Tumour necrosis factor is a compact trimer

Recombinant produced human tumour necrosis factor (TNF) has been studied to characterise the subunit structure of the protein. TNF is shown to be a trimer Mr 52000 in which the subunits are associated in a compact, triangular form. In secondary structure it belongs to the all-beta class of proteins. It has high thermodynamic stability and the unfolded subunits can fold and associate spontaneously to form native, biologically active TNF.     

Crystallization and preliminary X-ray investigation reveals that tumor necrosis factor is a compact trimer furnished with 3-fold symmetry

Crystals of recombinant human tumor necrosis factor produced by Escherichia coli have been obtained under different conditions. Crystals suitable for X-ray studies are produced by a vapor diffusion technique using sodium phosphate as both precipitant and buffer at pH 6.5. The crystals belong to the cubic space group, P2(1)3 with unit cell dimensions a = b = c = 95.7 A (1 A = 0.1 nm). Preliminary photography reveals that the crystals are moderately stable to X-rays and diffract to at least 3 A resolution. The diffraction data for native crystals have been collected on a diffractometer at 3 A resolution. Another crystal form, which appeared in a solution containing sodium phosphate at pH 8.0, has the trigonal space group P3 with unit cell dimensions a = b = 63.8 A and c = 54.4 A, and produces measurable reflections to a resolution of 3 A. Hexagonal crystals also have been obtained by the use of polyethylene glycol as precipitant in the range pH 7.6 to 8.0; however, the crystals are fragile and unstable to X-rays. Conservation of 3-fold symmetry in the different crystal forms obtained could reflect the ability of tumor necrosis factor molecules to form trimers in solution and probably the nature of binding of the molecules to cellular receptors.     

Retroviral restriction factor TRIM5alpha is a trimer

The retrovirus restriction factor TRIM5alpha targets the viral capsid soon after entry. Here we show that the TRIM5alpha protein oligomerizes into trimers. The TRIM5alpha coiled-coil and B30.2(SPRY) domains make important contributions to the formation and/or stability of the trimers. A functionally defective TRIM5alpha mutant with the RING and B-box 2 domains deleted can form heterotrimers with wild-type TRIM5alpha, accounting for the observed dominant-negative activity of the mutant protein. Trimerization potentially allows TRIM5alpha to interact with threefold pseudosymmetrical structures on retroviral capsids.     

The tumor necrosis factor family member LIGHT is a target for asthmatic airway remodeling

Individuals with chronic asthma show a progressive decline in lung function that is thought to be due to structural remodeling of the airways characterized by subepithelial fibrosis and smooth muscle hyperplasia. Here we show that the tumor necrosis factor (TNF) family member LIGHT is expressed on lung inflammatory cells after allergen exposure. Pharmacological inhibition of LIGHT using a fusion protein between the IgG Fc domain and lymphotoxin β receptor (LTβR) reduces lung fibrosis, smooth muscle hyperplasia and airway hyperresponsiveness in mouse models of chronic asthma, despite having little effect on airway eosinophilia. LIGHT-deficient mice also show a similar impairment in fibrosis and smooth muscle accumulation. Blockade of LIGHT suppresses expression of lung transforming growth factor-β (TGF-β) and interleukin-13 (IL-13), cytokines implicated in remodeling in humans, whereas exogenous administration of LIGHT to the airways induces fibrosis and smooth muscle hyperplasia, Thus, LIGHT may be targeted to prevent asthma-related airway remodeling.     

Selection of tumor cell variants for resistance to tumor necrosis factor also induces a form of pleiotropic drug resistance

Summary This study has addressed the question of whether there may be some common mechanism underlying the induction or expression of acquired cytokine and drug resistance in a tumor cell line. This study employed the tumor-necrosis-factor(TNF)-sensitive U937 tumor cell line as a model system to determine if selection of a tumor cell variant for cytokine resistance would also result in drug resistance and vice versa. Variants were selected by culturing in the presence of purified recombinant TNF or a mixed-lymphokine-containing supernatant derived from concanavalin-A-stimulated peripheral blood lymphocytes. The resulting variants were resistant not only to TNF, but also to certain chemotherapeutic drugs. The variants were most resistant to colchicine and theVinca alkaloids, requiring drug concentrations 50- to 5000-fold higher to mediate levels of cytotoxicity comparable to that seen with the parental U937. The variants were moderately resistant to cycloheximide, actinomycin D, and mitomycin C. In contrast, these lines were relatively sensitive to doxorubicin or daunomycin. This phenomenon was not unique to U937 cells since we obtained a similar pattern of drug resistance by selecting TNF-resistant variants of the WEHI-164 tumor cell line. The cytokine-selected U937 variants were still lysed by NK cells, although they were somewhat less sensitive than the parental U937. Both variants were relatively resistant to lysis by activated macrophages, probably because of their TNF resistance. In an alternative selection procedure, U937 variants were derived by culturing in the presence of increasing concentrations of colchicine. The resulting variants were relatively resistant to TNF, providing further support for the existence of some common mechanism operating in induction or expression of acquired cytokine and drug resistance. The resistance mechanism apparently does not involve the P glycoprotein since the cytokine-selected U937 variants do not overexpress the mdr gene. This study has demonstrated that selection of TNF-resistant variants results in coexpression of a unique form of drug resistance that is characterized by resistance to microtubule-active drugs but not to the anthracycline antibiotics and is not associated with overexpression of the mdr gene.This work was supported by grant CA 47 669-01 awarded by the National Cancer Institute Nomenclature of variants: U9-LKR, U937 variant selected by lymphokines; U9-TR, U937 variant selected by tumor necrosis factor (TNF); WEHI-TR, WEHI-164 variant selected by TNF     

Antibodies to a soluble form of a tumor necrosis factor (TNF) receptor have TNF-like activity.

Immunological cross-reactivity between tumor necrosis factor (TNF) binding proteins which are present in human urine (designated TBPI and TBPII) and two molecular species of the cell surface receptors for TNF is demonstrated. The two TNF receptors are shown to be immunologically distinct, to differ in molecular weight (58,000 and 73,000), and to be expressed differentially in different cells. It is further shown that polyclonal antibodies against one of the TNF binding proteins (TBPI) display, by virtue of their ability to bind the TNF receptor, activities which are very similar to those of TNF. These antibodies are cytotoxic to cells which are sensitive to TNF toxicity, induce resistance to TNF toxicity, enhance the incorporation of thymidine into normal fibroblasts, inhibit the growth of chlamydiae, and induce the synthesis of prostaglandin E2. Monovalent F(ab) fragments of the polyclonal antibodies lack TNF-like activities, but acquire them upon cross-linking with anti-F(ab)2 antibodies, suggesting that the ability of the anti-TBPI antibodies to mimic TNF correlates with their ability to cross-link the TNF receptors. This notion was further supported by data obtained in a comparative study of the TNF-like cytotoxicity of a panel of monoclonal antibodies against TBPI. The induction of TNF-like effects by antibodies to a TNF receptor suggests that TNF is not directly involved in intracellular signalling. Rather, it is the receptors to this cytokine which, when properly triggered in a process which appears to involve clustering of these receptors, transduce the signal for response to TNF into the cell's interior.     

TRAF1 is a substrate of caspases activated during tumor necrosis factor receptor-alpha-induced apoptosis

TRAF family proteins are signal-transducing adapter proteins that interact with the cytosolic domains of tumor necrosis factor (TNF) family receptors. Here we show that TRAF1 (but not TRAF2-6) is cleaved by certain caspases in vitro and during TNF-alpha- and Fas-induced apoptosis in vivo. (160)LEVD(163) was identified as the caspase cleavage site within TRAF1, generating two distinct fragments. Significant enhancement of TNF receptor-1 (CD120a)- and, to a lesser extent, Fas (CD95)-mediated apoptosis was observed when overexpressing the C-terminal TRAF1 fragment in HEK293T and HT1080 cells. The same fragment was capable of potently suppressing TNF receptor-1- and TRAF2-mediated nuclear factor-kappaB activation in reporter gene assays, providing a potential mechanism for the enhancement of TNF-mediated apoptosis. Cell death induced by other death receptor-independent stimuli such as cisplatin, staurosporine, and UV irradiation did not result in cleavage of TRAF1, and overexpression of the C-terminal TRAF1 fragment did not enhance cell death in these cases. TRAF1 cleavage was markedly reduced in cells that contain little procaspase-8 protein, suggesting that this apical protease in the TNF/Fas death receptor pathway is largely responsible. These data identify TRAF1 as a specific target of caspases activated during TNF- and Fas-induced apoptosis and illustrate differences in the repertoire of protease substrates cleaved during activation of different apoptotic pathways.     

Efficient purification of recombinant human tumor necrosis factor beta from Escherichia coli yields biologically active protein with a trimeric structure that binds to both tumor necrosis factor receptors.

A fast and efficient method for medium scale purification of recombinant human tumor necrosis factor beta (rTNF-beta) from Escherichia coli cells is described. The purified rTNF-beta displayed biological activity similar to rTNF-alpha in a WEHI 164 cell cytotoxicity assay. The titration curve of rTNF-beta and elution profiles of rTNF-beta in gel filtration experiments were different from those of rTNF-alpha. However, light scattering and ultra-centrifugation studies showed that both cytokines have trimeric structures in solution at 0.5 mg/ml, with minor differences in the distribution of nontrimeric species. rTNF-beta bound to purified 55- and 75-kDa TNF receptors with high affinity. The binding of rTNF-beta to either receptor was analyzed on Scatchard plots and compared with that of rTNF-alpha.     

Expression of synthetic human tumor necrosis factor is toxic to Escherichia coli

The overlap forward-primer-walk polymerase chain reaction method was used to synthesize the human tumor necrosis factor α (hTNF) gene in Escherichia coli cells. Growth curves for hTNF and pET23d vector cultures exhibited slower doubling rates than cultures containing the pET23d vector alone. Cell cultures transformed with hTNF reached peak densities (0.4-0.6 OD₆₀₀) 3 to 4 h post-induction, then decreased prior to growth recovery. This inhibition occurred in the BL21DE3 strain of E. coli, whereas no inhibition of growth and no expression of hTNF were observed in the JM109 strain of E. coli containing hTNF. Induced hTNF cultures hyperexpressed the hTNF-histidine fusion protein for the first 3 to 4 h of induction; subsequently, growth retardation was observed. Hyperexpression and continuous growth were observed in the extracellular expression system. Electron microscopy revealed that accumulation of hTNF inclusion bodies was apparent only in the intracellular expression system — no accumulation was observed with regard to the secretory system. The hTNF-pET23d vector was purified from cells expressing the fusion protein and from cells with recovered growth curves. Sequencing of the vector demonstrated the complete hTNF gene and T7 promoter in cells expressing the fusion protein and mutations of the T7 promoter site from recovered cells.     

Basic fibroblast growth factor is released from endothelial extracellular matrix in a biologically active form

Basic fibroblast growth factor (bFGF) binds to heparin-like molecules present in the extracellular matrix (ECM) of transformed fetal bovine aortic endothelial GM 7373 cells. Binding of bFGF to ECM can be competed by heparin or heparan sulfate, and ECM-bound bFGF can be released by treating the cells with heparinase or heparatinase. After binding to ECM, bFGF is slowly released into the medium in a biologically active form, as shown by its capacity to induce an increase of cell-associated plasminogen activator activity and cell proliferation. The increase is prevented upon removal of ECM-bound bFGF by a neutral 2 M NaCl wash. Soluble heparin and heparan sulfate reduce the amount of ECM-bound bFGF released into the medium, possibly competing with ECM polysaccharides for heparinase-like enzymes produced by endothelial cells, suggesting that these enzymes are involved in the mobilization of ECM-bound bFGF.     

Ubiquitination of RIP is required for tumor necrosis factor alpha-induced NF-kappaB activation

Stimulation of cells with tumor necrosis factor (TNFalpha) triggers a recruitment of various signaling molecules, such as RIP, to the TNFalpha receptor 1 complex, leading to activation of NF-kappaB. Previous studies indicate that RIP plays an essential role for TNFalpha-induced NF-kappaB activation, but the molecular mechanism by which RIP mediates TNFalpha signals to activate NF-kappaB is not fully defined. Earlier studies suggest that RIP undergoes a ligand-dependent ubiquitination. However, it remains to be determined whether the ubiquitination of RIP is required for TNFalpha-induced NF-kappaB activation. In this study, we have identified Lys377 of RIP as the functional ubiquitination site, because mutating this residue to arginine completely abolished RIP-mediated NF-kappaB activation. The K377R mutation of RIP cannot undergo ligand-dependent ubiquitination and fails to recruit its downstream signaling components into the TNFalpha receptor 1 complex. Together, our studies provide the first genetic evidence that the ubiquitination of RIP is required for TNFalpha-induced NF-kappaB activation.     

Capacity of tumor necrosis factor to bind and penetrate membranes is pH-dependent

Studies with human U937 cells as targets established that a 15-min exposure to rTNF at pH 5.3 caused a significant increase in TNF-mediated cytolysis when compared to cells exposed to TNF at pH 7.4. A detailed examination of TNF-membrane interactions revealed that although TNF bound avidly to model membrane targets, no damage was generated under any condition tested. Binding of TNF, monitored with 125I-labeled as well as unlabeled protein, was enhanced at low pH. In the pH range tested (i.e., 4 to 8), target membrane permeability actually decreased in the presence of TNF. This membrane stabilization may be a consequence of TNF insertion into the target bilayer, a process we detected through use of an intramembranous photolabeling assay; interestingly, the efficiency of TNF insertion into membranes increased dramatically with decreasing pH. We conclude that native TNF does not cause pore formation directly and that its ability to induce cell lysis, as monitored by 51Cr release, is a consequence of some as yet obscure signaling event or intracellular activity. Parallel studies were carried out with diphtheria toxin, a protein with a more thoroughly characterized pH-dependent intoxification pathway. This toxin displayed acid-enhanced activities with both biologic and artificial targets.     

Purification of rabbit tumor necrosis factor

Rabbit tumor necrosis factor (TNF) was purified and shown by SDS-PAGE to be a single protein of 18 kDa. TNF in 355 ml rabbit serum was precipitated with ammonium sulfate, and purified by repeated DEAE-Sephadex and Sephacryl S-200 chromatographies, and the final fractionation on Blue-Sepharose 6B. By this procedure its yield was 22% and its specific activity was 2.4 × 10⁷ U/mg protein. The sequence of the N-terminal 20 amino acids was determined.