UNIPROT:P05231 - FACTA Search

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Query: UNIPROT:P05231 (interleukin-6)
23,907 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The high affinity interleukin-6 (IL-6) signaling complex consists of IL-6 and two membrane-associated receptor components: a low affinity but specific IL-6 receptor and the affinity converter/signal transducing protein gp130. Monomeric (IL-6M) and dimeric (IL-6D) forms of Escherichia coli-derived human IL-6 and the extracellular ("soluble") portions of the IL-6 receptor (sIL-6R) and gp130 have been purified in order to investigate the effect of IL-6 dimerization on binding to the receptor complex. Although IL-6D has a higher binding affinity for immobilized sIL-6R, as determined by biosensor analysis employing surface plasmon resonance detection, IL-6M is more potent than IL-6D in a STAT3 phosphorylation assay. The difference in potency is significantly less pronounced when measured in the murine 7TD1 hybridoma growth factor assay and the human hepatoma HepG2 bioassay due to time-dependent dissociation at 37 degrees C of IL-6 dimers into active monomers. The increased binding affinity of IL-6D appears to be due to its ability to cross-link two sIL-6R molecules on the biosensor surface. Studies of the IL-6 ternary complex formation demonstrated that the reduced biological potency of IL-6D resulted from a decreased ability of the IL-6D (sIL-6R)2 complex to couple with the soluble portion of gp130. These data imply that IL-6-induced dimerization of sIL-6R is not the driving force in promoting formation of the hexameric (IL-6 IL-6R gp130)2 complex. A model is presented whereby the trimeric complex of IL-6R, gp130, and IL-6M forms before the functional hexamer. Due to its increased affinity for the IL-6R but its decreased ability to couple with gp130, we suggest that a stable IL-6 dimer may be an efficient IL-6 antagonist.
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PMID:Influence of interleukin-6 (IL-6) dimerization on formation of the high affinity hexameric IL-6.receptor complex. 870 37

Lung epithelial cells (A549) synthesize and secrete fibrinogen (FBG) in vitro when stimulated with interleukin-6 and dexamethasone. This FBG secretion is polarized in the basolateral direction, suggesting that FBG is a component of the extracellular matrix (ECM). Immunofluorescent staining of A549 cells showed a fibrillar pattern of FBG, similar to the staining detected using antibodies against the matrix constituents, collagen type IV and fibronectin (FN). The same pattern of staining was detected using antibodies against fibrinopeptides A and B, as well as with the T2G1 monoclonal antibody against the fibrin-specific epitope, beta15-21. Matrix staining was unaltered in the presence of the thrombin inhibitor, hirudin, or the plasmin inhibitor, aprotinin, consistent with the interpretation that matrix deposition of FBG does not require such enzymatic action. Metabolic labeling studies confirmed that FBG secreted from A549 cells or deposited into the ECM showed no evidence of thrombin or plasmin proteolytic processing or of transglutaminase-mediated covalent cross-linking (gamma-gamma dimers or alpha-polymers). Incubation of either A549 cell-derived or purified plasma FBG with cultures of human foreskin fibroblasts resulted in FBG deposition in the ECM that colocalized with matrix fibrils containing endogenously produced FN and laminin (LN). Binding of FBG to this exogenously produced matrix was unaltered by inhibition of thrombin and plasmin action, yet also exhibited exposure of the fibrin-specific epitope, beta15-21. The majority (approximately 70%) of newly synthesized and secreted FBG is bound to the cell surface as determined by its trypsin-sensitivity. Cell surface-bound FBG is initially deoxycholate-soluble, which, over time, becomes incorporated in the deoxycholate-insoluble ECM in a similar fashion to FN. These data suggest that matrix incorporation requires the binding of secreted FBG to cell-associated matrix assembly sites. However, unlike FN, FBG in the ECM is composed of the dimeric protamer (A alpha/B beta/gamma gamma) and not high molecular weight polymers indicative of fibrin. This study provides evidence that deposition of FBG in both endogenous and exogenously produced matrices results in conformational changes that occur independently of thrombin cleavage. This matrix-bound FBG, on which unique cell-reactive domains are likely exposed, could augment cellular response mechanisms evoked during injury and inflammation.
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PMID:Thrombin cleavage-independent deposition of fibrinogen in extracellular matrices. 932 31

A noncovalently bound dimeric form of recombinant human IL-6 interleukin-6 (IL-6D) was shown to be an antagonist for IL-6 activity, in a STAT3 tyrosine phosphorylation assay using HepG2 cells, under conditions where it does not dissociate into monomeric IL-6 (IL-6M). The fluorescence from Trp157, the single tryptophan residue in the primary sequence of IL-6, is altered in IL-6D, where the wavelength maximum is blue-shifted by 3 nm and the emission intensity is reduced by 30%. These data suggest that Trp157 is close to, but not buried by, the dimer interface. Both IL-6D and IL-6M are compact molecules, as determined by sedimentation velocity analysis, and contain essentially identical levels of secondary and tertiary structure, as determined by far- and near-UV CD, respectively. IL-6D and IL-6M show the same susceptibility to limited proteolytic attack, and exhibit identical far-UV CD-monitored urea-denaturation profiles with the midpoint of denaturation occurring at 6.0 +/- 0.1 M urea. However, IL-6D was found to dissociate prior to the complete unfolding of the protein, with a midpoint of dissociation of 3 M urea, suggesting that dissociation and dimerization occur when the protein is in a partially unfolded state. Based on these results, we suggest that IL-6D is a metastable domain-swapped dimer, comprising two monomeric units where identical helices from each protein chain are swapped through the loop regions at the "top" of the protein (i.e., the region of the protein most distal from the N- and C-termini). Such an arrangement would account for the antagonistic activity of IL-6D. In this model, receptor binding site I, which comprises residues in the A/B loop and the C-terminus of the protein, is free to bind the IL-6 receptor. However, site III, which includes Trp157 and residues in the C/D loop and N-terminal end of helix D, and perhaps site II, which comprises residues in the A and C helices, are no longer able to bind the signal transducing component of the IL-6 receptor complex, gp130.
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PMID:Physicochemical characterization of an antagonistic human interleukin-6 dimer. 969 57

Macrophage inflammatory protein (MIP)-1alpha and MIP-1beta regulate leukocyte activation and trafficking. To assess the role of MIP-1alpha and MIP-1beta in human inflammation, healthy subjects were studied during experimental endotoxemia with prior administration of ibuprofen, a cyclooxygenase inhibitor, or dimeric p75 tumor necrosis factor (TNF)-alpha receptor, a TNF antagonist; septic patients were also studied. Following endotoxin, blood levels of both MIP-1 molecules rose acutely and fell to baseline by 6 h (P=. 001). While MIP-1 mediates fever in animals independent of cyclooxygenase blockade, in subjects given endotoxin and ibuprofen, MIP-1 levels increased and fever was suppressed. MIP-1 levels were not diminished by inhibiting circulating TNF-alpha in humans. In septic patients, elevated levels of MIP-1alpha and MIP-1beta were detected within 24 h of sepsis and fell in parallel with TNF-alpha and interleukin-6 (P<.01). MIP-1alpha and MIP-1beta increase during acute inflammation but are not associated with fever in endotoxemic humans during cyclooxygenase blockade.
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PMID:Detection of macrophage inflammatory protein (MIP)-1alpha and MIP-1beta during experimental endotoxemia and human sepsis. 984 32

Signal transduction in response to interleukin-6 (IL-6) results from homodimerization of gp130. This dimerization occurs after binding of IL-6 to its surface receptor (IL-6R) and can also be triggered by the complex of soluble IL-6R and IL-6. We fused IL-6 to the constant region of a human IgG1 heavy chain (Fc). IL-6Fc was expressed in COS-7 cells and purified via Protein A Sepharose. Using three different assays we found that the biological activity of this dimeric IL-6 protein is comparable with monomeric IL-6. Recently, we described the designer cytokine Hyper-IL-6 (H-IL-6) in which soluble IL-6R and IL-6 are connected via a flexible peptide linker. This molecule turned out to be 100-1000 times more effective than unlinked IL-6 and soluble IL-6R. Hyper-IL-6 acts on cells only expressing gp130 and is a potent stimulator of in vitro expansion of early hematopoietic precursors. Here we show that a Fc fusion protein of H-IL-6 (H-IL-6Fc) has the same biological activity on BAF/gp130 cells as H-IL-6. Furthermore, both H-IL-6 forms have a similar ability to induce the synthesis of acute phase proteins in human hepatoma cells HepG2 and in mice in vivo. The introduction of a thrombin cleavage site between H-IL-6 and the Fc portion of H-IL-6Fc made it possible to specifically recover biologically active monomeric H-IL-6 by limited proteolysis of the fusion protein. A more general use of cleavable immunoadhesins expressed in mammalian cells is discussed.
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PMID:Immunoadhesins of interleukin-6 and the IL-6/soluble IL-6R fusion protein hyper-IL-6. 1008 96

Recombinant human interleukin-6 (hIL-6), a pleiotropic cytokine containing two intramolecular disulfide bonds, was expressed in Escherichia coli as an insoluble inclusion body, before being refolded and purified in high yield providing sufficient qualities for clinical use. Quantitative reconstitution of the native disulfide bonds of hIL-6 from the fully denatured E. coli extracts could be performed by glutathione-assisted oxidation in a completely denaturating condition (6M guanidinium chloride) at protein concentrations higher than 1 mg/mL, preventing aggregation of reduced hIL-6. Oxidation in 6M guanidinium chloride (GdnHCl) required remarkably low concentrations of glutathione (reduced form, 0.01 mM; oxidized form, 0.002 mM) to be added to the solubilized hIL-6 before the incubation at pH 8.5, and 22 degrees C for 16 h. After completion of refolding by rapid transfer of oxidized hIL-6 into acetate buffer by gel filtration chromatography, residual contaminants including endotoxin and E. coli proteins were efficiently removed by successive steps of chromatography. The amount of dimeric hIL-6s, thought to be purification artifacts, was decreased by optimizing the salt concentrations of the loading materials in the ion-exchange chromatography, and gradually removing organic solvents from the collected fractions of the preparative reverse-phase HPLC. These refolding and purification processes, which give an overall yield as high as 17%, seem to be appropriate for the commercial scale production of hIL-6 for therapeutic use.
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PMID:High yield refolding and purification process for recombinant human interleukin-6 expressed in Escherichia coli. 1009 41

The utility of isotachophoresis-capillary zone electrophoresis (ITP-CZE) and high-performance size-exclusion chromatography (HPSEC) was investigated for determination of dimeric and monomeric recombinant human interleukin-6 (rhIL-6). Using ITP-CZE heterogeneity of dimeric rhIL-6 could be revealed resolving two peaks in the electropherograms, while with HPSEC dimeric rhIL-6 eluted as one homogeneous fraction. Both protein forms were monitored during incubation of monomeric rhIL-6 at different pH and temperature. The selectivity of counterflow ITP-CZE in conjunction with the low concentration determination limits enabled reanalysis of HPSEC fractions for identification of the dimer in the electropherograms. Both ITP-CZE and HPSEC were shown to be suitable to monitor the dimerization of rhIL-6, similar monomer-to-dimer peak area ratios were obtained throughout the incubation. Dimer formation kinetics increased with decreasing pH and with increasing temperature, it was entirely suppressed at neutral pH and room temperature. In contrast to HPSEC, ITP-CZE enabled separation of further still unidentified artifacts apparently formed during incubation of rhIL-6. CZE analysis in conjunction with electrospray ionization mass spectrometry revealed the non-covalent binding character of the dimeric rhIL-6 complex and facilitated interpretation of the electropherograms.
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PMID:Utility of isotachophoresis-capillary zone electrophoresis, mass spectrometry and high-performance size-exclusion chromatography for monitoring of interleukin-6 dimer formation. 1036 Mar 28

Placenta growth factor (PlGF) is a dimeric glycoprotein, structurally and functionally related to the vascular endothelial growth factor, a potent angiogenic/permeability factor known to play a role in the neoangiogenesis during wound repair. In this study we evaluated the expression of PlGF in human keratinocytes and investigated its possible role in wound healing. Northern blot analysis on cultured keratinocytes revealed a 1.7 kb mRNA transcript and reverse transcriptase-polymerase chain reaction allowed the detection of two PlGF isoforms generated by alternative RNA splicing. PlGF and vascular endothelial growth factor homodimers as well as vascular endothelial growth factor/PlGF heterodimers could be detected in keratinocyte conditioned medium. Increased expression of both PlGF mRNA and protein was observed upon treatment of keratinocytes with epidermal growth factor, transforming growth factor-alpha, transforming growth factor-beta, and interleukin-6, all cytokines present at the wound site during the early phase of repair. The analysis of human full-thickness healing wounds revealed appreciable levels of PlGF mRNA and protein in the migrating keratinocytes starting from day 3 after injury, and increasing at day 5. At day 7 PlGF mRNA was no longer detectable, while the protein was still expressed by migrating suprabasal keratinocytes. At day 13, when the wound had reepithelialized, PlGF immunostaining was completely negative. By in situ hybridization an intense signal for PlGF was also found on endothelial capillaries adjacent to the wound. These data demonstrate that keratinocytes are a source of PlGF during wound healing in vivo and indicate a role for this factor in the neoangiogenesis process associated with cutaneous wound repair.
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PMID:Placenta growth factor is induced in human keratinocytes during wound healing. 1095 Dec 73

The granulocyte colony-stimulating factor receptor (G-CSF-R) forms a tetrameric complex with G-CSF containing two ligand and two receptor molecules. The N-terminal Ig-like domain of the G-CSF-R is required for receptor dimerization, but it is not known whether it binds G-CSF or interacts elsewhere in the complex. Alanine scanning mutagenesis was used to show that residues in the Ig-like domain of the G-CSF-R (Phe(75), Gln(87), and Gln(91)) interact with G-CSF. This binding site for G-CSF overlapped with the binding site of a neutralizing anti-G-CSF-R antibody. A model of the Ig-like domain showed that the binding site is very similar to the viral interleukin-6 binding site (site III) on the Ig-like domain of gp130, a related receptor. To further characterize the G-CSF-R complex, exposed and inaccessible regions of monomeric and dimeric ligand-receptor complexes were mapped with monoclonal antibodies. The results showed that the E helix of G-CSF was inaccessible in the dimeric but exposed in the monomeric complex, suggesting that this region binds to the Ig-like domain of the G-CSF-R. In addition, the N terminus of G-CSF was exposed to antibody binding in both complexes. These data establish that the dimerization interface of the complete receptor complex is different from that in the x-ray structure of a partial complex. A model of the tetrameric G-CSF.G-CSF-R complex was prepared, based on the viral interleukin-6.gp130 complex, which explains these and previously published data.
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PMID:Identification of ligand-binding site III on the immunoglobulin-like domain of the granulocyte colony-stimulating factor receptor. 1146 84

The gp130-cytokine system has been fertile ground for protein structure-function studies aimed at elucidating the basis of ligand recognition and receptor activation. A number of longstanding questions involve the mechanism of the stepwise assembly of the active signaling complexes, as well as the structure of the gp130-cytokine complexes. It has been clear from functional studies that the paradigm of gp130-cyokine recognition will differ substantially from the classical homo-dimeric systems, typified by human growth hormone (hGH) and its receptor. Recently, a crystal structure of a viral interleukin-6 (vIL-6), complexed with the D1D2D3 domains of the gp130 extracellular domain, has resolved many of these questions, and reconciled much of the functional and mutagenesis data which have existed for a variety of gp130-cytokines. In this review, we discuss the structure of the vIL-6/gp130 complex in some detail and suggest that the geometry of this complex will be a common structural template utilized by other gp130-cytokines, as well as cytokines from distinct signaling systems.
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PMID:A structural template for gp130-cytokine signaling assemblies. 1242 68

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