Biomedical subjects

G Ciliberto

Publications and source records attributed to G Ciliberto.

At least 91 records · Page 5Linked to original sources

PubMed1995

Two distinct and independent sites on IL-6 trigger gp 130 dimer formation and signalling.

The helical cytokine interleukin (IL) 6 and its specific binding subunit IL-6R alpha form a 1:1 complex which, by promoting homodimerization of the signalling subunit gp130 on the surface of target cells, triggers intracellular responses. We expressed differently tagged forms of gp130 and used them in solution-phase binding assays to show that the soluble extracellular domains of gp130 undergo dimerization in the absence of membranes. In vitro receptor assembly reactions were also performed in the presence of two sets of IL-6 variants carrying amino acid substitutions in two distinct areas of the cytokine surface (site 2, comprising exposed residues in the A and C helices, and site 3, in the terminal part of the CD loop). The binding affinity to IL-6R alpha of these variants is normal but their biological activity is poor or absent. We demonstrate here that both the site 2 and site 3 IL-6 variants complexed with IL-6R alpha bind a single gp130 molecule but are unable to dimerize it, whereas the combined site 2/3 variants lose the ability to interact with gp130. The binding properties of these variants in vitro, and the result of using a neutralizing monoclonal antibody directed against site 3, lead to the conclusion that gp130 dimer is formed through direct binding at two independent and differently oriented sites on IL-6. Immunoprecipitation experiments further reveal that the fully assembled receptor complex is composed of two IL-6, two IL-6R alpha and two gp130 molecules. We propose here a model representing the IL-6 receptor complex as hexameric, which might be common to other helical cytokines.

Amino Acid Sequence↗

PubMed1995

Synergistic stimulation of interleukin 6 release and gene expression by phorbol esters and interleukin 1 beta in rat cortical astrocytes: role of protein kinase C activation and blockade.

The involvement of protein kinase C and its interaction with interleukin 1 beta in the control of interleukin 6 release by cortical astrocytes was studied. The blockade of protein kinase C catalytic domain, by staurosporine, as well as the desensitization of protein kinase C by short-term phorbol 12-myristate 13-acetate pretreatment, increased the basal release of interleukin 6 by rat cortical astrocytes, whereas calphostin C, an antagonist of phorbol ester binding on protein kinase C regulatory domain, did not affect the basal release of the cytokine. The activation of protein kinase C by phorbol 12-myristate 13-acetate enhanced concentration- and time-dependently interleukin 6 release. This stimulatory action of phorbol 12-myristate 13-acetate was significantly reduced by staurosporine, by calphostin C and by the desensitization of protein kinase C. Interleukin 1 beta increased interleukin 6 release in a concentration-related manner. Protein kinase C inhibition, by staurosporine or desensitization, potentiated severalfold, whereas calphostin C reduced interleukin 1 beta stimulation of interleukin 6 release. The treatment of cortical astrocytes with both interleukin 1 beta (3 ng/ml) and phorbol 12-myristate 13-acetate (10 nM) caused a synergistic stimulation of interleukin 6 release and its gene expression, an effect that was not relieved by either 20 nM staurospine or by calphostin C but was slightly affected by protein kinase C desensitization. In conclusion, our data show that in rat cortical astrocytes the basal release of interleukin 6 is under a tonic inhibition exerted by a protein kinase C isoform or isoforms sensitive to blockade by staurosporine and desensitization but insensitive to calphostin C.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Rational design of a receptor super-antagonist of human interleukin-6.

Interleukin-6 (IL-6) is a differentiation and growth factor for a variety of cell types and its excessive production plays a major role in the pathogenesis of multiple myeloma and post-menopausal osteoporosis. IL-6, a four-helix bundle cytokine, is believed to interact sequentially with two transmembrane receptors, the low-affinity IL-6 receptor (IL-6R alpha) and the signal transducer gp130, via distinct binding sites. In this paper we show that combined mutations in the predicted A and C helices, previously suggested to establish contacts with gp130, give rise to variants with no bioactivity but unimpaired binding to IL-6R alpha. These mutants behave as full and selective IL-6 receptor antagonists on a variety of human cell lines. Furthermore, a bifacial mutant was generated (called IL-6 super-antagonist) in which the antagonist mutations were combined with amino acid substitutions in the predicted D helix that increase binding for IL-6R alpha. The IL-6 super-antagonist has no bioactivity, but improved first receptor occupancy and, therefore, fully inhibits the wild-type cytokine at low dosage. The demonstration of functionally independent receptor binding sites on IL-6 suggests that it could be possible to design super-antagonists of other helical cytokines which drive the assembly of structurally related multisubunit receptor complexes.

Carcinoma, Hepatocellular↗

PubMed1994

The affinity-selection of a minibody polypeptide inhibitor of human interleukin-6.

A major challenge in basic and applied biological research is the engineering of small proteins with pre-determined structures and novel functions. In a limited number of cases, this has been achieved by de novo design. An alternative combinatorial approach is based on the construction of large libraries of random peptides and on methods for the selection of the desired molecules. Here we describe a successful combination of both the rational design and the combinatorial approaches for developing proteins with useful biological functions, in this case the construction of a specific inhibitor of the cytokine human interleukin-6. In previous work, the 'minibody', a 61 residue polypeptide consisting of a beta-pleated framework and two hypervariable regions, was designed, synthesized and expressed on f1 phage surface. We report the construction of a repertoire of 50 million minibodies displayed on phage in which the hypervariable regions have been randomized. One polypeptide which binds tightly and specifically to human interleukin-6 was isolated from this collection of minibody mutants. This particular minibody is an effective inhibitor of the cytokine's biological activity. The approach described here could in principle be applied to other molecular targets.

Amino Acid Sequence↗

PubMed1994

Development of progressive kidney damage and myeloma kidney in interleukin-6 transgenic mice.

Interleukin-6 (IL-6) is a pleiotropic cytokine that has been postulated as playing a role in the pathogenesis of multiple myeloma, chronic autoimmune diseases, and alcoholic liver cirrhosis. We generated transgenic mice carrying a fusion between the mouse metallothionein-I (MT-I) gene promoter and the human IL-6 cDNA. MT-I/IL-6 transgenics express IL-6 constitutively in the liver and secrete the cytokine in the blood. They show initially activation of acute-phase response genes and accumulation of alpha 2- and beta-globulins in the plasma, which is followed by polyclonal hypergammaglobulinemia. MT-I/IL-6 transgenics die between 12 to 20 weeks of age. Histologic examination of transgenic animals at different ages and after necropsy showed, as expected from previous studies of IL-6 disregulation in vivo, an increase in the number of megakaryocytes in the spleen and bone marrow and, at later stages, IgG plasmacytosis in the spleen, lymph nodes, and thymus. However, no plasma cell infiltration was detected in other organs. The distinguishing feature of MT-I/IL-6 transgenics is the development of a progressive kidney pathology, in which the initial membranous glomerulonephritis is followed by focal glomerulosclerosis and finally by extensive tubular damage that reproduces the damage observed in patients at terminal stages of multiple myeloma (myeloma kidney). The pathogenetic role of IL-6 overproduction and of the resulting serum protein overload in the kidney damage is discussed.

Acute-Phase Proteins↗

PubMed1994

Oncostatin M binds directly to gp130 and behaves as interleukin-6 antagonist on a cell line expressing gp130 but lacking functional oncostatin M receptors.

Oncostatin M (OM) and interleukin 6 (IL-6) are functionally related cytokines, which trigger similar biological responses because they share gp130 as a common signal transducing transmembrane receptor. While IL-6 recruits gp130 only upon binding to its specific receptor subunit (IL-6R alpha), reconstitution and cross-linking experiments on cell membranes suggest that OM can directly interact with gp130 and that this interaction is necessary but not sufficient to stimulate cells. However, the issue of the direct binding between gp130 and OM, in the absence of any additional membrane component, remained essentially unclarified. In this paper we show that, uniquely among the family of cytokines that transduce through gp130, OM directly binds in vitro with a 10(-8) M affinity sgp130, a soluble form of gp130. Moreover, titration of sgp130 with OM inhibits the formation of a ternary complex comprising IL-6, sIL-6R alpha, and sgp130. These in vitro properties of OM are consistent with the additional finding that on human hepatoma Hep3B cells, which express gp130 but not functional OM receptors, OM does not mimic IL-6 activity, but rather behaves, at high doses, as an IL-6 antagonist.

Animals↗

PubMed1994

A fast and sensitive colorimetric assay for IL-6 in hepatoma cells based on the production of a secreted form of alkaline phosphatase (SEAP).

Structure-function studies of cytokines require that simple, sensitive and reliable biological assays are available. A well known property of interleukin-6 (IL-6) is that of being able to induce transcription from several liver-specific promoters in human hepatoma cells. However, the available assays of IL-6 in hepatoma cells, which are either based on the detection of increased expression of endogenous acute phase response genes or on the activation of reporter genes transfected under the control of IL-6 responsive promoters, are not very sensitive and are time consuming. We have established a new assay for IL-6 in hepatoma cells which is based on the transfection of an IL-6 inducible promoter/secreted alkaline phosphatase (SEAP) gene fusion and which measures the inducible production and release of SEAP in the culture medium. SEAP activity is measured with a simple colorimetric assay that requires no cell manipulation, thus allowing a large set of samples to be analysed simultaneously. The CRP/SEAP assay can be used in studies on the structure-function relationships of human IL-6.

Alkaline Phosphatase↗

PubMed1994

Generation of interleukin-6 receptor antagonists by molecular-modeling guided mutagenesis of residues important for gp130 activation.

Interleukin-6 (IL-6) drives the sequential assembly of a receptor complex formed by the IL-6 receptor (IL-6R alpha) and the signal transducing subunit, gp130. A model of human IL-6 (hIL-6) was constructed by homology using the structure of bovine granulocyte colony stimulating factor. The modeled cytokine was predicted to interact sequentially with the cytokine binding domains of IL-6R alpha and gp130 bridging them in a way similar to that of the interaction between growth hormone and its homodimeric receptor. Several residues on helices A and C which were predicted as contact points between IL-6 and gp130 and therefore essential for IL-6 signal transduction, were subjected to site-directed mutagenesis individually or in combined form. Interestingly, while single amino acid changes never produced major alterations in IL-6 bioactivity, a subset of double mutants of Y31 and G35 showed a considerable reduction of biological activity and were selectively impaired from associating with gp130 in binding assays in vitro, while they maintained wild-type affinity towards hIL-6-R alpha. More importantly, we demonstrated the antagonistic effect of mutant Y31D/G35F versus wild-type IL-6.

Amino Acid Sequence↗

PubMed1994

Interleukin-6 deficient mice are protected from bone loss caused by estrogen depletion.

Interleukin-6 (IL-6) is a multifunctional cytokine whose circulating levels are under physiological conditions below detection, but whose production is rapidly and strongly induced by several pathological and inflammatory stimuli. IL-6 has been implicated in a number of cell functions connected to immunity and hematopoiesis. Recently, it has been proposed to act as a stimulator of osteoclast formation and activity, in particular following estrogen depletion. The purpose of this study was to gain additional insights into the role of IL-6 during development, as well as in physiological and pathological conditions. We report here that IL-6 deficient mice generated by gene targeting are viable and do not present any evident phenotypic abnormality. However, analysis of bone metabolism revealed a specific bone phenotype. IL-6 deficient female mice have a normal amount of trabecular bone, but higher rates of bone turnover than control littermates. Estrogen deficiency induced by ovariectomy causes in wild type animals a significant loss of bone mass together with an increase in bone turnover rates. Strikingly, ovariectomy does not induce any change in either bone mass or bone remodeling rates in the IL-6 deficient mice. These findings indicate that IL-6 plays an important role in the local regulation of bone turnover and, at least in mice, appears to be essential for the bone loss caused by estrogen deficiency.

Animals↗

PubMed1994

High yield expression and purification of human endothelin-1.

A DNA construct encoding human big endothelin (Big ET) preceded by the factor Xa protease recognition site (Ile-Glu-Gly-Arg), fused in frame to the maltose binding protein sequence, has been introduced in DH5-alpha cells. The fusion product (MBP-Big ET) was expressed at a concentration close to 100 micrograms/ml of culture broth and constituted approximately 50% of the total protein content. Crude cell extracts containing the fusion product have been directly treated with trypsin under mild denaturing conditions in order to release big endothelin (1-37) from the adduct. Cleavage yield of the MBP-Big ET adduct was close to 70%. Big ET(1-37) was separated from unrelated peptides derived from the tryptic digest of the bacterial extract by affinity chromatography. The affinity column was prepared by immobilizing a protease resistant peptide ligand able to recognize Big ET with sufficient affinity, selectivity, and specificity. From the affinity step (recovery, 90%), recombinant Big ET(1-37) was obtained with a purity close to 80%. The affinity-purified recombinant product was then digested with alpha-chymotrypsin in order to release endothelin (1-21), which was then purified by RP-HPLC. With this two-step purification protocol, 3 micrograms of endothelin was recovered from 1 ml of bacterial broth, with a purity close to 95%.

ATP-Binding Cassette Transporters↗

PubMed1993

Saturation mutagenesis of the human interleukin 6 receptor-binding site: implications for its three-dimensional structure.

Interleukin 6 is a 184-aa polypeptide postulated to belong to the class of helical cytokines. We built a three-dimensional model of human interleukin 6 based on the similarity of its hydrophobicity pattern with that of other cytokines and on the x-ray structure of growth hormone, interleukin 2, interleukin 4, interferon beta, and granulocyte-macrophage colony-stimulating factor. The resulting model is a bundle of four alpha-helices and suggests possible alternative conformations for the 9 C-terminal amino acids; in this region, the importance of Arg-182 and Met-184 for biological activity has been demonstrated [Lutticken, C., Kruttgen, A., Moller, C., Heinrich, P.C. & Rose-John, S. (1991) FEBS Lett. 282, 265-267]. Therefore, we generated a large collection of single-amino acid variants in residues 175-181. Analysis of their biological activity in two systems and the receptor binding properties of a subset of the mutants indicates that the entire region is involved in forming the receptor binding surface and supports the hypothesis that this region does not assume an alpha-helical conformation. Remarkably, we also found a mutant with receptor affinity and biological activity much higher than wild type; the potential therapeutical value of this finding is discussed.

Amino Acid Sequence↗

PubMed1993

Involvement of the Arg179 in the active site of human IL-6.

Three internal-amino acid deletions of amino acids 171-179 of human interleukin 6 (IL-6) were introduced at the cDNA level. While all deletion proteins were biologically inactive, immunoprecipitations with a set of conformation-specific anti-(IL-6) monoclonal antibodies showed that only mutant delta 177-179 does not present major alterations in folding. This finding, together with the observation that delta 177-179 is not able to compete with IL-6 for binding to the soluble human IL-6 receptor, suggested that some or all of these three residues participate to the composition of the receptor-binding site of human IL-6. A large number of single-amino-acid-substitution mutants were generated in residues 177, 178 and 179. Their detailed analysis revealed that Arg179 is crucial for activity in mouse cells, because all amino acid substitutions in this position cause a dramatic drop of biological activity on murine hybridoma cells without affecting the overall protein folding. The only substitution which preserved some residual activity was the conservative Arg to Lys change. This demonstrates the absolute requirement for a positive charge in position 179 for the interaction of human IL-6 with its receptor.

Arginine↗

PubMed1993

The two C/EBP isoforms, IL-6DBP/NF-IL6 and C/EBP delta/NF-IL6 beta, are induced by IL-6 to promote acute phase gene transcription via different mechanisms.

The promoter regions of three IL-6 inducible genes, hemopexin (Hpx), haptoglobin (Hp) and C-reactive protein (CRP) contain cis-acting IL-6 responsive elements (IL-6REs) which are necessary and sufficient to induce IL-6 transcription activation. Transcription factors of the C/EBP family interact with IL-6REs. Among these, IL-6DBP/NF-IL6 plays a key role in IL-6 signal transduction because its trans-activation potential is induced by IL-6 in the human hepatoma cell line Hep3B. We show here that a different C/EBP-related factor, C/EBP delta/NF-IL6 beta, is the major IL-6 induced protein interacting with IL-6REs in the nuclei of Hep3B cells. In contrast to IL-6DBP/NF-IL6, whose activity in Hep3B cells is modulated by IL-6 via a post-translational mechanism, C/EBP delta/NF-IL6 beta is transcriptionally induced by IL-6. Another contrasting feature is that the C/EBP delta cDNA transfected in Hep3B cells activates transcription from an IL-6RE synthetic promoter in a constitutive manner which is not further enhanced by IL-6. Therefore, in Hep3B cells, two distinct members of the C/EBP family are recruited in the IL-6 signal transduction pathway via different mechanisms.

Acute-Phase Proteins↗

PubMed1993

Structural characterization of a biologically active human lipocortin 1 expressed in Escherichia coli.

Lipocortin or annexin 1 is a calcium-dependent phospholipid-binding protein which probably acts as a glucocorticoid- regulated anti-inflammatory factor. cDNA for human lipocortin 1 was cloned in the pT7.7 expression plasmid under the control of the inducible bacteriophage T7 RNA polymerase promoter. Upon induction with isopropyl thio-beta-D-galactoside, large amounts of the protein were produced and accumulated in Escherichia coli in a soluble form. The recombinant protein was purified to homogeneity by means of two subsequent ion-exchange chromatographic steps. The final yield was about 30 mg/l bacterial culture. Electrospray mass spectrometric analysis of the purified protein demonstrated that the recombinant product corresponds to the native human lipocortin 1, without the initial methionine and with a free N-terminal alanine; tryptic peptide mapping by fast-atom-bombardment mass spectrometry showed that the recombinant protein contains cysteine residues at positions 263 and 324 with free thiol groups, whereas Cys270 and Cys343 are probably involved in an intrachain disulfide bridge. Recombinant human lipocortin 1 reduces the carrageenin-induced paw oedema in rat in vivo and inhibits porcine pancreatic phospholipase A2 activity in vitro; in both cases, a dose-related response is observed.

Amino Acid Sequence↗

PubMed1993

A bipartite activation domain is responsible for the activity of transcription factor HNF1/LFB1 in cells of hepatic and nonhepatic origin.

HNF1/LFB1 is a transcription factor that controls the expression of several liver-specific genes. Previous in vitro experiments allowed us to identify two different regions in the carboxy-terminal portion of the protein responsible for most of the transcription activation potential: the first, ADI, between amino acids 546 and 628 and the second, ADII, between amino acids 281 and 318. To characterize the molecular anatomy of HNF1/LFB1 better, we have analyzed its trans-activating properties in vivo. Several HNF1/LFB1 deletion mutants were tested for their ability to induce transcription from HNF1/LFB1-dependent synthetic promoters in cells of hepatic and nonhepatic origin. These last recipient cells provide an HNF1/LFB1-deficient environment that is useful for a precise quantification of the recombinant protein. Our results confirm the importance of ADI and indicate that no activating property can be assigned to ADII in vivo. Moreover, a novel glutamine/proline-rich activation domain (ADIII) has been identified between amino acids 440 and 506. These findings are confirmed by domain-swapping experiments, carried out with the heterologous GAL4 DNA-binding domain, which also show that the activity of each individual activation domain is influenced by combining adjacent HNF1/LFB1 sequences. The data presented indicate that HNF1/LFB1 transcription activating potential relies on a complex structure and also provide important clues to understanding the different functions exerted by transcription factors of this family.

Amino Acid Sequence↗

PubMed1993

Anti-inflammatory actions of an N-terminal peptide from human lipocortin 1.

An acetylated polypeptide corresponding to residues 2-26 of human lipocortin 1 was synthesized and the anti-inflammatory activity assessed in three models of acute inflammation in rat and mouse. In the carrageenin rat paw oedema test, the peptide produced a maximal inhibition of approximately 41% at the 3 h time point with a 10 micrograms dose. When rat paw oedema was induced by the injection of venom phospholipase A2, the peptide produced a significant inhibition (31%) at the top dose of 20 micrograms per paw. In the mouse air-pouch model, systemic treatment with the peptide produced a dramatic reduction in cytokine-induced leukocyte migration with an ID50 of approximately 40 micrograms per mouse. The N-terminal peptide 2-26 shares the actions of lipocortin 1 in these acute models of inflammation.

Amino Acid Sequence↗

PubMed1993

A Zn-finger protein, Xfin, is expressed during cone differentiation in the retina of the frog Xenopus laevis.

Xfin is a member of a Zn-finger multigene family that shares homology with the Drosophila segmentation gene, Krüppel. This paper reports on our identification and cellular localization, through the utilization of specific antibodies, of the expression product of Xfin in the Xenopus laevis retina, and its pattern of expression during the retinal developmental stages. By immunostaining sections of the retina, we show that the major staining is localized in the cytoplasm of the cones. The protein appears at an early differentiation stage of the cones, when they can not be univocally identified by morphological criteria, and is maintained up to the adult retina. The same antigenicity pattern is detectable in the retina of the anuran genus Bufo. The immunostaining data are confirmed by Western blot analysis on Xenopus eye protein extracts. Because of its cytoplasmic localization, and because of the ability of Zn-finger proteins to bind nucleic acids, we think that Xfin may be involved in the terminal differentiation of the cones through RNA-protein interactions.

Animals↗

PubMed1993

Inducible formation of liver tumors in transgenic mice.

Transgenic mice have been generated with an inducible SV40 t/T antigen construct with the aim of analysing the early changes that take place in the course of liver tumorigenesis. The strictly liver-specific human C-reactive protein (CRP) gene promoter was chosen for the control of the transgene expression because this promoter can be turned on transiently by injection of bacterial lipopolysaccharide. Among 10 independently derived CRP-Tag mouse lines five showed inducible expression of the CRP-Tag transgene in liver. However, only one had a tight control of the transgene with virtually no expression under physiological conditions and high levels of Tag expression after stimulation. Females of this line were used to analyse the progression of liver alterations upon repeated induction of the t/T antigen for different lengths of time. The first signs of transgene-induced liver alterations could be monitored by the activation of the marker enzyme gamma-glutamyltranspeptidase 30 days after the start of the induction program. After 90 days hepatocellular carcinomas were already detectable. Thus, CRP-Tag mice constitute an excellent system to analyse the sequential events that take place during liver carcinogenesis.

Animals↗