Necrosis Factor Receptor-associated Death Research Articles

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.

The ORF3 Protein of Genotype 1 Hepatitis E Virus Suppresses TLR3-induced NF-κB Signaling via TRADD and RIP1.

Hepatitis E virus (HEV) genotype 1 infection is common and can emerge as outbreaks in developing areas, thus posing a threat to public health. However, due to the absence of feasible animal models, the mechanism of HE pathogenesis remains obscure. The HEV pathogenic mechanism has been suggested to be mediated by the immune system and not by direct viral duplication. We firstly discovered that the open reading frame 3 (ORF3) protein of genotype 1 HEV downregulates TLR3-mediated NF-κB signaling in Human A549 Lung Epithelial Cells (A549 cells) which were exposed to different TLR agonists associated with viral nucleic acids. Additionally, we identified the P2 domain of ORF3 as being responsible for this inhibition. Intriguingly, tumor necrosis factor receptor 1-associated death domain protein (TRADD) expression and receptor-interacting protein kinase 1 (RIP1) K63-ubiquitination were reduced in the presence of both ORF3 and Poly(I:C). Furthermore, we found that Lys377 of RIP1 acts as the functional ubiquitination site for ORF3-associated inhibition. Overall, we found that ORF3 protein downregulates TLR3-mediated NF-κB signaling via TRADD and RIP1. Our findings provide a new perspective on the cellular response in HEV infection and expand our understanding of the molecular mechanisms of HEV pathogenesis in innate immunity.

N-terminal and C-terminal domains of calmodulin mediate FADD and TRADD interaction.

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.

Outer membrane proteins of wild-type and intimin-deficient enteropathogenic Escherichia coli induce Hep-2 cell death through intrinsic and extrinsic pathways of apoptosis

Enteropathogenic Escherichia coli cause protracted diarrhoea and malnutrition in infants by cytoskeletal depolymerisation and effacement of enterocyte microvilli. In this study, outer membrane proteins of wild-type enteropathogenic E. coli and an intimin-deficient mutant are shown to induce apoptosis by up-regulation of tumour necrosis factor α and activation of c-jun N-terminal kinase. Fluorescence-activated cell sorter analysis revealed apoptosis of cells treated with outer membrane proteins of wild-type and intimin-deficient strains. Proteinase K treatment of outer membrane proteins reduced apoptosis significantly, as did neutralising tumour necrosis factor α with specific antibodies. Elevated tumour necrosis factor receptor 1-associated death domain and caspase-3 expression were also observed on treatment with both types of outer membrane proteins. Furthermore, apoptosis was associated with suppression of Bcl-2 protein expression, up-regulation of Bax mRNA levels and increased cytochrome c release from mitochondria. Elevated phospho-c-jun N-terminal kinase, c-jun mRNA and activator protein-1 expression were observed, and phosphorylation of activator protein-1 was also observed by DNA-binding assays. Inhibition of c-jun N-terminal kinase, but not inhibition of p38 mitogen-activated protein kinase, resulted in reduction of tumour necrosis factor α mRNA levels and caspase-3 protein levels, and a reduction in apoptosis as observed by fluorescence-activated cell sorter analysis. From the host point of view, this study suggests a possible interplay between the death receptor and mitochondrial pathways when cell-free bacterial outer membrane preparations are used to trigger apoptosis.

Beyond tumor necrosis factor receptor: TRADD signaling in toll-like receptors

Tumor necrosis factor receptor 1-associated death domain protein (TRADD) is the core adaptor recruited to TNF receptor 1 (TNFR1) upon TNFalpha stimulation. In cells from TRADD-deficient mice, TNFalpha-mediated apoptosis and TNFalpha-stimulated NF-kappaB, JNK, and ERK activation are defective. TRADD is also important for germinal center formation, DR3-mediated costimulation of T cells, and TNFalpha-mediated inflammatory responses in vivo. TRADD deficiency does not enhance IFNgamma-induced signaling. Importantly, TRADD has a novel role in TLR3 and TLR4 signaling. TRADD participates in the TLR4 complex formed upon LPS stimulation, and TRADD-deficient macrophages show impaired cytokine production in response to TLR ligands in vitro. Thus, TRADD is a multifunctional protein crucial both for TNFR1 signaling and other signaling pathways relevant to immune responses.

Significant inhibition of TRAIL-mediated fibroblast-like synovial cell apoptosis by IFN-γ through JAK/STAT pathway by translational regulation

The pathway of interferon-gamma (IFN-gamma)-induced suppression in tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-mediated apoptosis of fibroblast-like synovial cells (FLS) was investigated. rTRAIL triggered FLS apoptosis in a type II cell death manner, whereas IFN-gamma pretreatment significantly inhibited TRAIL-mediated apoptosis. As disruption of mitochondrial transmembrane potential (DeltaPsim), Leu-Glu-His-Asp ase (IETD ase) activity, and the appearance of hypodiploid DNA + cells were markedly suppressed in IFN-gamma-treated FLS in response to TRAIL, IFN-gamma-induced suppression was supposed to achieve at upstream of caspase-8. IFN-gamma rapidly phosphorylated signal transducers and activators of transcription 1 (STAT1), STAT3, and STAT6 as well as ERK, whereas enhanced neither phosphorylation of Akt nor nuclear translocation of nuclear factor kappaB (NF-kappaB) p65. Janus kinase (JAK)-induced phosphorylation of STAT1/3/6, which acts at translational regulation, seemed to be crucial because chemical inhibition of JAK as well as cycloheximide (CHX) abolished both the phosphorylation of STAT1/3/6 and the IFN-gamma-induced inhibitory effect. Although ERK was phosphorylated through IFN-gamma, chemical inhibition of ERK by PD98059 did not abolish the IFN-gamma-induced inhibitory effect. The authors tried to determine the responsible molecules; however, expression of TRAIL receptors; pro-caspase-3/-8/-9; Fas-associated death domain protein (FADD); tumor necrosis factor receptor 1-associated death domain protein (TRADD); silencer of death domain (SODD); FLICE inhibitory protein (FLIP); and Bcl-2, Bcl-xL, and Bax in FLS was not modulated by IFN-gamma. Although the authors have not yet clarified the precise mechanism, these data suggest that IFN-gamma/JAK/STAT pathway, which is supposed to be activated in inflammatory rheumatoid arthritis (RA) synovial tissues, contributes to form apoptosis resistance phenotype of the cells in situ, leading to a marked increase in cellularity of synovial cells.