scholarly journals Single-molecule imaging reveals the oligomeric state of functional TNFα-induced plasma membrane TNFR1 clusters in cells

2020 ◽  
Vol 13 (614) ◽  
pp. eaax5647 ◽  
Author(s):  
Christos Karathanasis ◽  
Juliane Medler ◽  
Franziska Fricke ◽  
Sonja Smith ◽  
Sebastian Malkusch ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Cancer Progression ◽  
Single Molecule ◽  
Tumor Necrosis Factor Receptor ◽  
Wild Type ◽  
Oligomeric State ◽  
Superresolution Microscopy ◽  
Ligand Free ◽  
Light Chain Enhancer ◽  
Necrosis Factor

Ligand-induced tumor necrosis factor receptor 1 (TNFR1) activation controls nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) signaling, cell proliferation, programmed cell death, and survival and is crucially involved in inflammation, autoimmune disorders, and cancer progression. Despite the relevance of TNFR1 clustering for signaling, oligomerization of ligand-free and ligand-activated TNFR1 remains controversial. At present, models range from ligand-independent receptor predimerization to ligand-induced oligomerization. Here, we used quantitative, single-molecule superresolution microscopy to study TNFR1 assembly directly in native cellular settings and at physiological cell surface abundance. In the absence of its ligand TNFα, TNFR1 assembled into monomeric and dimeric receptor units. Upon binding of TNFα, TNFR1 clustered predominantly not only into trimers but also into higher-order oligomers. A functional mutation in the preligand assembly domain of TNFR1 resulted in only monomeric TNFR1, which exhibited impaired ligand binding. In contrast, a form of TNFR1 with a mutation in the ligand-binding CRD2 subdomain retained the monomer-to-dimer ratio of the unliganded wild-type TNFR1 but exhibited no ligand binding. These results underscore the importance of ligand-independent TNFR1 dimerization in NF-κB signaling.

scholarly journals Identification of Amino Acid Residues Important for Ligand Binding to Fas

1997 ◽  
Vol 185 (8) ◽  
pp. 1487-1492 ◽  
Author(s):  
Gary C. Starling ◽  
Jürgen Bajorath ◽  
John Emswiler ◽  
Jeffrey A. Ledbetter ◽  
Alejandro Aruffo ◽  
...  
Keyword(s):  
Amino Acid ◽  
Immune Responses ◽  
Ligand Binding ◽  
Fusion Proteins ◽  
Molecular Model ◽  
Tumor Necrosis Factor Receptor ◽  
Jurkat Cells ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Necrosis Factor

The interaction of Fas (CD95), a member of the tumor necrosis factor receptor (TNFR) family, and its ligand (FasL) triggers programmed cell death (apoptosis) and is involved in the regulation of immune responses. Although the Fas–FasL interaction is conserved across species barriers, little is currently known about the molecular details of this interaction. Our aim was to identify residues in Fas that are important for ligand binding. With the aid of a Fas molecular model, candidate amino acid residues were selected in the Fas extracellular domain 2 (D2) and D3 and subjected to serine-scanning mutagenesis to produce mutant Fas molecules in the form of Ig fusion proteins. The effects of these mutations on FasL binding was examined by measuring the ability of these proteins to inhibit FasL-mediated apoptosis of Jurkat cells and bind FasL in ELISA and BIAcore™ assays. Mutation of two amino acids, R86 and R87 (D2), to serine totally abolished the ability of Fas to interact with its ligand, whereas mutants K84S, L90S, E93S (D2), or H126S (D3) showed reduced binding compared with wild-type Fas. Two mutants (K78S and H95S) bound FasL comparably to wild type. Therefore, the binding of FasL involves residues in two domains that correspond to positions critical for ligand binding in other family members (TNFR and CD40) but are conserved between murine and human Fas.

scholarly journals Role of TNFR1 in the innate airway hyperresponsiveness of obese mice

2012 ◽  
Vol 113 (9) ◽  
pp. 1476-1485 ◽  
Author(s):  
Ming Zhu ◽  
Alison S. Williams ◽  
Lucas Chen ◽  
Allison P. Wurmbrand ◽  
Erin S. Williams ◽  
...  
Keyword(s):  
Airway Hyperresponsiveness ◽  
Bronchoalveolar Lavage Fluid ◽  
Pulmonary Inflammation ◽  
Tumor Necrosis Factor Receptor ◽  
Lavage Fluid ◽  
Forced Oscillation Technique ◽  
Obese Mice ◽  
Wild Type ◽  
Necrosis Factor

The purpose of this study was to examine the role of tumor necrosis factor receptor 1 (TNFR1) in the airway hyperresponsiveness characteristic of obese mice. Airway responsiveness to intravenous methacholine was measured using the forced oscillation technique in obese Cpe fat mice that were either sufficient or genetically deficient in TNFR1 ( Cpe fat and Cpe fat/TNFR1−/− mice) and in lean mice that were either sufficient or genetically deficient in TNFR1 [wild-type (WT) and TNFR1−/− mice]. Compared with lean WT mice, Cpe fat mice exhibited airway hyperresponsiveness. Airway hyperresponsives was also greater in Cpe fat/TNFR1−/− than in Cpe fat mice. Compared with WT mice, Cpe fat mice had increases in bronchoalveolar lavage fluid concentrations of several inflammatory moieties including eotaxin, IL-9, IP-10, KC, MIG, and VEGF. These factors were also significantly elevated in Cpe fat/TNFR1−/− vs. TNFR1−/− mice. Additional moieties including IL-13 were also elevated in Cpe fat/TNFR1−/− vs. TNFR1−/− mice but not in Cpe fat vs. WT mice. IL-17A mRNA expression was greater in Cpe fat/TNFR1−/− vs. Cpe fat mice and in TNFR1−/− vs. WT mice. Analysis of serum indicated that obesity resulted in systemic as well as pulmonary inflammation, but TNFR1 deficiency had little effect on this systemic inflammation. Our results indicate that TNFR1 is protective against the airway hyperresponsiveness associated with obesity and suggest that effects on pulmonary inflammation may be contributing to this protection.

scholarly journals Apparently Normal Tumor Necrosis Factor Receptor 1 Signaling in the Absence of the Silencer of Death Domains

2003 ◽  
Vol 23 (18) ◽  
pp. 6609-6617 ◽  
Author(s):  
Robert Endres ◽  
Georg Häcker ◽  
Inge Brosch ◽  
Klaus Pfeffer
Keyword(s):  
Tumor Necrosis Factor ◽  
Tumor Necrosis ◽  
Tumor Necrosis Factor Receptor ◽  
Death Receptor ◽  
High Dose ◽  
Wild Type ◽  
Induced Apoptosis ◽  
Necrosis Factor

ABSTRACT The silencer of death domains (SODD) has been proposed to prevent constitutive signaling of tumor necrosis factor receptor 1 (TNFR1) in the absence of ligand. Besides TNFR1, death receptor 3 (DR3), Hsp70/Hsc70, and Bcl-2 have been characterized as binding partners of SODD. In order to investigate the in vivo role of SODD, we generated mice congenitally deficient in expression of the sodd gene. No spontaneous inflammatory infiltrations were observed in any organ of these mice. Consistent with this finding, in the absence of SODD no alteration in the activation patterns of nuclear factor κB (NF-κB), stress kinases, or ERK1 or -2 was observed after stimulation with tumor necrosis factor (TNF). Activation of NF-κB by DR3 was also unchanged. The extents of DR3- and TNF-induced apoptosis were comparable in gene-deficient and wild-type cells. Protection of cells against heat shock as mediated by the Hsp70 system and against staurosporine-induced apoptosis was independent of SODD. Furthermore, resistance to high-dose lipopolysaccharide (LPS) injections, LPS-d-GalN injections, and infection with listeriae was similar in wild-type and gene-deficient mice. In conclusion, our data do not support the concept of a unique, nonredundant role of SODD for the functions of TNFR1, Hsp70, and DR3.

scholarly journals Role of Flagellin and the Two-Component CheA/CheY System of Listeria monocytogenes in Host Cell Invasion and Virulence

2004 ◽  
Vol 72 (6) ◽  
pp. 3237-3244 ◽  
Author(s):  
Lone Dons ◽  
Emma Eriksson ◽  
Yuxuan Jin ◽  
Martin E. Rottenberg ◽  
Krister Kristensson ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Wild Type Strain ◽  
Tumor Necrosis Factor Receptor ◽  
Soft Agar ◽  
Initial Contact ◽  
Wild Type ◽  
Chemotaxis Proteins ◽  
Deficient Mice ◽  
Necrosis Factor

ABSTRACT The flagellum protein flagellin of Listeria monocytogenes is encoded by the flaA gene. Immediately downstream of flaA, two genes, cheY and cheA, encoding products with homology to chemotaxis proteins of other bacteria, are located. In this study we constructed deletion mutants with mutations in flaA. cheY, and cheA to elucidate their role in the biology of infection with L. monocytogenes. The ΔcheY, ΔcheA, and double-mutant ΔcheYA mutants, but not ΔflaA mutant, were motile in liquid media. However, the ΔcheA mutant had impaired swarming and the ΔcheY and ΔcheYA mutants were unable to swarm on soft agar plates, suggesting that cheY and cheA genes encode proteins involved in chemotaxis. The ΔflaA, ΔcheY, ΔcheA, and ΔcheYA mutants (grown at 24°C) showed reduced association with and invasion of Caco-2 cells compared to the wild-type strain. However, spleens from intragastrically infected BALB/c and C57BL/6 mice showed larger and similar numbers of the ΔflaA and ΔcheYA mutants, respectively, compared to the wild-type controls. Such a discrepancy could be explained by the fact that tumor necrosis factor receptor p55 deficient mice showed dramatically exacerbated susceptibility to the wild-type but unchanged or only slightly increased levels of the ΔflaA or ΔcheYA mutant. In summary, we show that listerial flaA. cheY, and cheA gene products facilitate the initial contact with epithelial cells and contribute to effective invasion but that flaA could also be involved in the triggering of immune responses.

scholarly journals Reduced Tumor Necrosis Factor Receptor–Associated Death Domain Expression Is Associated with Prostate Cancer Progression

2009 ◽  
Vol 69 (24) ◽  
pp. 9448-9456 ◽  
Author(s):  
Diping Wang ◽  
R. Bruce Montgomery ◽  
Lucy J. Schmidt ◽  
Elahe A. Mostaghel ◽  
Haojie Huang ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Tumor Necrosis Factor ◽  
Cancer Progression ◽  
Tumor Necrosis ◽  
Tumor Necrosis Factor Receptor ◽  
Death Domain ◽  
Prostate Cancer Progression ◽  
Necrosis Factor

scholarly journals Essential Roles of Receptor-Interacting Protein and TRAF2 in Oxidative Stress-Induced Cell Death

2004 ◽  
Vol 24 (13) ◽  
pp. 5914-5922 ◽  
Author(s):  
Han-Ming Shen ◽  
Yong Lin ◽  
Swati Choksi ◽  
Jamie Tran ◽  
Tian Jin ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Death ◽  
Tumor Necrosis Factor Receptor ◽  
Membrane Lipid ◽  
Critical Event ◽  
Wild Type ◽  
Embryonic Fibroblasts ◽  
Interacting Protein ◽  
Necrosis Factor

ABSTRACT Oxidative stress and reactive oxygen species (ROS) can elicit and modulate various physiological and pathological processes, including cell death. However, the mechanisms controlling ROS-induced cell death are largely unknown. Data from this study suggest that receptor-interacting protein (RIP) and tumor necrosis factor receptor (TNFR)-associated factor 2 (TRAF2), two key effector molecules of TNF signaling, are essential for ROS-induced cell death. We found that RIP−/− or TRAF2−/− mouse embryonic fibroblasts (MEF) are resistant to ROS-induced cell death when compared to wild-type cells, and reconstitution of RIP and TRAF2 gene expression in their respective deficient MEF cells restored their sensitivity to H2O2-induced cell death. We also found that RIP and TRAF2 form a complex upon H2O2 exposure, but without the participation of TNFR1. The colocalization of RIP with a membrane lipid raft marker revealed a possible role of lipid rafts in the transduction of cell death signal initiated by H2O2. Finally, our results demonstrate that activation of c-Jun NH2-terminal kinase 1 is a critical event downstream of RIP and TRAF2 in mediating ROS-induced cell death. Therefore, our study uncovers a novel signaling pathway regulating oxidative stress-induced cell death.

scholarly journals Polymorphism on human aromatase affects protein dynamics and substrate binding: spectroscopic evidence

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Giovanna Di Nardo ◽  
Almerinda Di Venere ◽  
Chao Zhang ◽  
Eleonora Nicolai ◽  
Silvia Castrignanò ◽  
...  
Keyword(s):  
Amino Acid ◽  
Ligand Binding ◽  
Exchange Rates ◽  
Protein Dynamics ◽  
Substrate Binding ◽  
Single Amino Acid ◽  
Aromatase Activity ◽  
Wild Type ◽  
Ligand Free ◽  
Human Aromatase

AbstractHuman aromatase is a member of the cytochrome P450 superfamily, involved in steroid hormones biosynthesis. In particular, it converts androgen into estrogens being therefore responsible for the correct sex steroids balance. Due to its capacity in producing estrogens it has also been considered as a promising target for breast cancer therapy. Two single-nucleotide polymorphisms (R264C and R264H) have been shown to alter aromatase activity and they have been associated to an increased or decreased risk for estrogen-dependent pathologies. Here, the effect of these mutations on the protein dynamics is investigated by UV/FTIR and time resolved fluorescence spectroscopy. H/D exchange rates were measured by FTIR for the three proteins in the ligand-free, substrate- and inhibitor-bound forms and the data indicate that the wild-type enzyme undergoes a conformational change leading to a more compact tertiary structure upon substrate or inhibitor binding. Indeed, the H/D exchange rates are decreased when a ligand is present. In the variants, the exchange rates in the ligand-free and –bound forms are similar, indicating that a structural change is lacking, despite the single amino acid substitution is located in the peripheral shell of the protein molecule. Moreover, the fluorescence lifetimes data show that the quenching effect on tryptophan-224 observed upon ligand binding in the wild-type, is absent in both variants. Since this residue is located in the catalytic pocket, these findings suggest that substrate entrance and/or retention in the active site is partially compromised in both mutants. A contact network analysis demonstrates that the protein structure is organized in two main clusters, whose connectivity is altered by ligand binding, especially in correspondence of helix-G, where the amino acid substitutions occur. Our findings demonstrate that SNPs resulting in mutations on aromatase surface modify the protein flexibility that is required for substrate binding and catalysis. The cluster analysis provides a rationale for such effect, suggesting helix G as a possible target for aromatase inhibition.

scholarly journals Synergistic Effects of Nanomedicine Targeting TNFR2 and DNA Demethylation Inhibitor—An Opportunity for Cancer Treatment

Cells ◽  
2019 ◽  
Vol 9 (1) ◽  
pp. 33 ◽  
Author(s):  
Mohammad A. I. Al-Hatamleh ◽  
Engku Nur Syafirah E.A.R. ◽  
Jennifer C. Boer ◽  
Khalid Ferji ◽  
Jean-Luc Six ◽  
...  
Keyword(s):  
Cancer Treatment ◽  
Cancer Progression ◽  
Synergistic Effects ◽  
Tumor Necrosis Factor Receptor ◽  
Dna Demethylation ◽  
Cancer Models ◽  
Anti Cancer ◽  
Cancer Immunotherapeutic ◽  
Immunosuppressive Cells ◽  
Necrosis Factor

Tumor necrosis factor receptor 2 (TNFR2) is expressed on some tumor cells, such as myeloma, Hodgkin lymphoma, colon cancer and ovarian cancer, as well as immunosuppressive cells. There is increasingly evidence that TNFR2 expression in cancer microenvironment has significant implications in cancer progression, metastasis and immune evasion. Although nanomedicine has been extensively studied as a carrier of cancer immunotherapeutic agents, no study to date has investigated TNFR2-targeting nanomedicine in cancer treatment. From an epigenetic perspective, previous studies indicate that DNA demethylation might be responsible for high expressions of TNFR2 in cancer models. This perspective review discusses a novel therapeutic strategy based on nanomedicine that has the capacity to target TNFR2 along with inhibition of DNA demethylation. This approach may maximize the anti-cancer potential of nanomedicine-based immunotherapy and, consequently, markedly improve the outcomes of the management of patients with malignancy.

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