopeptide immunoprecipitation and mass spectrometry to identify host proteins GlcNAcylated by endogenous levels of SseK1 and SseK3 during Salmonella infection. We observed that SseK1 modified the mammalian signaling protein TRADD, but not FADD as previously reported. Overexpression of SseK1 greatly broadened substrate specificity, whereas ectopic co-expression of SseK1 and TRADD increased the range of modified arginine residues within the death domain of TRADD. In contrast, endogenous levels of SseK3 resulted in modification of the death domains of receptors of the mammalian TNF superfamily, TNFR1 and TRAILR, at residues Arg376 and Arg293 respectively. Structural studies on SseK3 showed that the enzyme displays a classic GT-A glycosyltransferase fold and binds UDP-GlcNAc in a narrow and deep cleft with the GlcNAc facing the surface. Together our data suggest that salmonellae carrying sseK1 and sseK3 employ the glycosyltransferase effectors to antagonise different components of death receptor signaling.
Subject(s)
Bacterial Proteins/metabolism , Salmonella/metabolism , Signal Transduction , TNF-Related Apoptosis-Inducing Ligand/metabolism , Tumor Necrosis Factor-alpha/metabolism , Acetylglucosamine/metabolism , Animals , Bacterial Proteins/chemistry , Conserved Sequence , Glutamic Acid/metabolism , Glycosylation , HEK293 Cells , HeLa Cells , Humans , Mice , Mice, Inbred C57BL , Mutagenesis , Mutation/genetics , Protein Domains , RAW 264.7 Cells , Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism , Receptors, Tumor Necrosis Factor, Type I/metabolism , Substrate Specificity , TNF Receptor-Associated Death Domain Protein/chemistry , TNF Receptor-Associated Death Domain Protein/metabolismABSTRACT
The tumor necrosis factor receptor-associated death domain protein, TRADD, is a multifunctional intracellular molecule participating in divergent signaling pathways, such as NF-κB and apoptosis. TRADD consists of two structurally distinct domains. Its N-terminal domain displays an α-ß plaits fold while its C-terminal domain belongs to the death domain (DD) superfamily. TRADD DD is a central component in the tumor necrosis factor receptor 1 signaling. It interacts with other DD-containing proteins through homotypic interactions. TRADD DD is also involved in p75NTR-mediated signalling in MCF-7 human breast cancer cells. Here we report backbone and sidechain 1H, 13C and 15N chemical shift assignments of TRADD DD in pure water as a basis for further structural and functional studies.
Subject(s)
Nuclear Magnetic Resonance, Biomolecular , TNF Receptor-Associated Death Domain Protein/chemistry , Amino Acid Sequence , Protein DomainsABSTRACT
The TNFR1-associated death domain protein (TRADD) is an intracellular adaptor protein involved in various signaling pathways, such as antiapoptosis. Its C-terminal death domain (DD) is responsible for binding other DD-containing proteins including the p75 neurotrophin receptor (p75NTR). Here we present a solution structure of TRADD DD derived from high-resolution NMR spectroscopy. The TRADD DD comprises two super-secondary structures, an all-helix Greek key motif and a ß-hairpin motif flanked by two α helices, which make it unique among all known DD structures. The ß-hairpin motif is essential for TRADD DD to fold into a functional globular domain. The highly-charged surface suggests a critical role of electrostatic interactions in TRADD DD-mediated signaling. This novel structure represents a new class within the DD superfamily and provides a structural basis for studying homotypic DD interactions. NMR titration revealed a direct weak interaction between TRADD DD and p75NTR DD monomers. A binding site next to the p75NTR DD homodimerization interface indicates that TRADD DD recruitment to p75NTR requires separation of the p75NTR DD homodimer, explaining the mechanism of NGF-dependent activation of p75NTR-TRADD-mediated antiapoptotic pathway in breast cancer cell.
Subject(s)
Death Domain Superfamily , TNF Receptor-Associated Death Domain Protein/chemistry , Binding Sites , Magnetic Resonance Spectroscopy , Nerve Tissue Proteins/metabolism , Protein Binding , Protein Conformation , Protein Folding , Receptors, Nerve Growth Factor/metabolism , TNF Receptor-Associated Death Domain Protein/metabolismABSTRACT
FADD (Fas-associated death domain) and TRADD (Tumor Necrosis Factor Receptor 1-associated death domain) proteins are important regulators of cell fate in mammalian cells. They are both involved in death receptors mediated signaling pathways and have been linked to the Toll-like receptor family and innate immunity. Here we identify and characterize by database search analysis, mutagenesis and calmodulin (CaM) pull-down assays a calcium-dependent CaM binding site in the α-helices 1-2 of TRADD death domain. We also show that oxidation of CaM methionines drastically reduces CaM affinity for FADD and TRADD suggesting that oxidation might regulate CaM-FADD and CaM-TRADD interactions. Finally, using Met-to-Leu CaM mutants and binding assays we show that both the N- and C-terminal domains of CaM are important for binding.
Subject(s)
Calmodulin/chemistry , Calmodulin/metabolism , Fas-Associated Death Domain Protein/metabolism , TNF Receptor-Associated Death Domain Protein/metabolism , Amino Acid Sequence , Binding Sites , Calcium/metabolism , Cell Line , Humans , Methionine/metabolism , Methionine Sulfoxide Reductases/pharmacology , Molecular Sequence Data , Mutation , Oxidation-Reduction , Protein Binding/drug effects , Protein Structure, Tertiary , TNF Receptor-Associated Death Domain Protein/chemistry , TNF Receptor-Associated Death Domain Protein/geneticsABSTRACT
Apoptosis can be induced by an extrinsic pathway involving the ligand-mediated activation of death receptors such as tumor necrosis factor receptor-1 (TNFR-1). TNFR-1-associated death domain (TRADD) protein is an adapter molecule that bridges the interaction between TNFR-1 and receptor-interacting serine/threonine-protein kinase 1 (RIP1). However, the molecular mechanism of the complex formation of these proteins has not yet been identified. Here, the binding among TNFR-1, TRADD, and RIP1 was identified using a GST pull-down assay and Biacore biosensor experiment. This study showed that structural characterization and formation of the death-signaling complex could be predicted using TNFR-1, TRADD, and RIP1. In addition, we found that the structure-based mutations of TNFR-1 (P367A and P368A), TRADD (F266A), and RIP1 (M637A and R638A) disrupted formation of the death domain (DD) complex and prevented stable interactions among those DDs.
Subject(s)
Receptor-Interacting Protein Serine-Threonine Kinases/chemistry , Receptors, Tumor Necrosis Factor, Type I/chemistry , TNF Receptor-Associated Death Domain Protein/chemistry , Amino Acid Sequence , Amino Acid Substitution , Apoptosis , Biosensing Techniques , Humans , Kinetics , Models, Molecular , Molecular Sequence Data , Multiprotein Complexes/chemistry , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Mutagenesis, Site-Directed , Protein Binding , Protein Interaction Domains and Motifs , Receptor-Interacting Protein Serine-Threonine Kinases/genetics , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Receptors, Tumor Necrosis Factor, Type I/genetics , Receptors, Tumor Necrosis Factor, Type I/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Signal Transduction , Static Electricity , TNF Receptor-Associated Death Domain Protein/genetics , TNF Receptor-Associated Death Domain Protein/metabolismABSTRACT
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.
Subject(s)
Enteropathogenic Escherichia coli/metabolism , Escherichia coli Infections/metabolism , Escherichia coli Infections/microbiology , Escherichia coli Proteins/metabolism , Gastrointestinal Tract/microbiology , Signal Transduction , Virulence Factors/metabolism , Animals , Caspase 8/metabolism , Cell Death , Citrobacter rodentium/pathogenicity , Citrobacter rodentium/physiology , Enteropathogenic Escherichia coli/pathogenicity , Enzyme Activation , Escherichia coli Infections/pathology , Fas Ligand Protein/antagonists & inhibitors , Fas Ligand Protein/metabolism , Fas-Associated Death Domain Protein/chemistry , Fas-Associated Death Domain Protein/metabolism , Female , HEK293 Cells , HeLa Cells , Humans , Male , Mice , N-Acetylglucosaminyltransferases/metabolism , Protein Structure, Tertiary , Receptor-Interacting Protein Serine-Threonine Kinases/chemistry , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , TNF Receptor-Associated Death Domain Protein/chemistry , TNF Receptor-Associated Death Domain Protein/metabolism , fas Receptor/deficiency , fas Receptor/metabolismSubject(s)
Arginine/metabolism , Enteropathogenic Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , N-Acetylglucosaminyltransferases/metabolism , Signal Transduction , TNF Receptor-Associated Death Domain Protein/chemistry , TNF Receptor-Associated Death Domain Protein/metabolism , Virulence Factors/metabolism , Animals , Humans , MaleABSTRACT
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. 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). 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. 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.
Subject(s)
Arginine/metabolism , Enteropathogenic Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , N-Acetylglucosaminyltransferases/metabolism , Signal Transduction , TNF Receptor-Associated Death Domain Protein/chemistry , TNF Receptor-Associated Death Domain Protein/metabolism , Virulence Factors/metabolism , Acylation , Animals , Apoptosis , Death Domain Receptor Signaling Adaptor Proteins/metabolism , Disease Models, Animal , Enteropathogenic Escherichia coli/pathogenicity , Escherichia coli Infections/metabolism , Escherichia coli Infections/microbiology , Escherichia coli Infections/pathology , Fas-Associated Death Domain Protein/chemistry , Fas-Associated Death Domain Protein/metabolism , HeLa Cells , Humans , Male , Mice , Mice, Inbred C57BL , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , NF-kappa B/metabolism , Protein Biosynthesis , Protein Structure, Tertiary , Receptor-Interacting Protein Serine-Threonine Kinases/chemistry , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Receptors, Tumor Necrosis Factor, Type I/chemistry , Receptors, Tumor Necrosis Factor, Type I/metabolism , TNF-Related Apoptosis-Inducing Ligand/metabolism , Tumor Necrosis Factor-alpha/metabolism , Virulence , fas Receptor/metabolismABSTRACT
EVER1 and 2 confer resistance to cutaneous oncogenic human papillomavirus infections by downregulating the activating protein 1 (AP-1) signaling pathway. Defects in their expression are associated with susceptibility to epidermodysplasia verruciformis, which is characterized by persistent ß-HPV infection, tumor necrosis factor alpha (TNF-α) overproduction in keratinocytes and the development of skin cancers. TNF-α-induced apoptosis is a key defense strategy, preventing the persistence of the virus within cells, but the role of EVER proteins in this cell death mechanism triggered by extrinsic stimuli is unknown. We show here that EVER2 induces TNF-α- and TRAIL-dependant apoptosis. It interacts with the N-terminal domain of TRADD, impairs the recruitment of TRAF2 and RIPK1 and promotes apoptosis. The skin cancer-associated EVER2 I306 allele results in an impaired TRADD-EVER2 interaction, with lower levels of cell death following treatment with TNF-α. These data highlight a new, critical function of EVER2 in controlling cell survival in response to death stimuli.
Subject(s)
Apoptosis/drug effects , Membrane Proteins/metabolism , TNF Receptor-Associated Death Domain Protein/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Alleles , Caspase 8/metabolism , Fas Ligand Protein/genetics , Fas Ligand Protein/metabolism , Fas Ligand Protein/pharmacology , HEK293 Cells , Humans , Inhibitor of Apoptosis Proteins/metabolism , Jurkat Cells , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/genetics , MicroRNAs/metabolism , NF-kappa B/metabolism , Protein Binding , Protein Structure, Tertiary , RNA Interference , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/pharmacology , TNF Receptor-Associated Death Domain Protein/chemistry , TNF Receptor-Associated Death Domain Protein/genetics , TNF Receptor-Associated Factor 2/metabolism , TNF-Related Apoptosis-Inducing Ligand/genetics , TNF-Related Apoptosis-Inducing Ligand/metabolism , TNF-Related Apoptosis-Inducing Ligand/pharmacology , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Tumor necrosis factor (TNF) is an important cytokine in immunity and inflammation and induces many cellular responses, including apoptosis and necrosis. TNF signaling enables the generation of superoxide in phagocytic and vascular cells through the activation of the NADPH oxidase Nox2/gp91. Here we show that TNF also activates the Nox1 NADPH oxidase in mouse fibroblasts when cells undergo necrosis. TNF treatment induces the formation of a signaling complex containing TRADD, RIP1, Nox1, and the small GTPase Rac1. TNF-treated RIP1-deficient fibroblasts fail to form such a complex, indicating that RIP1 is essential for Nox1 recruitment. Moreover, the prevention of TNF-induced superoxide generation with dominant-negative mutants of TRADD or Rac1, as well as knockdown of Nox1 using siRNA, inhibits necrosis. Thus our study suggests that activation of Nox1 through forming a complex with TNF signaling components plays a key role in TNF-induced necrotic cell death.
Subject(s)
Cell Death/drug effects , Cell Death/physiology , NADH, NADPH Oxidoreductases/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Animals , Antioxidants/pharmacology , Butylated Hydroxyanisole/pharmacology , Cell Line , GTPase-Activating Proteins/chemistry , GTPase-Activating Proteins/metabolism , Humans , Mice , Multiprotein Complexes , NADH, NADPH Oxidoreductases/antagonists & inhibitors , NADH, NADPH Oxidoreductases/chemistry , NADH, NADPH Oxidoreductases/genetics , NADPH Oxidase 1 , Necrosis , Neuropeptides/chemistry , Neuropeptides/metabolism , RNA, Small Interfering/genetics , Signal Transduction , Superoxides/metabolism , TNF Receptor-Associated Death Domain Protein/chemistry , TNF Receptor-Associated Death Domain Protein/metabolism , rac GTP-Binding Proteins/chemistry , rac GTP-Binding Proteins/metabolism , rac1 GTP-Binding ProteinABSTRACT
Necrosis is a caspase-independent cell death process involving the generation of reactive oxygen species (ROS). In a recent issue of Molecular Cell, Kim et al. (2007) reported on a novel TNF receptor 1 necrotic signaling complex inducing TRADD- and RIP1-dependent recruitment and activation of the ROS-generating Nox1 NADPH oxidase complex.