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

When murine interleukin-6 is overexpressed in Escherichia coli, a small population of molecules exhibits a novel C-terminal modification. Peptide mapping, electrospray ionization-mass spectrometry, and automated N- and C-terminal sequencing identified a peptide ("tag" peptide), -Ala-Ala-Asn-Asp-Glu-Asn-Tyr-Ala-Leu-Ala-Ala-COOH, encoded by a small metabolically stable RNA of E. coli (10Sa RNA) attached to truncated C termini of the recombinant protein. A mutant strain of E. coli in which the chromosomal 10Sa RNA gene (ssrA) is disrupted does not produce this C-terminal modification, confirming that the tag peptide originates from the ssrA gene.
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PMID:C-terminal extension of truncated recombinant proteins in Escherichia coli with a 10Sa RNA decapeptide. 753 43

Interleukin-6 (IL-6) is a multifunctional cytokine that plays an important role in host defense. It has been predicted that IL-6 may fold as a 4 alpha-helix bundle structure with up-up-down-down topology. Despite a high degree of sequence similarity (42%) the human and mouse IL-6 polypeptides display distinct species-specific activities. Although human IL-6 (hIL-6) is active in both human and mouse cell assays, mouse IL-6 (mIL-6) is not active on human cells. Previously, we demonstrated that the 5 C-terminal residues of mIL-6 are important for activity, conformation, and stability (Ward LD et al., 1993, Protein Sci 2:1472-1481). To further probe the structure-function relationship of this cytokine, we have constructed several human/mouse IL-6 hybrid molecules. Restriction endonuclease sites were introduced and used to ligate the human and mouse sequences at junction points situated at Leu-62 (Lys-65 in mIL-6) in the putative connecting loop AB between helices A and B, at Arg-113 (Val-117 in mIL-6) at the N-terminal end of helix C, at Lys-150 (Asp-152 in mIL-6) in the connecting loop CD between helices C and D, and at Leu-178 (Thr-180 in mIL-6) in helix D. Hybrid molecules consisting of various combinations of these fragments were constructed, expressed, and purified to homogeneity. The conformational integrity of the IL-6 hybrids was assessed by far-UV CD. Analysis of their biological activity in a human bioassay (using the HepG2 cell line), a mouse bioassay (using the 7TD1 cell line), and receptor binding properties indicates that at least 2 regions of hIL-6, residues 178-184 in helix D and residues 63-113 in the region incorporating part of the putative connecting loop AB through to the beginning of helix C, are critical for efficient binding to the human IL-6 receptor. For human IL-6, it would appear that interactions between residues Ala-180, Leu-181, and Met-184 and residues in the N-terminal region may be critical for maintaining the structure of the molecule; replacement of these residues with the corresponding 3 residues in mouse IL-6 correlated with a significant loss of alpha-helical content and a 200-fold reduction in activity in the mouse bioassay. A homology model of mIL-6 based on the X-ray structure of human granulocyte colony-stimulating factor is presented.
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PMID:Structure-function analysis of human IL-6: identification of two distinct regions that are important for receptor binding. 753 47

Interleukin-6 (IL-6) triggers the formation of a high affinity receptor complex constituted by the ligand-binding subunit IL-6 receptor alpha (IL-6R alpha) and the signal-transducing beta chain gp130. Since the cytoplasmic region of IL-6R alpha is not required for signal transduction, soluble forms of IL-6R alpha (sIL-6R alpha) show agonistic properties because they are still able to originate IL-6.sIL-6R alpha complexes, which in turn associate with gp130. A three-dimensional model of the human IL-6.IL-6R alpha.gp130 complex has been constructed and verified by site-directed mutagenesis of regions in shIL-6R alpha (where "h" is human) anticipated to contact hgp130, with the final goal of generating receptor variants with antagonistic properties. In good agreement with our structural model, substitutions at Asn-230, His-280, and Asp-281 selectively impaired the capability of shIL-6R alpha to associate with hgp130 both in vitro and on the cell surface, without affecting its affinity for hIL-6. Moreover, the multiple substitution mutant A228D/N230D/H280S/D281V expressed as a soluble protein partially antagonized hIL-6 bioactivity on hepatoma cells.
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PMID:Interleukin-6 (IL-6) antagonism by soluble IL-6 receptor alpha mutated in the predicted gp130-binding interface. 774 75

Nuclear factor-interleukin-6 (NF-IL6), a member of the CCAAT box/enhancer-binding protein (C/EBP) family, contains a basic domain-leucine zipper (bZIP) DNA binding motif. Controlled protease digestion was used to probe free and DNA-complexed NF-IL6 protein. Digestion with trypsin in the absence of DNA produced the leucine zipper domain (containing residues 303-345). In contrast, digestion of NF-IL6.DNA complexes produced a stable domain, spanning residues 266-345, termed the tryptic core domain (TCD). The NH2-terminal boundary of the TCD is longer than tryptic peptides reported from C/EBP alpha.DNA complexes. Digestion of NF-IL6 with endoprotease Asp-N produced a domain smaller than the TCD (NF-IL6 bZIP domains (NFBD) (272-345)), a domain identified either in the absence or the presence of DNA. Both recombinant peptides bind acute-phase response element DNA in a sequence-specific fashion. The equilibrium disassociation constant (Kd) for the TCD was 36 +/- 8 nM, whereas the Kd for NFBD (272-345) was 283 +/- 160 nM. Moreover, in comparison with the TCD, NFBD (272-345) formed unstable DNA complexes with a 15-fold faster off-rate. We conclude that the amino acids represented between 266 and 272 termed the complex stabilizing subdomain, influences DNA complex formation independent of DNA binding specificity, and may be one mechanism for heterogeneity of DNA interaction by C/EBP family members.
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PMID:Identification of a novel determinant for basic domain-leucine zipper DNA binding activity in the acute-phase inducible nuclear factor-interleukin-6 transcription factor. 814 15

Sixty-four kinds of cell lines were examined for their ability to produce megakaryocyte potentiating activity by means of conditioned media obtained from a protein-free culture system. Six human tumor cell lines were shown to produce this activity, and the cell line HPC-Y5, established from human pancreatic cancer, was shown to have the highest level of activity. The megakaryocyte potentiating factor (MPF) was purified from an HPC-Y5 conditioned medium by a combination of ion-exchange chromatography, gel filtration and reverse-phase HPLC. The purified MPF showed a megakaryocyte potentiating activity almost equal to human interleukin-6 in the presence of murine interleukin-3 in a colony formation assay with mouse bone marrow cells. The apparent molecular weight of MPF is 32,000 when determined by SDS-polyacrylamide gel electrophoresis. Glycopeptidase F digestion, and amino sugar analysis of the factor demonstrated that MPF is a glycoprotein carrying at least one N-linked sugar chain. The N-terminal amino acid sequence of MPF was determined to be Leu-Ala-Gly-Glu-Thr-Gly-Gln-Glu-Ala-Ala-Pro-Leu- Asp-Gly-Val-Leu-Ala-Asn. The same or homologous amino acid sequence has not been found in known proteins, demonstrating that MPF is a novel cytokine that has megakaryocyte potentiating activity in the murine assay system.
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PMID:A novel cytokine exhibiting megakaryocyte potentiating activity from a human pancreatic tumor cell line HPC-Y5. 828 29

Three forms of interleukin-6 (IL-6) have been constructed and stably transfected into human hepatoma cells (HepG2). Wild type IL-6 containing a signal peptide was rapidly secreted as a biologically active protein. IL-6 lacking the signal peptide accumulated within the cytoplasm of transfected cells. Surprisingly, IL-6 carrying a COOH-terminal extension of the amino acids Lys-Asp-Glu-Leu (KDEL) was not completely retained in the endoplasmic reticulum (ER). Complete retention in the ER was achieved when the 14 COOH-terminal amino acids of protein disulfide isomerase which include the KDEL signal were added to the COOH terminus of IL-6. This finding clearly demonstrates that the addition of the protein sorting signal KDEL alone is not sufficient for full retention of IL-6 in the ER. IL-6 accumulated in the cytoplasm and IL-6 retained in the ER failed to induce liver-specific acute-phase protein synthesis in the host cells, indicating that there is no intracellular role for IL-6 in signal transduction. Retention of IL-6 in the ER led to the prevention of surface expression of the IL-6 receptor protein gp80, making these cells unresponsive to IL-6. This phenomenon can be exploited in the future to generate transgenic animals which will become completely cytokine unresponsive in the tissues in which they express an ER retained cytokine.
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PMID:Intracellular retention of interleukin-6 abrogates signaling. 840 66

Interleukin-6 (IL-6), a four-helix bundle protein, is a multifunctional cytokine which plays an important role in the regulation of the immune system, hematopoiesis, and inflammatory response, as well as in the pathogenesis of multiple myeloma. We have previously shown that a single-disulfide variant of human IL-6, lacking 22 N-terminal amino acids and the disulfide bond connecting Cys-45 and Cys-51 in the 185-residue chain of the wild-type protein, fully retains the conformational, stability, and functional properties of the full-length human IL-6 [Breton et al. (1995) Eur. J. Biochem. 227, 573-581]. In this study, we have investigated the conformational and stability properties of mutant IL-6 at acidic pH (A-state). Using far- and near-ultraviolet (UV) circular dichroism (CD), fluorescence emission, and second-derivative absorption spectroscopy, we have established that mutant IL-6 at pH 2.0 fully retains the helical secondary structure of the native protein at pH 7.5, while the tertiary interactions are much weaker. At variance from the native species, mutant IL-6 in the A-state binds 1-anilinonaphthalene-8-sulfonic acid (ANS), a property considered most typical of a protein in the molten globule state. The pH-induced conformational change from the native to the A-state, monitored either by near-UV CD or by ANS-binding measurements, shows a transition midpoint at pH approximately 4.5, thus indicating that the partial unfolding of the protein is mediated by the titration of glutamic and/or aspartic acid residues. At pH 2.0, the thermal denaturation of mutant IL-6 occurs as a broad process of low cooperativity with a transition at 50-60 degrees C, whereas at pH 7.5 the thermal unfolding is cooperative and characterized by a transition midpoint at 65 degrees C. Of interest, the unfolding of the A-state is not complete even up to approximately 85 degrees C. The urea-mediated unfolding profile of mutant IL-6, measured by far-UV CD, is essentially identical at both pH 7.5 and 2.0, with a midpoint of the cooperative unfolding transition at 5.5 +/- 0.1 M denaturant. Both thermal and urea denaturations of the A-state are complex and cannot fit to a two-state model for unfolding. The unusual stability of mutant IL-6 in acid is also reflected by the resistance to proteolysis at pH 3.6-4.0 by Staphylococcus aureus V8 protease or cathepsin D, an acid protease released by machrophages upon inflammatory stimulation. It is suggested that the molten globule state of IL-6 at acidic pH can play a role in the biological activity of this cytokine, which can exert its activity also at mildly acidic pH, as in inflammation sites.
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PMID:Acid-induced molten globule state of a fully active mutant of human interleukin-6. 878 6

We have previously shown that, after peripheral administration, different cytokines affect cognitive functions in mice. In this study, we evaluated the effects of mouse interleukin-6 (IL-6) on the classical behavioural test of scopolamine-induced amnesia for a passive avoidance response in the mouse. Pretraining i.p. administration of this cytokine (0.125 and 0.5 microgram/mouse) significantly reduced the amnesic action of the muscarinic receptor antagonist. As it is well known that brain amino acids are deeply involved in the modulation of cognitive processes we measured the levels of glutamine, aspartic acid, glutamic acid and GABA in the hippocampus and hypothalamus of mice treated with IL-6. At both doses which affected the cognitive functions, this cytokine had no effect on brain levels of measured amino acids. Neither nociceptive thresholds to a thermal stimulus, nor spontaneous locomotor activity were modified by the acute administration of IL-6 (0.5 microgram/mouse). Our data confirm previous observations indicating that peripheral administration of cytokines affects some, but not other brain functions and suggest the involvement of IL-6 in the central modifications induced by the immune activation.
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PMID:Interleukin-6 affects scopolamine-induced amnesia, but not brain amino acid levels in mice. 942 97

We have identified a preparation of recombinant murine interleukin-6 (mIL-6) that, in addition to the anticipated product, also contained approximately equal amounts of mIL-6 with a C-terminal pentapeptide extension. The extension mutant was generated by readthrough of the stopcodon, and termination at a second in-frame stopcodon 12 base pairs 3' in the expression vector. Aliquots of the preparation were subjected to proteolytic digestion with Asp-N and Lys-C-endopeptidase. The resultant peptides were separated by reversed-phase capillary HPLC, and analysed using a combination of mass spectrometry and N-terminal sequence analysis. These data revealed a C-terminal pentapeptide (Gln-Gly-Ser-Val-Asp) extension, with the authentic stopcodon being translated as glutamine. The extension mutant was isolated by reversed-phase HPLC and shown to have similar mitogenic activity to mIL-6 on murine hybridoma 7TD1 cells.
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PMID:Identification and characterization of recombinant murine interleukin-6 with a C-terminal pentapeptide extension using capillary reversed phase HPLC-MS and edman degradation. 941 11

Loss of long-term hematopoietic stem cell function in vitro is associated with cell cycle progression. To determine whether cytokine-induced proliferation also limits the rate of short-term engraftment and potential clinical utility of ex vivo expanded hematopoietic cells, murine Sca-1(+)c-kit(+)Lin(-) cells were cultured in interleukin-6 (IL-6), IL-11, granulocyte colony-stimulating factor (G-CSF), stem cell factor, flk-2 ligand, and thrombopoietin for 7 days. Cells amplified 2000-fold were then stained with Hoechst 33342, separated into G(0)/G(1) (72% +/- 3%) or S/G(2)/M (27% +/- 3%) fractions by flow sorting, and injected into lethally irradiated mice. Although long-term (more than 6 months) engraftment of lymphoid and myeloid lineages was greater in primary and secondary recipients of expanded cells residing in G(0)/G(1) at the time of transplantation, there were no noted differences in the short-term (less than 6 weeks) recovery kinetics of circulating blood cells. When hematopoietic cells were expanded in cultures containing the tetrapeptide stem cell inhibitor N-Acetyl-Ser-Asp-Lys-Pro (AcSDKP) to reduce progenitor cycling prior to transplantation, again there were no differences observed in short-term reconstitution by inhibited or uninhibited cells. Interestingly, AcSDKP significantly accelerated engraftment by expanded hematopoietic cells when administered in vivo at the time of transplantation. Leukocytes recovered to 20% of normal levels approximately 1 week faster, and thrombocytopenia was largely abrogated in AcSDKP-treated versus untreated mice. Therefore, while AcSDKP can accelerate the engraftment of ex vivo expanded hematopoietic progenitors, which suggests a relatively simple approach to improve their clinical utility, its effects appear unrelated to cell cycle arrest. (Blood. 2000;95:2829-2837)
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PMID:Effects of cell cycle activation on the short-term engraftment properties of ex vivo expanded murine hematopoietic cells. 1077 28


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