Oligomeric tumour necrosis factor alpha slowly converts into inactive forms at bioactive levels.
ABSTRACT: The stability of oligomeric human tumour necrasis factor alpha (TNF) at bioactive levels has been studied by two immunoenzymatic assays: one able to specifically detect oligomeric and not monomeric TNF (O-e.l.i.s.a.) and the other able to detect both forms (OM-e.l.i.s.a.). The selectivity of O-e.l.i.s.a. and OM-e.l.i.s.a. for oligomeric and monomeric TNF was demonstrated with isolated forms prepared by partial dissociation of recombinant TNF with 10% (v/v) dimethyl sulphoxide and gel-filtration h.p.l.c. Evidence for instability of oligomeric TNF were obtained in physiological buffers, as well as in serum and cell-culture supernatants, as a function of TNF concentration. In particular, only a half of the TNF antigen was recovered in the oligomeric form after 72 h incubation (37 degrees C) at 0.12 nM, whereas no apparent dissociation was detected at 4 nM. The structural changes observed at picomolar concentrations were rapidly reversed by raising the concentration of TNF to about 2 nM by ultrafiltration, suggesting that subunit dissociation and reassociation reactions occur in the picomolar and nanomolar range respectively. The cytolytic activity of L-M cells correlates with oligomeric-TNF levels after incubation at picomolar concentrations. Moreover, isolated oligomeric TNF was cytotoxic towards L-M cells, whereas monomeric TNF was virtually inactive. In conclusion, the results suggest that bioactive oligomeric TNF is unstable at picomolar levels and slowly converts into inactive monomers, supporting the hypothesis that quaternary-structure changes in TNF may contribute to the fine regulation of TNF cytotoxicity.
Project description:We reported [Ransnäs, Svoboda, Jasper & Insel (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7900-7903] that in intact S49 lymphoma cells the beta-adrenergic-receptor agonist isoprenaline dissociates the stimulatory guanine-nucleotide-binding protein, Gs, into its alpha s and beta gamma subunits, leading to redistribution of alpha s from plasma membranes to the cytoplasm. In the present studies we investigated the kinetics of Gs dissociation and membrane release in plasma membranes from S49 lymphoma cells. We analysed cholate extracts of membranes for alpha s levels by a competitive e.l.i.s.a. with a polyclonal antibody that selectively recognizes monomeric alpha s and we assayed supernatant fractions using both competitive e.l.i.s.a. and immunoblotting. The plasma membranes contained 19.3 +/- 1.4 pmol of alpha s/mg of membrane protein and lacked significant dissociation of Gs and activity of adenylate cyclase in the absence of guanine nucleotides. Mg2+ ions were obligatorily required for isoprenaline-induced dissociation of Gs in plasma membranes and for membrane release of alpha s. At a physiological concentration of free Mg2+ ions (100 microM), 100 microM-GTP induced a slow first-order (k = 0.038 +/- 0.004 min-1) dissociation of 17.8 +/- 1.2 pmol of Gs/mg of membrane protein. A substantial increase in the dissociation rate of Gs was achieved by addition of 1 microM-isoprenaline and 100 microM-GTP; 18.4 +/- 0.9 pmol of Gs/mg of membrane protein was dissociated, with a kappa of 1.49 +/- 0.22 min-1. The effect of isoprenaline on the dissociation rate and on membrane release of Gs was completely blocked by the beta-adrenergic receptor antagonist propranolol. The concentration-response relationship for isoprenaline-induced dissociation during the first 1 min after addition of hormone yielded a kappa act. of 16 +/- 5 nM, whereas the kappa act. for isoprenaline-induced membrane release was 10 nM. We conclude that release of alpha s from plasma membranes is likely to accompany Gs-subunit dissociation and constitutes a potentially important facet of Gs action.
Project description:We examined the interaction between the stimulatory guanine-nucleotide-binding protein, Gs, and the inhibitory guanine-nucleotide-binding protein, Gi, in cell membranes of S49 lymphoma cells. In these cells, beta-adrenergic receptors stimulate the activity of adenylate cyclase via Gs, whereas inhibition via somatostatin receptors is transduced by an inhibitory G-protein, Gi. Using an antibody that selectively recognizes alpha s, the monomeric, but not the heterotrimeric, alpha-subunit of Gs, we quantified the extent of dissociation of Gs in a competitive e.l.i.s.a. Incubation of S49-cell plasma membranes with 0.1 microM-isoprenaline, 100 microM free Mg2+ and 100 microM-GTP produced substantial subunit dissociation of Gs, which was reversible by addition of purified beta gamma-subunit dimer or somatostatin. Somatostatin produced an immediate (without a lag) time- and concentration-dependent decrease in the concentration of dissociated Gs (kinhib. for somatostatin = 51 +/- 12 nM) and in the activity of adenylate cyclase (kinhib. = 121 +/- 20 nM). By contrast, after addition of a 10-fold molar excess of beta gamma-dimer relative to alpha s, there was a 2-3 min lag, after which the beta gamma-dimer re-associated Gs. Isoprenaline-induced dissociation of Gs was accompanied by a release of alpha s from the incubated membranes to a post-100,000 g supernatant, and somatostatin could reverse this release. Immunoblot analysis with both a C-terminal anti-peptide antibody and an antibody directed against a sequence near the N-terminal also showed release of alpha s by the beta-agonist and reversal by somatostatin. Membrane release of Gs by isoprenaline that could be blocked by somatostatin was also confirmed in reconstitution studies of supernatant fraction into cyc- S49-cell membranes. We conclude that in native cell membranes somatostatin-induced activation of Gi dissociates Gi and interferes with the Gs activation cycle by providing beta gamma-dimer, which acts to prevent or reverse formation of monomeric alpha s. Because alpha s can be released from the cell membrane, regulation of the local concentration of GTP-liganded dissociated alpha s is likely to be an important factor in modulating the activity of adenylate cyclase.
Project description:The movement of nucleophosmin from nucleoli to nucleoplasm in HeLa cells induced by cytotoxic drugs and detected by immunofluorescence is inhibited by concomitant treatment with antimycin A in glucose-free medium. Incubation of HeLa cells with antimycin A (300 nM; 30 min) and glucose-free medium resulted in an approximately 90% decrease in cellular ATP pools. To study the biochemical events involved in nucleophosmin translocation, we used an in vitro system consisting of Triton-permeabilized HeLA cells. Incubation of permeabilized cells with ATP (0.5 mM; 1 h) resulted in the translocation of nucleophosmin from nucleoli to nucleoplasm and cytoplasm. Similarly to drug-induced nucleophosmin translocation in whole cultured cells, there is no reduction (measured by e.l.i.s.a.) or degradation of nucleophosmin or change in the ratio of the high-molecular-mass form to the monomeric form (ascertained by Western blotting) during ATP treatment of permeabilized cells. Together, these results indicate a requirement for ATP for redistribution of nucleophosmin from nucleoli to nucleoplasm. Because this permeabilized cell model is simple and efficient and works effectively with exogenous factors, it should provide a powerful tool for investigating the biochemical features of nucleophosmin translocation from nucleoli to nucleoplasm.
Project description:The [PSI+] determinant of Saccharomyces cerevisiae, consisting of the cytosolic translation termination factor Sup35, is a prion-type genetic element that induces an inheritable conformational change and converts the Sup35 protein into amyloid fibers. The molecular chaperone Hsp104 is required to maintain self-replication of [PSI+]. We observe in vitro that addition of catalytic amounts of Hsp104 to the prion-determining region of the NM domain of Sup35, Sup355-26, results in the dissociation of oligomeric Sup35 into monomeric species. Several intermediates of Sup355-26 could be detected during this process. Strong interactions are found between Hsp104 and hexameric/tetrameric Sup355-26, whereas the intermediate and monomeric "release" forms show a decreased affinity with respect to Hsp104, as monitored by saturation transfer difference and diffusion-ordered NMR spectroscopic experiments. Interactions are mediated mostly by the side chains of Gln, Asn, and Tyr residues in Sup355-26. No interaction can be detected between Hsp104 and higher oligomeric states (>/=8) of Sup355-26. Taking into account the fact that Hsp104 is required for maintenance of [PSI+], we suggest that low-oligomeric-weight species of Sup35 are important for prion propagation in yeast.
Project description:Hepatitis C virus NS3 helicase can unwind double-stranded DNA and RNA and has been proposed to form oligomeric structures. Here we examine the DNA unwinding activity of monomeric NS3. Oligomerization was measured by preparing a fluorescently labeled form of NS3, which was titrated with unlabeled NS3, resulting in a hyperbolic increase in fluorescence anisotropy and providing an apparent equilibrium dissociation constant of 236 nm. To evaluate the DNA binding activity of individual subunits within NS3 oligomers, two oligonucleotides were labeled with fluorescent donor or acceptor molecules and then titrated with NS3. Upon the addition of increasing concentrations of NS3, fluorescence energy transfer was observed, which reached a plateau at a 1:1 ratio of NS3 to oligonucleotides, indicating that each subunit within the oligomeric form of NS3 binds to DNA. DNA unwinding was measured under multiple turnover conditions with increasing concentrations of NS3; however, no increase in specific activity was observed, even at enzyme concentrations greater than the apparent dissociation constant for oligomerization. An ATPase-deficient form of NS3, NS3(D290A), was prepared to explore the functional consequences of oligomerization. Under single turnover conditions in the presence of excess concentration of NS3 relative to DNA, NS3(D290A) exhibited a dominant negative effect. However, under multiple turnover conditions in which DNA concentration was in excess to enzyme concentration, NS3(D290A) did not exhibit a dominant negative effect. Taken together, these data support a model in which monomeric forms of NS3 are active. Oligomerization of NS3 occurs, but subunits can function independently or cooperatively, dependent upon the relative concentration of the DNA.
Project description:DNA replication is a central process in all living organisms. Polyomavirus DNA replication serves as a model system for eukaryotic DNA replication and has considerably contributed to our understanding of basic replication mechanisms. However, the details of the involved processes are still unclear, in particular regarding lagging strand synthesis. To delineate the complex mechanism of coordination of various cellular proteins binding simultaneously or consecutively to DNA to initiate replication, we investigated single-stranded DNA (ssDNA) interactions by the SV40 large T antigen (Tag). Using single molecule imaging by atomic force microscopy (AFM) combined with biochemical and spectroscopic analyses we reveal independent activity of monomeric and oligomeric Tag in high affinity binding to ssDNA. Depending on ssDNA length, we obtain dissociation constants for Tag-ssDNA interactions (KD values of 10-30 nM) that are in the same order of magnitude as ssDNA binding by human replication protein A (RPA). Furthermore, we observe the formation of RPA-Tag-ssDNA complexes containing hexameric as well as monomeric Tag forms. Importantly, our data clearly show stimulation of primase function in lagging strand Okazaki fragment synthesis by monomeric Tag whereas hexameric Tag inhibits the reaction, redefining DNA replication initiation on the lagging strand.
Project description:Gram-negative bacteria such as Escherichia coli have an inner membrane and an asymmetric outer membrane (OM) that together protect the cytoplasm and act as a highly selective permeability barrier. Lipopolysaccharide (LPS) is the major component of the outer leaflet of the OM and is essential for the survival of nearly all Gram-negative bacteria. Recent advances in understanding the proteins involved in the transport of LPS across the periplasm and into the outer leaflet of the OM include the identification of seven proteins suggested to comprise the LPS transport (Lpt) system. Crystal structures of the periplasmic Lpt protein LptA have recently been reported and show that LptA forms oligomers in either an end-to-end arrangement or a side-by-side dimer. It is not known if LptA oligomers bridge the periplasm to form a large, connected protein complex or if monomeric LptA acts as a periplasmic shuttle to transport LPS across the periplasm. Therefore, the studies presented here focus specifically on the LptA protein and its oligomeric arrangement and concentration dependence in solution using experimental data from several biophysical approaches, including laser light scattering, crosslinking, and double electron electron resonance spectroscopy. The results of these complementary techniques clearly show that LptA readily associates into stable, end-to-end, rod-shaped oligomers even at relatively low local protein concentrations and that LptA forms a continuous array of higher order oligomeric end-to-end structures as a function of increasing protein concentration.
Project description:The effect of tumor necrosis factor-? (TNF?) on cartilage matrix degradation is mediated by its transport and binding within the extracellular matrix (ECM) of the tissue, which mediates availability to cell receptors. Since the bioactive form of TNF? is a homotrimer of monomeric subunits, conversion between trimeric and monomeric forms during intratissue transport may affect binding to ECM and, thereby, bioactivity within cartilage. We studied the transport and binding of TNF? in cartilage, considering the quaternary structure of this cytokine. Competitive binding assays showed significant binding of TNF? in cartilage tissue, leading to an enhanced uptake. However, studies in which TNF? was cross-linked to remain in the trimeric form revealed that the binding of trimeric TNF? was negligible. Thus, binding of TNF? to ECM was associated with the monomeric form. Binding of TNF? was not disrupted by pre-treating cartilage tissue with trypsin, which removes proteoglycans and glycoproteins but leaves the collagen network intact. Therefore, proteoglycan loss during osteoarthritis should only alter the passive diffusion of TNF? but not its binding interaction with the remaining matrix. Our results suggest that matrix binding and trimer-monomer conversion of TNF? both play crucial roles in regulating the accessibility of bioactive TNF? within cartilage.
Project description:Oligomeric gastric mucin was isolated from the fundic part of the rat stomach. Previously we have shown by biochemical analysis that this oligomeric mucin consists of disulphide-linked homo-oligomers, which contain no other covalently attached proteins [Dekker, Aelmans & Strous (1991) Biochem. J. 277, 423-427]. Electron-microscopic images of the oligomeric mucin revealed a heterogenous population of long filamentous molecules of 300-3000 nm length. After reduction and carboxymethylation the monomeric mucins displayed a length distribution with a single peak at about 279 nm. Length-distribution analysis of oligomeric molecules with length up to 1000 nm revealed three subpopulations with one, two or three times the length of the monomeric mucin. The oligomers displayed small globular domains of about 15 nm, which were equally spaced along the molecule's length. As the distance between these globular domains was similar to the monomer length, these domains most likely indicate attachment sites of the monomers. These results show that the mucin monomers attached end-to-end in the oligomer. Biosynthesis of the mucin oligomers was studied by labelling of stomach explants in vitro with [35S]methionine, [3H]galactose or [35S]sulphate and subsequent immunoprecipitation of the mucin with a specific antiserum. Analysis by electrophoresis and gel filtration revealed that the oligomerization takes place by formation of disulphide bonds between the 300 kDa mucin precursors. The mucin was exclusively synthesized and secreted as fully glycosylated oligomers, as neither precursor proteins nor monomeric mucin were detected in the culture medium. A model for the biosynthesis of rat gastric mucin is proposed in which the filamentous mucin monomers are linked end-to-end by disulphide bonds.
Project description:Conventional local treatment for medullary osteomyelitis (OM) includes insertion of antibiotic-loaded polymethylmethacrylate (PMMA) cement. Nevertheless, PMMA may delivery irregular concentration of antibiotic to surrounding tissue. We aimed to compare the in vitro antibacterial activity of Bioactive Glass (BAG) S53P4, which is a compound showing local antibacterial activity, to that of antibiotic-loaded PMMA against multidrug resistant bacteria from OM isolates.We studied convenience samples of multidrug resistant (MDR) microorganisms obtained from patients presenting OM and prosthetic joint infection (PJI). Mixtures containing tryptic soy broth (TSB) and inert glass beads (2 mm), BAG-S53P4 granules (0.5-0.8 mm and?<?45 mm) and Gentamicin or Vancomycin-loaded PMMA beads were inoculated with methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant coagulase-negative Staphylococcus (MR-CoNS), Pseudomonas aeruginosa or Klebsiella pneumoniae isolates. Glass beads (2.0 mm) were used as a control. Antibacterial activity was evaluated by means of time-kill curve, through seeding the strains on blood agar plates, and subsequently performing colony counts after 24, 48, 72, 96, 120 and 168 h of incubation. Differences between groups were evaluated by means of two-way analysis of variance (ANOVA) and Bonferroni's t test.Inhibition of bacterial growth started soon after 48 h of incubation, reached zero CFU/ml between 120 and 168 h of incubation for both antibiotic-loaded PMMA and BAG S53P4 groups, in comparison with inert glass (p?<?0.05). No difference regarding time-kill curves between antibiotic-loaded PMMA and BAG S53P4 was observed.BAG S53P4 presented antibacterial properties as much as antibiotic-loaded PMMA for MDR bacteria producing OM and PJI.