The structure of tumor necrosis factor-alpha at 2.6 A resolution. Implications for receptor binding

The three-dimensional structure of tumor necrosis factor (TNF-alpha), a protein hormone secreted by macrophages, has been determined at 2.6 A resolution by x-ray crystallography. Phases were determined by multiple isomorphous replacement using data collected from five heavy atom derivatives. The multiple isomorphous replacement phases were further improved by real space symmetry averaging, exploiting the noncrystallographic 3-fold symmetry of the TNF-alpha trimer. An atomic model corresponding to the known amino acid sequence of TNF-alpha was readily built into the electron density map calculated with these improved phases. The 17,350-dalton monomer forms an elongated, antiparallel beta-pleated sheet sandwich with a "jelly-roll" topology. Three monomers associate intimately about a 3-fold axis of symmetry to form a compact bell-shaped trimer. Examination of the model and comparison to known protein structures reveals striking structural homology to several viral coat proteins, particularly satellite tobacco necrosis virus. Locations of residues conserved between TNF-alpha and lymphotoxin (TNF-beta, a related cytokine known to bind to the same receptors as TNF-alpha) suggest that lymphotoxin, like TNF-alpha, binds to the receptor as a trimer and that the general site of interaction with the receptor is at the "base" of the trimer.     

Mutational analysis of structure--activity relationships in human tumor necrosis factor-alpha

To determine the region of human tumor necrosis factor-alpha (TNF-alpha), essential for cytotoxic activity against mouse L-M cells, single amino-acid-substituted TNF-alpha mutant proteins (muteins) were produced in Escherichia coli by protein engineering techniques. An expression plasmid for TNF-alpha was mutagenized by passage through an E. coli mutD5 mutator strain and by oligonucleotide-directed mutagenesis. Approximately 100 single amino-acid-substituted TNF-alpha muteins were produced and assayed for cytotoxic activity. The cytotoxic activities of purified TNF-alpha muteins, e.g. TNF-31T, -32Y, -82D, -85H, -115L, -141Y, -144K and -146E, were less than 1% of that of parent TNF-alpha. These results indicate that the integrity of at least four distinct regions of the TNF-alpha molecule is required for full biological activity. These regions are designated as follows: region I, from position 30 to 32; region II, from position 82 to 89; region III, from position 115 to 117; region IV, from position 141 to 146. In addition, TNF-141Y could not completely compete with parent TNF-alpha for binding to the receptor. This demonstrates that region IV, and at least aspartic acid at position 141, must be involved in the TNF receptor binding site.     

Molecular weight of recombinant human tumor necrosis factor-alpha

Recombinant DNA-derived human tumor necrosis factor-alpha from Escherichia coli was examined by equilibrium ultracentrifugation under conditions similar to those where gel filtration experiments suggested an oligomeric structure. Short-column equilibrium experiments at concentrations in the range 0.015-0.12% at pH 8.5 in 0.04 M Tris/Tris-HCl gave molecular weights corresponding to 3 times the sequence molecular weight both in the presence and absence of 0.1 M NaCl. Long (2.6 mm)-column experiments under the same solvent conditions indicated molecular weights of 51,900 +/- 900 in the absence of added NaCl and 52,600 +/- 700 in the presence of added 0.1 M NaCl. No evidence of any species other than the trimer was found.     

Lipopolysaccharide-induced inhibition of scavenger receptor expression in human monocyte-macrophages is mediated through tumor necrosis factor-alpha.

The exposure of mononuclear cells to LPS results in a variety of cellular alterations including changes in the expression of various membrane receptors. In human monocyte-macrophages the development of the scavenger receptor, which mediates the uptake and internalization of chemically modified proteins, was suppressed by 100 ng/ml of LPS, concomitant with a reduction in receptor mRNA. Removal of LPS from the media resulted in a rapid increase in scavenger-receptor activity and mRNA that was further enhanced by macrophage-CSF and granulocyte/macrophage-CSF. However, neither macrophage-CSF nor granulocyte/macrophage-CSF could overcome the suppression of scavenger-receptor activity in the presence of LPS. The LPS-induced suppression of the scavenger receptor could be overcome by the co-addition of neutralizing antibody to TNF-alpha. TNF-alpha added to human monocyte-macrophages in the absence of LPS suppressed scavenger receptor activity to the same extent as did LPS. In contrast, the co-addition of LPS and neutralizing antibodies to either IFN-gamma or to IL-1 beta did not overcome the inhibitory effects of LPS on scavenger receptor activity. We conclude that the LPS-induced suppression of the scavenger receptor is mediated primarily through TNF-alpha.     

Tumor-stimulated release of tumor necrosis factor-alpha by human monocyte-derived macrophages.

Tumor necrosis factor-alpha (TNF) release by monocytes and macrophages may be an important determinant of the physiologic response of the host to neoplastic disease; however, the mechanisms which regulate TNF release by macrophages in hosts with neoplastic diseases are poorly understood. The purpose of this study was to determine if cell membranes and growth medium from human leukemia cell lines and solid tumor cell lines induced TNF release by cultured human blood monocyte-derived macrophages. The capacity for TNF release and direct tumor killing was highest in monocytes cultured for 7 to 11 days. Cell membranes and culture media from K562 erythroleukemia and several small cell lung carcinoma cell lines, including H82, induced the release of up to 1500 TNF units per 10(6) macrophages over 24 hr. By contrast, allogeneic peripheral blood lymphocytes, cell membranes from normal mixed donor peripheral blood leukocytes, or growth medium from normal embryonic lung fibroblasts induced the release of little or no TNF during culture up to 24 hr, suggesting that this macrophage response was specific for tumor cells. Release of TNF by tumor-stimulated macrophages was gradual, peaking 24 hr following the addition of stimuli. Induction of macrophage TNF release was concentration dependent, with half-maximal TNF levels induced by 12.5 and 25 micrograms/ml cell membranes prepared from K562 and H82, respectively. Pretreatment of tumor cell membranes with polymixin B, which inhibits many of the actions of endotoxin, failed to neutralize tumor induction of TNF, suggesting that endotoxin was not responsible for this activity. Depletion of macrophages by treatment with 3C10 monoclonal antibody and complement abrogated tumor-induced TNF release, indicating that macrophages were the source of the secreted TNF. HPLC analysis of H82 growth medium demonstrated a single peak of macrophage activating activity with approximate 40-kDa molecular weight. We have demonstrated that cell membranes and growth medium from some human leukemia and solid tumor cell lines, but not from normal human cells, induce human peripheral blood monocytes and monocyte-derived macrophages to release functionally active TNF. This process may contribute to the host response to some neoplastic diseases.     

Identification of the binding site of 55kDa tumor necrosis factor receptor by synthetic peptides.

We have synthesized a series of peptides, which cover almost the whole range of the N-terminal extracellular domain of human 55kDa TNF receptor (55kDa TNF-R). The peptides were examined for the binding activity to TNF by solid phase binding assay and for the inhibition of TNF cytotoxicity to mouse L-M cells. The peptide 159-178 exhibited remarkably higher binding activity to TNF than other peptides did. The specificity of the TNF binding to the peptides was confirmed by their inability to bind other cytokines. The peptide 159-178 also inhibited TNF cytotoxicity. These results indicate that the specific binding site of 55kDa TNF-R to TNF might reside within the peptide segment of amino acid numbers 159 to 178 in the N-terminal extracellular domain.     

Localization of the active site of human tumour necrosis factor (hTNF) by mutational analysis.

In order to define the active site(s) of human tumour necrosis factor (hTNF), we mutagenized its gene at random and directly screened the resulting population for loss of cytotoxic activity on L929 cells. Four biologically inactive mutant proteins (Arg32----Trp, Leu36----Phe, Ser86----Phe and Ala84----Val) behaved similar to the wild-type in various physico-chemical assays. The residues were positioned on a 3D structural model and were found to cluster together at the base of the molecule at each side of the groove that separates two monomers in the trimeric structure. A very conservative mutation at one of these sites (Ala84----Val) almost completely abolished cytotoxic activity. Amino acid alterations in three other residues in close proximity to this receptor binding site were introduced: replacements at positions 29 and 146 clearly reduced cytotoxicity only when non-conservative alterations were introduced (Leu29----Ser and Glu146----Lys), suggesting an indirect influence on the active site. However, a conservative mutation at position 91 (Val----Ala) caused a significant drop (500-fold) in bioactivity which suggests that Val91 may also play a direct role in receptor recognition. Our results favor a model in which each TNF molecule has three receptor-interaction sites (between the three subunits), thus allowing signal transmission by receptor clustering.     

Human tumor necrosis factor-alpha receptor. Purification by immunoaffinity chromatography and initial characterization

The receptor for human tumor necrosis factor-alpha (TNF-alpha) was isolated from a subclone of the human histiocytic lymphoma cell line U937. These cells exhibit a single class of high affinity receptors (Kd = 0.51 +/- 0.25 nM) with an average density of 55,000 +/- 5,000 binding sites/cell. After solubilization with detergent, the receptor retained its ability to bind free TNF-alpha but failed to bind to TNF-alpha immobilized on various solid supports. For receptor purification, 125I-TNF-alpha was covalently attached to the receptor on intact cells by the bifunctional cross-linking reagents ethylene glycolbis(succinimidylsuccinate) or 3,3-dithiobis(sulfosuccinimidylpropionate). The cells were then solubilized with the nonionic detergent Triton X-100, and the supernatants, clarified by centrifugation, were passed over an IgG-Sepharose column prepared from TNF-alpha antiserum. The receptor-rich fraction from the antibody column was further purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These two steps together provided approximately 165,000-fold purification of the TNF-alpha receptor. The TNF-alpha receptor-ligand complex obtained by this method had a subunit molecular weight of 100,000 +/- 5,000 when examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis but on gel filtration the complex migrated with an apparent molecular weight of 480,000 +/- 32,000. However, the receptor showed a molecular weight of 65,000 +/- 32,000 when gel filtration was performed in the absence of ligand. Additional characteristics of the receptor are discussed.     

Recycling of tumor necrosis factor-alpha receptor in MCF-7 cells

Kinetics of regulation of membrane receptors for tumor necrosis factor-alpha (TNF) in human breast adenocarcinoma MCF-7 cells was investigated. When MCF-7 cells were incubated with radioiodinated human recombinant TNF, they bound TNF specifically and accumulated it intracellularly. Preincubation of cells with native TNF up to 1 x 10(-9) M for 12 h stimulated specific binding of TNF, indicating that concentrations of membrane receptors for TNF were regulated by the ligand. Accumulation of radioactivity in cells incubated with [125I]TNF proceeded at a constant rate for up to 24 h. Kinetics of binding and internalization of TNF were similar in the presence and absence of protein synthesis for at least 1 h, suggesting that the TNF receptor was either replenished from an intracellular pool of receptors or was recycled (reutilized) during the course of TNF internalization. Data were analyzed kinetically by fitting equations of compartmental models of ligand-cell interactions with and without the term for receptor recycling. Fits were obtained only for the model with receptor recycling; absence of the term for receptor recycling resulted in physically impossible best-fit parameter values. Analysis of the best-fit parameters indicated that both internalization and recycling of the receptor were stimulated by the ligand.     

Site-directed mutational analysis of active site residues in the acetate kinase from Methanosarcina thermophila

Acetate kinase catalyzes the magnesium-dependent transfer of the gamma-phosphate of ATP to acetate. The recently determined crystal structure of the Methanosarcina thermophila enzyme identifies it as a member of the sugar kinase/Hsc70/actin superfamily based on the fold and the presence of five putative nucleotide and metal binding motifs that characterize the superfamily. Residues from four of these motifs in M. thermophila acetate kinase were selected for site-directed replacement and analysis of the variants. Replacement of Asp(148) and Asn(7) resulted in variants with catalytic efficiencies less than 1% of that of the wild-type enzyme, indicating that these residues are essential for activity. Glu(384) was also found to be essential for catalysis. A 30-fold increase in the magnesium concentration required for half-maximal activity of the E384A variant relative to that of the wild type implicated Glu(384) in magnesium binding. The kinetic analysis of variants and structural data is consistent with nonessential roles for active site residues Ser(10), Ser(12), and Lys(14) in catalysis. The results are discussed with respect to the acetate kinase catalytic mechanism and the relationship to other sugar kinase/Hsc70/actin superfamily members.     

The molecular action of tumor necrosis factor-alpha.

Tumor necrosis factor-alpha (TNF-alpha) is a polypeptide hormone newly synthesized by different cell types upon stimulation with endotoxin, inflammatory mediators (C5a anaphylatoxin), or cytokines such as interleukin-1 and, in an autocrine manner, TNF itself. The net biological effect of TNF-alpha may vary depending on relative concentration, duration of cell exposure and presence of other mediators which may act in synergism with this cytokine. TNF-alpha may be relevant either in pathological events occurring in cachexia and endotoxic shock and inflammation or in beneficial processes such as host defense, immunity and tissue homeostasis. The biological effects of TNF-alpha are triggered by the binding to specific cell surface receptors. The formation of TNF-alpha-receptor complex activates a variety of biochemical pathways that include the transduction of the signal at least in part controlled by guanine-nucleotide-binding regulatory proteins (G proteins), its amplification through activation of adenyl cyclase, phospholipases and protein kinases with the generation of second messenger pathways. The transduction of selected genes in different cell types determines the characteristics of the cell response to TNF-alpha. The full understanding of the molecular mechanisms of TNF-alpha will provide the basis for a pharmacological approach intended to inhibit or potentiate selected biological actions of this cytokine.     

Screening of the human tumor necrosis factor-alpha (TNF-alpha) gene promoter polymorphisms by PCR-DGGE analysis.

We have designed a new PCR-DGGE technique that enables detection of base changes in the TNF-alpha gene promoter. Screening of 130 samples from Spanish children has shown that this technique accurately detects the altered band patterns induced by the presence of the polymorphisms at positions -376, -308, -238 and -163 of the promoter sequence. Although further analysis are needed to fully characterise the alterations detected, we believe that this PCR-DGGE technique is a rapid and sensitive first approach to the genetic characterisation of the TNF-alpha promoter.     

Role of single disulfide in recombinant human tumor necrosis factor-alpha

Two analogs of tumor necrosis factor-alpha (TNF-alpha) were produced by in vitro site-directed mutagenesis. In these analogs, cysteine residues at positions 69 and 101, which form a disulfide bond, were changed to alanine or leucine. CD spectra showed that the analogs are apparently similar in secondary and tertiary structure to the natural sequence TNF-alpha. In addition, the molecular size of the analogs was identical to that of the natural sequence TNF-alpha as determined by gel filtration. However, fluorescence spectra and quenching indicated that the removal of the disulfide bond alters the local conformation around tryptophan residues. The cytolytic, macrophage activation, and lipogenic activities decreased in the order of the natural sequence TNF-alpha greater than the alanine analog greater than the leucine analog, suggesting that the surface involving the disulfide bond plays a role in these biological functions and the introduced modifications decrease the activity. Differential effect of the modifications was suggested in the antiviral activity, since in this assay only the leucine analog showed significantly lower activity.     

Characterization of a recombinant extracellular domain of the type 1 tumor necrosis factor receptor: evidence for tumor necrosis factor-alpha induced receptor aggregation.

An expression plasmid encoding the extracellular portion of the human tumor necrosis factor (TNF) type 1 receptor (TNF-R1) was constructed and used to generate a stable cell line secreting soluble TNF-R1 (sTNF-R1). The sTNF-R1 was purified, and its biochemical properties and its interactions with human TNF-alpha were examined. SDS-PAGE resolved the purified sTNF-R1 into three bands of approximate Mr 24,200, 28,200, and 32,800. Sedimentation equilibrium analysis gave a molecular weight of 25,000 for sTNF-R1 whereas the molecular weight obtained by gel filtration chromatography was approximately 55,000-60,000. Scatchard analysis of [125I]TNF-alpha binding to sTNF-R1 revealed high-affinity binding (Kd = 93 pM), comparable to that observed for the intact receptor on whole cells. Competitive binding experiments showed that sTNF-R1 has a 50-60-fold higher affinity for TNF-alpha than for TNF-beta, in contrast to the equal affinities of TNF-alpha and TNF-beta for the full-length TNF-R1 transiently expressed in mammalian cells. The sTNF-R1 was found to block the cytotoxicity of TNF-alpha and TNF-beta on a murine L-M cell assay. The sizes of the sTNF-R1.TNF-alpha complex determined by gel filtration chromatography and sedimentation equilibrium were approximately 141 and 115 kDa, respectively. The stoichiometry of the complex was examined by Scatchard analysis, size-exclusion chromatography, HPLC separation, amino acid composition, sequence analysis, and sedimentation equilibrium. The data from these studies suggest that at least two molecules of sTNF-R1 can bind to a single TNF-alpha trimer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serotonin inhibition of tumor necrosis factor-alpha synthesis by human monocytes

Serotonin inhibited in a concentration dependent way (10(-3) M to 10(-10) M) the LPS induced Tumor Necrosis Factor-alpha synthesis both, when added to the monocyte cultures from the beginning and when added together with the activating stimulus 8 hours before the end of the culture. The inhibitory effect was specifically blocked by the 5-HT1 and 5-HT2 serotonin antagonist methysergide and the 5-HT2 receptor antagonist ketanserin. This indicates that only the 5-HT2 receptor family (5-HT2 or 5-HT1C) may be involved in the inhibitory effect. Serotonin seems to play an important immunomodulatory role in macrophage functions.     

The actin binding site of thymosin beta 4 mapped by mutational analysis.

We characterized in detail the actin binding site of the small actin-sequestering protein thymosin beta 4 (T beta 4) using chemically synthesized full-length T beta 4 variants. The N-terminal part (residues 1-16) and a hexapeptide motif (residues 17-22) form separate structural entities. In both, we identified charged and hydrophobic residues that participate in the actin interaction using chemical cross-linking, complex formation in native gels and actin-sequestering experiments. Quantitative data on the activity of the variants and circular dichroism experiments allow to present a model in which the N-terminal part needs to adopt an alpha-helix for actin binding and interacts through a patch of hydrophobic residues (6M-I-F12) on one side of this helix. Also, electrostatic contacts between actin and lysine residues 18, in the motif, and 14, in the N-terminal alpha-helix, appear important for binding. The residues critical for contacting actin are conserved throughout the beta-thymosin family and in addition to this we identify a similar pattern in the C-terminal headpiece of villin and dematin.     

Zymosan-stimulated tumor necrosis factor-alpha production by human monocytes. Down-modulation by phorbol ester.

In this study, we showed that human monocytes produced TNF-alpha in response to zymosan, a particulate agonist. Protein kinase C (PKC) seems to play a regulatory role in zymosan-induced TNF-alpha secretion. The pretreatment of monocytes with PMA induced a dose-dependent inhibition of zymosan-stimulated TNF production. This inhibition was likely due to an activation of PKC because it was prevented by inhibitors of PKC, sphingosine, and staurosporine. Moreover, PMA elicited a profound down-modulation of zymosan binding to monocytes. The inhibition of zymosan binding and TNF production displayed similar dose-dependence, suggesting that both events were closely related. In addition, PMA did not modify the expression of CD11b/CD18 receptor that is involved in zymosan recognition. In view of these findings, qualitative changes of CD11b/CD18 molecules might account for the inhibition of zymosan binding and TNF production. Thus, PMA specifically increased the association of CD11b/CD18 with the detergent-insoluble cytoskeleton. Cytochalasin B but not microtubule disrupters, nocodazole and colchicine, partially prevented the inhibition of zymosan binding. Hence, the inhibitory action of PMA on zymosan binding seems to be mediated by an increase in attachment of zymosan receptor to cytoskeleton and more likely to microfilaments. The regulatory activity of PKC might represent a first way of limiting cytokine over-production in response to pathogens which interact with monocytes via CD11/CD18 molecules.     

Cytotoxic mechanism of tumor necrosis factor-alpha

Many intracellular pathways are set in motion by the binding of tumor necrosis factor (TNF) to its cell surface receptor. Major steps in the TNF-mediated cytotoxicity cascade include G protein-coupled activation of phospholipases, generation of free radicals, and damage to nuclear DNA by endonucleases. Ultimately the cells undergo apoptosis and die. Understanding how TNF initiates these pathways will facilitate the rational design of pharmaceuticals that can attenuate or potentiate the action of this important cytokine.