Roles of the adaptor protein tumor necrosis factor receptor type 1-associated death domain protein (TRADD) in human diseases.

Cells communication in response to extracellular or biophysical stimulus relies on elaborated systems of signal transduction. In the course of most signal pathway, the cascades involve signal protein complexes, which are often assembled by adaptor proteins. Tumor necrosis factor receptor type 1-associated death domain protein (TRADD) is an adaptor molecule involved in various signal pathways and mediating multiple biological activities, including cell survival, cell proliferation, cell differentiation, apoptosis, necroptosis and inflammation. TRADD contains an N terminal tumor necrosis factor receptor-associated factor 2 (TRAF2) binding domain and a C terminal death domain (DD) for interacting with multiple DD-containing proteins. Following activation of specific receptors, such as tumor necrosis factor receptor 1 (TNFR1), death receptor 3 (DR3), tumor necrosis factor-related apoptosis-inducing ligand receptor 1 (TRAILR1, DR4), TRAILR1 (DR5), DR6 and p75 neurotrophin receptor (p75NTR)，TRADD can bind to the receptors, serving as a platform for the recruitment of the downstream molecules for signal propagating and thus mediating various physiological and pathological processes. In this review, we provide a brief overview of the current knowledge on TRADD and discuss the roles of TRADD in infectious and inflammatory diseases, cardiovascular diseases, central nervous system diseases, cancer, endometriosis, hepatocyte proliferation, preterm birth and perinatal development.

RIPK1 and TRADD Regulate TNF-Induced Signaling and Ripoptosome Formation.

TNF is a proinflammatory cytokine that is critical for the coordination of tissue homeostasis. RIPK1 and TRADD are the main participants in the transduction of TNF signaling. However, data on the cell fate-controlling functions of both molecules are quite controversial. Here, we address the functions of RIPK1 and TRADD in TNF signaling by generating RIPK1- or TRADD-deficient human cell lines. We demonstrate that RIPK1 is relevant for TNF-induced apoptosis and necroptosis in conditions with depleted IAPs. In addition, TRADD is dispensable for necroptosis but required for apoptosis. We reveal a new possible function of TRADD as a negative regulator of NIK stabilization and subsequent ripoptosome formation. Furthermore, we show that RIPK1 and TRADD do not appear to be essential for the activation of MAPK signaling. Moreover, partially repressing NF-κB activation in both RIPK1 and TRADD KO cells does not result in sensitization to TNF alone due to the absence of NIK stabilization. Importantly, we demonstrate that RIPK1 is essential for preventing TRADD from undergoing TNF-induced ubiquitination and degradation. Taken together, our findings provide further insights into the specific functions of RIPK1 and TRADD in the regulation of TNF-dependent signaling, which controls the balance between cell death and survival.

Bacterial death and TRADD-N domains help define novel apoptosis and immunity mechanisms shared by prokaryotes and metazoans.

Several homologous domains are shared by eukaryotic immunity and programmed cell-death systems and poorly understood bacterial proteins. Recent studies show these to be components of a network of highly regulated systems connecting apoptotic processes to counter-invader immunity, in prokaryotes with a multicellular habit. However, the provenance of key adaptor domains, namely those of the Death-like and TRADD-N superfamilies, a quintessential feature of metazoan apoptotic systems, remained murky. Here, we use sensitive sequence analysis and comparative genomics methods to identify unambiguous bacterial homologs of the Death-like and TRADD-N superfamilies. We show the former to have arisen as part of a radiation of effector-associated α-helical adaptor domains that likely mediate homotypic interactions bringing together diverse effector and signaling domains in predicted bacterial apoptosis- and counter-invader systems. Similarly, we show that the TRADD-N domain defines a key, widespread signaling bridge that links effector deployment to invader-sensing in multicellular bacterial and metazoan counter-invader systems. TRADD-N domains are expanded in aggregating marine invertebrates and point to distinctive diversifying immune strategies probably directed both at RNA and retroviruses and cellular pathogens that might infect such communities. These TRADD-N and Death-like domains helped identify several new bacterial and metazoan counter-invader systems featuring underappreciated, common functional principles: the use of intracellular invader-sensing lectin-like (NPCBM and FGS), transcription elongation GreA/B-C, glycosyltransferase-4 family, inactive NTPase (serving as nucleic acid receptors), and invader-sensing GTPase switch domains. Finally, these findings point to the possibility of multicellular bacteria-stem metazoan symbiosis in the emergence of the immune/apoptotic systems of the latter.

The Odd Faces of Oligomers: The Case of TRAF2-C, A Trimeric C-Terminal Domain of TNF Receptor-Associated Factor.

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.

Knockdown of circROBO2 attenuates acute myocardial infarction through regulating the miR-1184/TRADD axis.

BACKGROUND: Studies have found that circular RNAs (circRNAs) play key roles in cardiovascular diseases. However, the function of circROBO2 in acute myocardial infarction (AMI) is unclear. This study aimed to investigate the pathogenesis of circROBO2 in AMI. METHODS: qRT-PCR and Western blot were used to determine the expression levels of circROBO2, miR-1184, and TRADD in AMI and sham-operated mouse models at mRNA and protein level, respectively. The relationship among miR-1184, circROBO2 and TRADD was evaluated by RNA immunoprecipitation (RIP) analysis and luciferase reporter gene analysis. The roles of circROBO2, miR-1184, and TRADD in myocardial cell apoptosis were evaluated using flow cytometry. Ultrasound echocardiography, serum creatine kinase MB (CK-MB) and lactate dehydrogenase (LDH), myocardial infarction area, and myocardial cell apoptosis were measured to examine the effects of circROBO2 on myocardial injury. RESULTS: The expression levels of miR-1184 were significantly reduced, and the expression levels of circROBO2 and TRADD were significantly increased in MI group. CircROBO2 acted as a sponge for miR-1184 by upregulating the expression of TRADD. In addition, overexpression of miR-1184 enhanced the protective effect of knockdown of circROBO2 by partially inhibiting the expression of TRADD in vivo and in vitro. CONCLUSION: Knockdown of circROBO2 reduced the apoptosis of cardiomyocytes by increasing the expression levels of miR-1184, which in turn decreased the expression levels of TRADD in the myocardium post-MI.

Roles of TNF receptor-associated and Fas-associated death domain proteins in the apoptosis of Eimeria tenella host cells.

The present study aimed to investigate the effects of death receptor adapter proteins, namely, TNF receptor-associated death domain (TRADD) and Fas-associated death domain (FADD) proteins, on Eimeria tenella-induced host cell apoptosis. Gene silencing, culture technique for primary chick embryo cecal epithelial cells, enzyme-linked immunosorbent assay, Hoechst-Annexin V/PI apoptosis staining, fluorescence quantitative PCR, and flow cytometry were used to detect the E. tenella host cell apoptotic rate, RIP1 and FADD protein expression levels, and caspase-8 activity of the TRADD siRNA-treated and FADD siRNA-treated groups. Results showed that the apoptotic rate in the TRADD siRNA group was significantly higher than that in the NC siRNA group at 4 h post-infection with E. tenella (P < 0.05). The RIP1 protein expression level in the TRADD siRNA group was significantly lower than that in the NC siRNA group at 4-24 h (P < 0.05). The FADD expression and apoptotic rates in the TRADD siRNA group were significantly lower than those in the NC siRNA group at 24-120 h (P < 0.05). The caspase-8 activity and apoptotic rates in the FADD siRNA group were significantly lower than those in the NC siRNA group (P < 0.05) at 24-120 h. These findings indicated that E. tenella inhibited the host cell apoptosis through the TRADD-RIP1 pathway at the early developmental stage and promoted host cell apoptosis via the TRADD-FADD-caspase-8 apoptotic pathway at the middle and late developmental stages.

Black carp TRADD suppresses MAVS/IFN signaling during the innate immune activation.

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.

Molecular characterization and functional analysis of tumor necrosis factor receptor-associated factor 2/7 and tumor necrosis factor receptor 1-associated death domain protein from Larimichthys crocea.

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.

Copper Induces Oxidative Stress and Apoptosis in the Mouse Liver.

Copper (Cu) is an essential trace element involved in the normal physiological processes of animals. However, excessive exposure to Cu can produce numerous detrimental impacts. The aim of this study was to investigate the effects of Cu on oxidative stress and apoptosis as well as their relationship in the mouse liver. Four-week-old ICR mice (n = 240) were randomly assigned to different Cu (Cu2+-CuSO4) treatment groups (0, 4, 8, and 16 mg/kg) for periods of 21 and 42 days. The high doses of Cu exposure could induce oxidative stress, by increasing the levels of reactive oxygen species (ROS) and protein carbonyls (PC) and decreasing the activities of antisuperoxide anion (ASA) and antihydroxyl radical (AHR) and content of glutathione (GSH), as well as activities and mRNA expression levels of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px). Moreover, high doses of Cu exposure induced hepatic apoptosis via the mitochondrial apoptotic pathway, as characterized by the depolarization of mitochondrial membrane potential (MMP); significantly increased mRNA and protein expression levels of cytosolic cytochrome (Cyt c), apoptosis-inducing factor (AIF), endonuclease G (Endo G), apoptosis protease-activating factor-1 (Apaf-1), cleaved caspase-9, cleaved caspase-3, cleaved PARP, Bcl-2 antagonist killer (Bak), Bcl-2-associated X protein (Bax), and Bcl-2-interacting mediator of cell death (Bim); and decreased mRNA and protein expression levels of B-cell lymphoma-2 (Bcl-2) and Bcl-extra-large (Bcl-xL). Furthermore, the activation of the tumor necrosis factor receptor-1 (TNF-R1) signaling pathway was involved in Cu-induced apoptosis, as characterized by the significantly increased mRNA and protein expression levels of TNF-R1, Fas-associated death domain (FADD), TNFR-associated death domain (TRADD), and cleaved caspase-8. These results indicated that exposure to excess Cu could cause oxidative stress triggered by ROS overproduction and diminished antioxidant function, which in turn promoted hepatic apoptosis via mitochondrial apoptosis and that the TNF-R1 signaling pathway was also involved in the Cu-induced apoptosis.

SNHG9, delivered by adipocyte-derived exosomes, alleviates inflammation and apoptosis of endothelial cells through suppressing TRADD expression.

Exosomes are membrane-derived vesicles and play a critical role in cell signaling by transferring RNAs and proteins to target cells through fusion with the cell membrane. Long non-coding RNA-small nucleolar RNA host gene 9 (lncRNA-SNHG9) was proven to be an important element in lncRNA-mRNA interaction networks during adipocyte differentiation, suggesting its potential involvement in the development of obesity, an important risk factor of cardiovascular and cerebrovascular endothelial dysfunction. However, the role of lncRNA-SNHG9 within the exosome in endothelial dysfunction of obese patients is largely unknown. In this study, we proved that adipocytes-derived exosomal SNHG9 were downregulated in obese persons and further decreased in obese individuals with endothelial dysfunction. Functional experimentations demonstrated that adipocytes-derived exosomal SNHG9 alleviated inflammation and apoptosis in endothelial cells. Bioinformatic analysis revealed that there was a potential interaction between SNHG9 and the TNF receptor type 1-associated death domain protein (TRADD) mRNA. Then, RNA-binding protein immunoprecipitation assay based on Ago2 antibody and ribonuclease protection assay demonstrated that exosomal SNHG9 directly bound to a specific region in TRADD mRNA sequence and formed an RNA dimeric inducible silencing complex. Moreover, knockdown of TRADD markedly inhibited inflammation and apoptosis in human umbilical vein endothelial cells (HUVECs), whereas overexpression of TRADD dramatically neutralized the protective effect of exosomal SNHG9 on epithelial dysfunction. Therefore, SNHG9 could prevent endothelial dysfunction in obese patients by suppressing inflammation and apoptosis, indicating that SNHG9 may be a potential therapeutic target for obese patients with endothelial dysfunction.

miR-149* Suppresses Liver Cancer Progression by Down-Regulating Tumor Necrosis Factor Receptor 1-Associated Death Domain Protein Expression.

Liver cancer is the third leading cause of cancer-related death worldwide. Herein, we show that miR-149* serves as a novel tumor suppressor for liver tumorigenesis. Mice with genetic deletion of miR-149* (miR-149*-/- mice), which caused loss of both miR-149 and miR-149*, were considerably more susceptible to acute liver injury and hepatic carcinogenesis induced by diethylnitrosamine than wild-type mice, accompanied by increased compensatory proliferation and up-regulated gene expression of certain inflammatory cytokines. miR-149* mimics dramatically impaired liver cancer cell proliferation and migration in vitro and blocked liver cancer progression in a xenograft model. Furthermore, miR-149* strongly suppressed NF-κB signaling and repressed tumor necrosis factor receptor type 1-associated death domain protein expression in the NF-κB signaling pathway. These results reveal that miR-149*, as a novel liver tumor suppressor, may serve as a potential therapeutic target for liver cancer treatment.

Grouper TRADD Mediates Innate Antiviral Immune Responses and Apoptosis Induced by Singapore Grouper Iridovirus (SGIV) Infection.

Tumor necrosis factor (TNF) receptor type 1-associated DEATH domain protein (TRADD) is a TNFR1-associated signal transducer and an essential component of the TNFR1 complex that is involved in activating both apoptotic and nuclear factor (NF)-κB pathways as an adaptor. It also is required for TNFR-1-initiated neuronal apoptosis following in vitro infection with virus as an essential component of the antiviral response. To date, few studies have investigated the function of TRADD in lower vertebrates and its antiviral response to DNA virus infection. In the present study, a TRADD gene (named as EcTRADD) from the orange-spotted grouper (Epinephelus coioides) was cloned and characterized. The full-length cDNA of EcTRADD consists of 1,370 base pairs (bp) and contains a 44 bp 5'-terminal untranslated region (UTR), a 450 bp 3'-UTR including a poly (A) tail, and an 876 bp open reading frame encoding a putative 291 amino acid protein. EcTRADD has two conserved domains of N-terminal domain (TRADD-N) and a death domain (DD). EcTRADD was detected in all examined tissues. EcTRADD was up-regulated in the spleen after infection with Singapore grouper iridovirus (SGIV). Subcellular localization analysis revealed that EcTRADD and EcTRADD-DD exhibited a clear pattern of discrete and interconnecting cytoplasmic filaments resembling the death-effector filaments, while EcTRADD-N was observed in the cytoplasm. After infection with SGIV, EcTRADD, and EcTRADD-DD were transferred to the nucleus. Overexpression of EcTRADD and its domains inhibited replication of SGIV in vitro. Both EcTRADD and EcTRADD-DD induced the caspase-dependent apoptosis in control and infected cells, while EcTRADD-N inhibited the apoptosis. Additionally, EcTRADD and EcTRADD-DD significantly promoted activation of NF-κB and reporter gene p53, whereas EcTRADD-N had no significant effect on p53. The results may provide new insights into the role of fish TRADD in fish virus infection.

Structural and Functional Insights into Host Death Domains Inactivation by the Bacterial Arginine GlcNAcyltransferase Effector.

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.

TRADD regulates perinatal development and adulthood survival in mice lacking RIPK1 and RIPK3.

TRADD is an adaptor for TNFR1-induced apoptosis and NFκB activation. However, TRADD-deficient mice undergo normal development and contain normal lymphoid populations, which contrasts with an embryonic defect in mice lacking FADD, the shared adaptor mediating apoptosis. Recent studies indicate FADD suppresses embryonic necroptosis mediated by RIPK1. TRADD was suggested to also mediate necroptosis. Here we report that targeting TRADD fails to rescue Fadd-/- embryos from necroptosis, and ablation of TRADD rescues Ripk1-/- mice from perinatal lethality when RIPK3-mediated necroptosis is disabled. The resulting Ripk1-/-Ripk3-/-Tradd-/- mice survive until early adulthood, but die thereafter. A single allele of Tradd is optimal for survival of Ripk1-/-Ripk3-/-Tradd+/- mice. We show that TRADD plays a more dominating role in NFκB-signaling than RIPK1. While RIPK1 protects thymocytes from TNFα-induced apoptosis, TRADD promotes this process. The data demonstrate that TRADD is critical in perinatal and adult mice lacking RIPK1 and RIPK3, which has not been appreciated in prior studies.

RIPK1 prevents TRADD-driven, but TNFR1 independent, apoptosis during development.

RIPK1 is an essential downstream component of many pattern recognition and death receptors. RIPK1 can promote the activation of caspase-8 induced apoptosis and RIPK3-MLKL-mediated necroptosis, however, during development RIPK1 limits both forms of cell death. Accordingly, Ripk1-/- mice present with systemic cell death and consequent multi-organ inflammation, which is driven through the activation of both FADD-caspase-8 and RIPK3-MLKL signaling pathways causing perinatal lethality. TRADD is a death domain (DD) containing molecule that mediates signaling downstream of TNFR1 and the TLRs. Following the disassembly of the upstream receptor complexes either RIPK1 or TRADD can form a complex with FADD-caspase-8-cFLIP, via DD-DD interactions with FADD, facilitating the activation of caspase-8. We show that genetic deletion of Ripk1 licenses TRADD to complex with FADD-caspase-8 and activates caspase-8 during development. Deletion of Tradd provided no survival advantage to Ripk1-/- animals and yet was sufficient to reduce the systemic cell death and inflammation, rescue the intestinal and thymic histopathologies, reduce cleaved caspases in most tissues and rescue the anemia observed in Ripk1-/- neonates. Furthermore, deletion of Ripk3 is sufficient to rescue the neonatal lethality of Ripk1-/-Tradd-/- animals and delays but does not completely prevent early mortality. Although Ripk3 deletion provides a significant survival advantage, Ripk1-/-Tradd-/-Ripk3-/- animals die between 22 and 49 days, are runty compared to littermate controls and present with splenomegaly. These findings reveal a new mechanism by which RIPK1 limits apoptosis through blocking TRADD recruitment to FADD and preventing aberrant activation of caspase-8.

From Mouse Models to Human Disease: An Approach for Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset neurodegenerative disorder. There are several genetic mutations that lead to ALS development, such as chromosome 9 hexanucleotide repeat 72 (C9ORF72), transactive response DNA-binding protein (TARDBP), superoxide dismutase 1 (SOD1) and fused in sarcoma (FUS). ALS is associated with disrupted gene homeostasis causing aberrant RNA processing or toxic pathology. Several animal models of ALS disease have been developed to understand whether TARDBP-mediated neurodegeneration results from a gain or a loss of function of the protein, however, none exactly mimic the pathophysiology and the phenotype of human ALS. Here, the pathophysiology of specific ALS-linked gene mutations is discussed. Furthermore, some of the generated mouse models, as well as the similarities and differences between these models, are comprehensively reviewed. Further refinement of mouse models will likely aid the development of a better form of model that mimics human ALS. However, disrupted gene homeostasis that causes mutation can result in an ALS-like syndrome, increasing concerns about whether neurodegeneration and other effects in these models are due to the mutation or to gene overexpression. Research on the pleiotropic role of different proteins present in motor neurons is also summarized. The development of better mouse models that closely mimic human ALS will help identify potential therapeutic targets for this disease.

TRADD mediates the tumor necrosis factor-induced apoptosis of L929 cells in the absence of RIP3.

Receptor-interacting protein kinase 3 (RIP3) is a critical initiator in mediating necroptosis induced by tumor necrosis factor alpha (TNFα) in L929 cells, so knockdown of RIP3 inhibits TNFα-induced L929 cell necroptosis. However, RIP3 knockdown was shown to switch TNFα-induced necroptosis to apoptosis in L929 cells in other studies. Therefore, whether RIP3 knockdown blocks the TNFα-induced death of L929 cells is controversial. In this study, TNFα activated caspase pathway and induced cell death in RIP3 knockdown L929 cells, and the RIP3-independent cell death had been blocked by Z-VAD-FMK (pan-caspase inhibitor) or caspase 8 knockdown, demonstrating that RIP3 knockdown switched TNFα-induced necroptosis to caspase-dependent apoptosis. Although both TNF receptor type 1-associated death domain protein (TRADD) and RIP1 have been reported to mediate TNFα-induced apoptosis, the knockdown of TRADD, but not RIP1, suppressed TNFα-induced activation of the caspase pathway and subsequent apoptosis in RIP3 knockdown L929 cells. In addition, TRADD bound and activated caspase 8 during the RIP3-independent apoptosis process, indicating that TRADD initiates RIP3-independent apoptosis by activating the caspase pathway. Collectively, we identified the target and mechanism underlying RIP3-independent apoptosis and elucidated the coordinated roles of RIP3 and TRADD in mediating the programmed cell death of L929 cells following TNFα stimulation.

The bacterial arginine glycosyltransferase effector NleB preferentially modifies Fas-associated death domain protein (FADD).

The inhibition of host innate immunity pathways is essential for the persistence of attaching and effacing pathogens such as enteropathogenic Escherichia coli (EPEC) and Citrobacter rodentium during mammalian infections. To subvert these pathways and suppress the antimicrobial response, attaching and effacing pathogens use type III secretion systems to introduce effectors targeting key signaling pathways in host cells. One such effector is the arginine glycosyltransferase NleB1 (NleBCR in C. rodentium) that modifies conserved arginine residues in death domain-containing host proteins with N-acetylglucosamine (GlcNAc), thereby blocking extrinsic apoptosis signaling. Ectopically expressed NleB1 modifies the host proteins Fas-associated via death domain (FADD), TNFRSF1A-associated via death domain (TRADD), and receptor-interacting serine/threonine protein kinase 1 (RIPK1). However, the full repertoire of arginine GlcNAcylation induced by pathogen-delivered NleB1 is unknown. Using an affinity proteomic approach for measuring arginine-GlcNAcylated glycopeptides, we assessed the global profile of arginine GlcNAcylation during ectopic expression of NleB1, EPEC infection in vitro, or C. rodentium infection in vivo NleB overexpression resulted in arginine GlcNAcylation of multiple host proteins. However, NleB delivery during EPEC and C. rodentium infection caused rapid and preferential modification of Arg117 in FADD. This FADD modification was extremely stable and insensitive to physiological temperatures, glycosidases, or host cell degradation. Despite its stability and effect on the inhibition of apoptosis, arginine GlcNAcylation did not elicit any proteomic changes, even in response to prolonged NleB1 expression. We conclude that, at normal levels of expression during bacterial infection, NleB1/NleBCR antagonizes death receptor-induced apoptosis of infected cells by modifying FADD in an irreversible manner.

Nuclear TRADD prevents DNA damage-mediated death by facilitating non-homologous end-joining repair.

TNF receptor-associated death domain (TRADD) is an essential mediator of TNF receptor signaling, and serves as an adaptor to recruit other effectors. TRADD has been shown to cycle between the cytoplasm and nucleus due to its nuclear localization (NLS) and export sequences (NES). However, the underlying function of nuclear TRADD is poorly understood. Here we demonstrate that cytoplasmic TRADD translocates to DNA double-strand break sites (DSBs) during the DNA damage response (DDR). Deficiency of TRADD or its sequestration in cytosol leads to accumulation of γH2AX-positive foci in response to DNA damage, which is reversed by nuclear TRADD expression. TRADD facilitates non-homologous end-joining (NHEJ) by recruiting NHEJ repair factors 53BP1 and Ku70/80 complex, whereas TRADD is dispensable for homologous recombination (HR) repair. Finally, an impaired nuclear localization of TRADD triggers cell death through the persistent activation of JNK and accumulation of reactive oxygen species (ROS). Thus, our findings suggest that translocation of TRADD to DSBs into the nucleus contributes to cell survival in response to DNA damage through an activation of DNA damage repair.

TRADD, TRAF2, RIP1 and TAK1 are required for TNF-α-induced pro-labour mediators in human primary myometrial cells.

PROBLEM: TNF-α plays a central role in the processes of human labour and delivery. This study sought to determine the role of the adaptor proteins TNFR1-associated death domain protein (TRADD), TNF receptor-associated factor 2 (TRAF2), receptor interacting protein 1 (RIP1) and transforming growth factor beta-activated kinase 1 (TAK1) in TNF-α-induced formation of pro-labour mediators. METHOD OF STUDY: Human primary myometrial cells were transfected with siRNA against TRADD (siTRADD), TRAF2 (siTRAF2), RIP1 (siRIP1) or TAK1 (siTAK1), treated with TNF-α, and assayed for pro-inflammatory mediators expression. RESULTS: siTRADD, siTRAF2, siRIP1 and siTAK1 significantly decreased TNF-α-induced IL-1α, IL-1ß, IL-6, IL-8, MCP-1 mRNA expression and release of IL-6, IL-8 and MCP-1; and cyclooxygenase (COX)-2 expression and release of prostaglandin PGF2α . There was a significant attenuation of TNF-α-induced expression of adhesion molecules ICAM-1 and VCAM-1 mRNA with siTRADD, siTRAF2 or siRIP1. siTRADD and siRIP1 significantly attenuated TNF-α-induced MMP-9 mRNA expression and release and nuclear factor κB (NF-κB) transcriptional activity. There was a significant increase in TNF-α-induced sVCAM-1 release, MMP-9 mRNA expression and NF-κB activity with siTAK1. CONCLUSION: TRADD, TRAF2, RIP1 and TAK1 are involved in TNF-α signalling in human myometrium. Further studies are required to determine whether inhibition of these proteins can prevent preterm birth.