52 results on '"Giacomo Paonessa"'
Search Results
2. Identification of novel multi-stage histone deacetylase (HDAC) inhibitors that impair Schistosoma mansoni viability and egg production
- Author
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Alessandra Guidi, Fulvio Saccoccia, Nadia Gennari, Roberto Gimmelli, Emanuela Nizi, Cristiana Lalli, Giacomo Paonessa, Giuliana Papoff, Alberto Bresciani, and Giovina Ruberti
- Subjects
Schistosoma mansoni ,HDAC inhibitors ,Parasite reproductive systems ,Infectious and parasitic diseases ,RC109-216 - Abstract
Abstract Background Novel anti-schistosomal multi-stage drugs are needed because only a single drug, praziquantel, is available for the treatment of schistosomiasis and is poorly effective on larval and juvenile stages of the parasite. Schistosomes have a complex life-cycle and multiple developmental stages in the intermediate and definitive hosts. Acetylation and deacetylation of histones play pivotal roles in chromatin structure and in the regulation of transcription in eukaryotic cells. Histone deacetylase (HDAC) inhibitors modulate acetylation of several other proteins localized both in the nucleus and in the cytoplasm and therefore impact on many signaling networks and biological processes. Histone post-translational modifications may provide parasites with the ability to readily adapt to changes in gene expression required for their development and adaptation to the host environment. The aim of the present study was to screen a HDAC class I inhibitor library in order to identify and characterize novel multi-stage hit compounds. Methods We used a high-throughput assay based on the quantitation of ATP in the Schistosoma mansoni larval stage (schistosomula) and screened a library of 1500 class I HDAC inhibitors. Subsequently, a few hits were selected and further characterized by viability assays and phenotypic analyses on adult parasites by carmine red and confocal microscopy. Results Three compounds (SmI-124, SmI-148 and SmI-558) that had an effect on the viability of both the schistosomula larval stage and the adult worm were identified. Treatment with sub-lethal doses of SmI-148 and SmI-558 also decreased egg production. Moreover, treatment of adult parasites with SmI-148, and to a lesser extent Sm-124, was associated with histone hyperacetylation. Finally, SmI-148 and SmI-558 treatments of worm pairs caused a phenotype characterized by defects in the parasite reproductive system, with peculiar features in the ovary. In addition, SmI-558 induced oocyte- and vitelline cell-engulfment and signs of degeneration in the uterus and/or oviduct. Conclusions We report the screening of a small HDAC inhibitor library and the identification of three novel compounds which impair viability of the S. mansoni larval stage and adult pairs. These compounds are useful tools for studying deacetylase activity during parasite development and for interfering with egg production. Characterization of their specificity for selected S. mansoni versus human HDAC could provide insights that can be used in optimization and compound design.
- Published
- 2018
- Full Text
- View/download PDF
3. Spiro-containing derivatives show antiparasitic activity against Trypanosoma brucei through inhibition of the trypanothione reductase enzyme.
- Author
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Lorenzo Turcano, Theo Battista, Esther Torrente De Haro, Antonino Missineo, Cristina Alli, Giacomo Paonessa, Gianni Colotti, Steven Harper, Annarita Fiorillo, Andrea Ilari, and Alberto Bresciani
- Subjects
Arctic medicine. Tropical medicine ,RC955-962 ,Public aspects of medicine ,RA1-1270 - Abstract
Trypanothione reductase (TR) is a key enzyme that catalyzes the reduction of trypanothione, an antioxidant dithiol that protects Trypanosomatid parasites from oxidative stress induced by mammalian host defense systems. TR is considered an attractive target for the development of novel anti-parasitic agents as it is essential for parasite survival but has no close homologue in humans. We report here the identification of spiro-containing derivatives as inhibitors of TR from Trypanosoma brucei (TbTR), the parasite responsible for Human African Trypanosomiasis. The hit series, identified by high throughput screening, was shown to bind TbTR reversibly and to compete with the trypanothione (TS2) substrate. The prototype compound 1 from this series was also found to impede the growth of Trypanosoma brucei parasites in vitro. The X-ray crystal structure of TbTR in complex with compound 1 solved at 1.98 Å allowed the identification of the hydrophobic pocket where the inhibitor binds, placed close to the catalytic histidine (His 461') and lined by Trp21, Val53, Ile106, Tyr110 and Met113. This new inhibitor is specific for TbTR and no activity was detected against the structurally similar human glutathione reductase (hGR). The central spiro scaffold is known to be suitable for brain active compounds in humans thus representing an attractive starting point for the future treatment of the central nervous system stage of T. brucei infections.
- Published
- 2020
- Full Text
- View/download PDF
4. SAR evolution towards potent C-terminal carboxamide peptide inhibitors of Zika virus NS2B-NS3 protease
- Author
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Stefania Colarusso, Federica Ferrigno, Simona Ponzi, Francesca Pavone, Immacolata Conte, Luigi Abate, Elisa Beghetto, Antonino Missineo, Jerome Amaudrut, Alberto Bresciani, Giacomo Paonessa, Licia Tomei, Christian Montalbetti, Elisabetta Bianchi, Carlo Toniatti, and Jesus M. Ontoria
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Dose-Response Relationship, Drug ,Molecular Structure ,Organic Chemistry ,Clinical Biochemistry ,Serine Endopeptidases ,Pharmaceutical Science ,Microbial Sensitivity Tests ,Zika Virus ,Dengue Virus ,Viral Nonstructural Proteins ,Biochemistry ,Antiviral Agents ,Structure-Activity Relationship ,Drug Discovery ,Molecular Medicine ,Humans ,Protease Inhibitors ,Peptides ,Molecular Biology ,West Nile virus ,RNA Helicases - Abstract
Zika virus (ZIKV) is a member of the Flaviviridae family that can cause neurological disorders and congenital malformations. The NS2B-NS3 viral serine protease is an attractive target for the development of new antiviral agents against ZIKV. We report here a SAR study on a series of substrate-like linear tripeptides that inhibit in a non-covalent manner the NS2B-NS3 protease. Optimization of the residues at positions P1, P2, P3 and of the N-terminal and C-terminal portions of the tripeptide allowed the identification of inhibitors with sub-micromolar potency with phenylglycine as arginine-mimicking group and benzylamide as C-terminal fragment. Further SAR exploration and application of these structural changes to a series of peptides having a 4-substituted phenylglycine residue at the P1 position led to potent compounds showing double digit nanomolar inhibition of the Zika protease (IC
- Published
- 2021
5. Optimization of 2-(1H-imidazo-2-yl)piperazines series of Trypanosoma brucei growth inhibitors as potential treatment for the second stage of HAT
- Author
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Nadia Gennari, Maria Vittoria Orsale, Vincenzo Summa, Rita Graziani, Simona Ponzi, Jesus Maria Ontoria Ontoria, Alina Ciammaichella, Steven J. Harper, Andreina Basta, Giacomo Paonessa, Ilaria Biancofiore, Valentina Nardi, Federica Ferrigno, Ivan Fini, Melania D'Amico, Ciammaichella, A., Ferrigno, F., Basta, A., D'Amico, M., Biancofiore, I., Nardi, V., Ponzi, S., Graziani, R., Gennari, N., Vittoria Orsale, M., Fini, I., Paonessa, G., Summa, V., Harper, S., and Ontoria, J. M.
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Antiparasitic ,Cell Survival ,medicine.drug_class ,Morpholines ,Trypanosoma brucei brucei ,Clinical Biochemistry ,Drug Evaluation, Preclinical ,Pharmaceutical Science ,Pharmacology ,Trypanosoma brucei ,01 natural sciences ,Biochemistry ,Piperazines ,Structure-Activity Relationship ,Isomerism ,In vivo ,Sleeping sickne ,Drug Discovery ,Human Umbilical Vein Endothelial Cells ,medicine ,Humans ,African trypanosomiasis ,Molecular Biology ,biology ,010405 organic chemistry ,Chemistry ,Organic Chemistry ,medicine.disease ,biology.organism_classification ,Human African Trypanosomiasis (HAT) ,Trypanocidal Agents ,Growth Inhibitors ,0104 chemical sciences ,010404 medicinal & biomolecular chemistry ,Trypanosomiasis, African ,Quinolines ,Molecular Medicine - Abstract
A previous publication from our laboratory reported the identification of a new class of 2-(1H-imidazo-2-yl)piperazines as potent T. brucei growth inhibitors as potential treatment for Human African Trypanosomiasis (HAT). This work describes the structure–activity relationship (SAR) around the hit compound 1, which led to the identification of the optimized compound 18, a single digit nanomolar inhibitor (EC50 7 nM), not cytotoxic and with optimal in vivo profile that made it a suitable candidate for efficacy studies in a mouse model mimicking the second stage of disease.
- Published
- 2020
6. Spiro-containing derivatives show antiparasitic activity against Trypanosoma brucei through inhibition of the trypanothione reductase enzyme
- Author
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Alberto Bresciani, Esther Torrente De Haro, Giacomo Paonessa, Gianni Colotti, Antonino Missineo, Annarita Fiorillo, Cristina Alli, Theo Battista, Andrea Ilari, Steven J. Harper, and Lorenzo Turcano
- Subjects
0301 basic medicine ,Protein Conformation ,highthroughput screening ,RC955-962 ,Glutathione reductase ,Trypanothione ,Drug Evaluation, Preclinical ,Plasma protein binding ,Crystallography, X-Ray ,Biochemistry ,chemistry.chemical_compound ,0302 clinical medicine ,Arctic medicine. Tropical medicine ,Zoonoses ,Medicine and Health Sciences ,NADH, NADPH Oxidoreductases ,Enzyme Inhibitors ,chemistry.chemical_classification ,Protozoans ,Crystallography ,biology ,Physics ,trypanothione reductase ,Eukaryota ,HTS ,neglected tropical diseases ,Condensed Matter Physics ,Glutathione ,Chemistry ,Infectious Diseases ,Physical Sciences ,Crystal Structure ,Public aspects of medicine ,RA1-1270 ,Protein Binding ,Research Article ,Neglected Tropical Diseases ,Chemical Elements ,Trypanosoma ,Antiparasitic ,medicine.drug_class ,030231 tropical medicine ,Trypanosoma brucei brucei ,Antiprotozoal Agents ,Trypanosoma brucei ,African Trypanosomiasis ,03 medical and health sciences ,Trypanosomiasis ,medicine ,Trypanosoma Brucei ,Parasitic Diseases ,Solid State Physics ,Histidine ,Binding Sites ,Protozoan Infections ,Public Health, Environmental and Occupational Health ,Organisms ,Biology and Life Sciences ,biology.organism_classification ,Tropical Diseases ,Parasitic Protozoans ,Carbon ,High-Throughput Screening Assays ,030104 developmental biology ,Enzyme ,chemistry ,Enzymology ,Peptides ,Toluene ,Trypanosoma Brucei Gambiense - Abstract
Trypanothione reductase (TR) is a key enzyme that catalyzes the reduction of trypanothione, an antioxidant dithiol that protects Trypanosomatid parasites from oxidative stress induced by mammalian host defense systems. TR is considered an attractive target for the development of novel anti-parasitic agents as it is essential for parasite survival but has no close homologue in humans. We report here the identification of spiro-containing derivatives as inhibitors of TR from Trypanosoma brucei (TbTR), the parasite responsible for Human African Trypanosomiasis. The hit series, identified by high throughput screening, was shown to bind TbTR reversibly and to compete with the trypanothione (TS2) substrate. The prototype compound 1 from this series was also found to impede the growth of Trypanosoma brucei parasites in vitro. The X-ray crystal structure of TbTR in complex with compound 1 solved at 1.98 Å allowed the identification of the hydrophobic pocket where the inhibitor binds, placed close to the catalytic histidine (His 461’) and lined by Trp21, Val53, Ile106, Tyr110 and Met113. This new inhibitor is specific for TbTR and no activity was detected against the structurally similar human glutathione reductase (hGR). The central spiro scaffold is known to be suitable for brain active compounds in humans thus representing an attractive starting point for the future treatment of the central nervous system stage of T. brucei infections., Author summary Trypanosoma brucei is a parasite responsible for neglected pathologies such as human African trypanosomiasis, also known as sleeping sickness. This disease is endemic in sub-Saharan Africa, with 70 million people at risk of infection. Current treatments for this type of disease are limited by their toxicity, administration in endemic countries and treatment resistance. Therapies against infectious diseases typically rely on targeting one or more components of the parasite that are not present in humans to ensure the best possible therapeutic window. In this case we aimed at targeting the Trypanosoma brucei trypanothione reductase (TR), one enzyme that synthesize the reduced trypanothione a key molecule for preserving the parasite redox balance. This enzyme does not exist in humans that have glutathione instead of trypanothione. Past attempts to identify novel inhibitors of this target has failed to generate drug-like molecules. To overcome this limitation we employed a recent, higher quality, TR activity assay to test a collection of compounds previously reported to be active against these parasites. This approach led to the identification and validation of a new chemotype with a unique mode of inhibition of TR. This chemical series is a drug-like starting point, in fact its core (spiro) is present in drugs approved for human use.
- Published
- 2020
7. Discovery of 2-(1H-imidazo-2-yl)piperazines as a new class of potent and non-cytotoxic inhibitors of Trypanosoma brucei growth in vitro
- Author
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Annalise Di Marco, Steven J. Harper, Savina Malancona, Nadia Gennari, Simona Ponzi, Jesus Maria Ontoria Ontoria, Marcel Kaiser, Rita Graziani, Ilaria Biancofiore, Giacomo Paonessa, Alberto Bresciani, Federica Ferrigno, Vincenzo Summa, Ferrigno, F., Biancofiore, I., Malancona, S., Ponzi, S., Paonessa, G., Graziani, R., Bresciani, A., Gennari, N., Di Marco, A., Kaiser, M., Summa, V., Harper, S., and Ontoria, J. M.
- Subjects
0301 basic medicine ,Antiparasitic ,medicine.drug_class ,030106 microbiology ,Clinical Biochemistry ,Plasmodium falciparum ,Trypanosoma brucei brucei ,Pharmaceutical Science ,Trypanosoma brucei ,HeLa Cell ,Biochemistry ,Piperazines ,03 medical and health sciences ,chemistry.chemical_compound ,Structure-Activity Relationship ,Sleeping sickne ,parasitic diseases ,Drug Discovery ,medicine ,Cytotoxic T cell ,Humans ,African trypanosomiasis ,Malaria, Falciparum ,Trypanosoma cruzi ,Molecular Biology ,Imidazole ,Piperazine ,biology ,2-(1H-imidazo-2-yl)piperazine ,Trypanocidal Agent ,Organic Chemistry ,Imidazoles ,Trypanosoma brucei rhodesiense ,medicine.disease ,biology.organism_classification ,Human African Trypanosomiasis (HAT) ,Virology ,Trypanocidal Agents ,030104 developmental biology ,Trypanosomiasis, African ,chemistry ,Molecular Medicine ,Growth inhibition ,Human ,HeLa Cells - Abstract
The identification of a new series of growth inhibitors of Trypanosoma brucei rhodesiense, causative agent of Human African Trypanosomiasis (HAT), is described. A selection of compounds from our in-house compound collection was screened in vitro against the parasite leading to the identification of compounds with nanomolar inhibition of T. brucei growth. Preliminary SAR on the hit compound led to the identification of compound 34 that shows low nanomolar parasite growth inhibition (T. brucei EC50 5 nM), is not cytotoxic (HeLa CC50 > 25,000 nM) and is selective over other parasites, such as Trypanosoma cruzi and Plasmodium falciparum (T. cruzi EC50 8120 nM, P. falciparum EC50 3624 nM).
- Published
- 2018
8. Identification of novel multi-stage histone deacetylase (HDAC) inhibitors that impair Schistosoma mansoni viability and egg production
- Author
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Giovina Ruberti, Fulvio Saccoccia, Alessandra Guidi, Nadia Gennari, Emanuela Nizi, Alberto Bresciani, Giuliana Papoff, Roberto Gimmelli, Cristiana Lalli, and Giacomo Paonessa
- Subjects
0301 basic medicine ,Male ,Histone Deacetylases ,lcsh:Infectious and parasitic diseases ,Histones ,03 medical and health sciences ,Mice ,HDAC inhibitors ,Gene expression ,Animals ,Humans ,Schistosomiasis ,lcsh:RC109-216 ,Ovum ,Anthelmintics ,Life Cycle Stages ,Mice, Inbred ICR ,biology ,Research ,fungi ,Acetylation ,Helminth Proteins ,Schistosoma mansoni ,biology.organism_classification ,Phenotype ,3. Good health ,Chromatin ,Cell biology ,Histone Deacetylase Inhibitors ,030104 developmental biology ,Infectious Diseases ,Histone ,biology.protein ,Parasitology ,Female ,Histone deacetylase ,Deacetylase activity ,Parasite reproductive systems - Abstract
Background Novel anti-schistosomal multi-stage drugs are needed because only a single drug, praziquantel, is available for the treatment of schistosomiasis and is poorly effective on larval and juvenile stages of the parasite. Schistosomes have a complex life-cycle and multiple developmental stages in the intermediate and definitive hosts. Acetylation and deacetylation of histones play pivotal roles in chromatin structure and in the regulation of transcription in eukaryotic cells. Histone deacetylase (HDAC) inhibitors modulate acetylation of several other proteins localized both in the nucleus and in the cytoplasm and therefore impact on many signaling networks and biological processes. Histone post-translational modifications may provide parasites with the ability to readily adapt to changes in gene expression required for their development and adaptation to the host environment. The aim of the present study was to screen a HDAC class I inhibitor library in order to identify and characterize novel multi-stage hit compounds. Methods We used a high-throughput assay based on the quantitation of ATP in the Schistosoma mansoni larval stage (schistosomula) and screened a library of 1500 class I HDAC inhibitors. Subsequently, a few hits were selected and further characterized by viability assays and phenotypic analyses on adult parasites by carmine red and confocal microscopy. Results Three compounds (SmI-124, SmI-148 and SmI-558) that had an effect on the viability of both the schistosomula larval stage and the adult worm were identified. Treatment with sub-lethal doses of SmI-148 and SmI-558 also decreased egg production. Moreover, treatment of adult parasites with SmI-148, and to a lesser extent Sm-124, was associated with histone hyperacetylation. Finally, SmI-148 and SmI-558 treatments of worm pairs caused a phenotype characterized by defects in the parasite reproductive system, with peculiar features in the ovary. In addition, SmI-558 induced oocyte- and vitelline cell-engulfment and signs of degeneration in the uterus and/or oviduct. Conclusions We report the screening of a small HDAC inhibitor library and the identification of three novel compounds which impair viability of the S. mansoni larval stage and adult pairs. These compounds are useful tools for studying deacetylase activity during parasite development and for interfering with egg production. Characterization of their specificity for selected S. mansoni versus human HDAC could provide insights that can be used in optimization and compound design.
- Published
- 2018
9. Peptidomimetic nitrile inhibitors of malarial protease falcipain-2 with high selectivity against human cathepsins
- Author
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Alberto Bresciani, Alessio Sferrazza, Matteo Andreini, Steven J. Harper, Rita Graziani, Danilo Fabbrini, Emanuela Nizi, Giacomo Paonessa, Valentina Nardi, and Nadia Gennari
- Subjects
Proteases ,Peptidomimetic ,medicine.medical_treatment ,Clinical Biochemistry ,Pharmaceutical Science ,Cysteine Proteinase Inhibitors ,01 natural sciences ,Biochemistry ,Antimalarials ,Structure-Activity Relationship ,parasitic diseases ,Drug Discovery ,Nitriles ,medicine ,Humans ,Malaria, Falciparum ,Molecular Biology ,Cathepsin ,chemistry.chemical_classification ,Protease ,biology ,Dose-Response Relationship, Drug ,Molecular Structure ,010405 organic chemistry ,Chemistry ,Organic Chemistry ,Plasmodium falciparum ,biology.organism_classification ,Cathepsins ,0104 chemical sciences ,010404 medicinal & biomolecular chemistry ,Cysteine Endopeptidases ,Enzyme ,Mechanism of action ,Molecular Medicine ,Peptidomimetics ,medicine.symptom ,Cysteine - Abstract
Falcipain-2 (FP2) is an essential enzyme in the lifecycle of malaria parasites such as Plasmodium falciparum, and its inhibition is viewed as an attractive mechanism of action for new anti-malarial agents. Selective inhibition of FP2 with respect to a family of human cysteine proteases (that include cathepsins B, K, L and S) is likely to be required for the development of agents targeting FP2. Here we describe a series of P2-modified aminonitrile based inhibitors of FP2 that provide a clear strategy toward addressing selectivity for the P. falciparum and show that it can provide potent FP2 inhibitors with strong selectivity against all four of these human cathepsin isoforms.
- Published
- 2017
10. Discovery of a Selective Series of Inhibitors of Plasmodium falciparum HDACs
- Author
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Nadia Gennari, Emanuela Nizi, Steven J. Harper, Savina Malancona, Sergio Altamura, Edith Monteagudo, David Roberts, Ilaria Biancofiore, Paul Willis, Maria Vittoria Orsale, Ottavia Cecchetti, Antonella Cellucci, Ralph Laufer, Rita Graziani, Alberto Bresciani, Simona Ponzi, Jesus Maria Ontoria Ontoria, Maria Veneziano, Annalise Di Marco, Giacomo Paonessa, Vincenzo Summa, Federica Ferrigno, Ontoriajm, G Paonessa, G, Ponzi, S, Ferrigno, F, Nizi, E, Biancofiore, I, Malancona, S, Graziani, R, Roberts, D, Willis, P, Bresciani, A, Nadia Gennari, N, Cecchetti, O, Monteagudo, E, Orsale, Mv, Veneziano, M, Di Marco, A, Cellucci, A, Laufer, R, Sergio Altamura, S, Summa, V, and Harper, S
- Subjects
0301 basic medicine ,biology ,Chemistry ,030106 microbiology ,Organic Chemistry ,Growth inhibitory ,Plasmodium falciparum ,biology.organism_classification ,Biochemistry ,03 medical and health sciences ,030104 developmental biology ,Mechanism of action ,Drug Discovery ,medicine ,medicine.symptom - Abstract
The identification of a new series of P. falciparum growth inhibitors is described. Starting from a series of known human class I HDAC inhibitors a SAR exploration based on growth inhibitory activity in parasite and human cells-based assays led to the identification of compounds with submicromolar inhibition of P. falciparum growth (EC50500 nM) and good selectivity over the activity of human HDAC in cells (up to50-fold). Inhibition of parasital HDACs as the mechanism of action of this new class of selective growth inhibitors is supported by hyperacetylation studies.
- Published
- 2015
11. Structural Basis for Resistance of the Genotype 2b Hepatitis C Virus NS5B Polymerase to Site A Non-Nucleoside Inhibitors
- Author
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Sergio Altamura, Giacomo Paonessa, Andrea Carfi, Marco Mattu, Linda Bartholomew, Giovanni Migliaccio, Antonella Cellucci, Raffaele De Francesco, Donald J. Graham, Steven W. Ludmerer, Gaetano Barbato, and Edwin H. Rydberg
- Subjects
Models, Molecular ,Genotype ,Hepatitis C virus ,Mutant ,Hepacivirus ,Viral Nonstructural Proteins ,Biology ,Crystallography, X-Ray ,medicine.disease_cause ,Antiviral Agents ,Protein Structure, Secondary ,chemistry.chemical_compound ,Structural Biology ,Drug Resistance, Viral ,medicine ,Replicon ,Binding site ,Molecular Biology ,NS5B ,Polymerase ,chemistry.chemical_classification ,Binding Sites ,Indoleacetic Acids ,Nucleosides ,DNA-Directed RNA Polymerases ,Molecular biology ,Kinetics ,Enzyme ,Amino Acid Substitution ,Biochemistry ,chemistry ,Structural Homology, Protein ,biology.protein ,Mutant Proteins - Abstract
Hepatitis C virus (HCV) exists in six major genotypes. Compared with the 1b enzyme, genotype 2b HCV polymerase exhibits a more than 100-fold reduction in sensitivity to the indole-N-acetamide class of non-nucleoside inhibitors. These compounds have been shown to bind in a pocket occupied by helix A of the mobile Lambda1 loop in the apoenzyme. The three-dimensional structure of the HCV polymerase from genotype 2b was determined to 1.9-A resolution and compared with the genotype 1b enzyme. This structural analysis suggests that genotypic variants result in a different shape of the inhibitor binding site. Mutants of the inhibitor binding pocket were generated in a 1b enzyme and evaluated for their binding affinity and sensitivity to inhibition by indole-N-acetamides. Most of the point mutants showed little variation in activity and IC(50), with the exception of 15- and 7-fold increases in IC(50) for Leu392Ile and Val494Ala mutants (1b--2b), respectively. Furthermore, a 1b replicon with 20-fold resistance to this class of inhibitors was selected and shown to contain the Leu392Ile mutation. Chimeric enzymes, where the 2b fingertip Lambda1 loop, pocket or both replaced the corresponding regions of the 1b enzyme, were also generated. The fingertip chimera retained 1b-like inhibitor binding affinity, whereas the other two chimeric constructs and the 2b enzyme displayed between 50- and 100-fold reduction in binding affinity. Together, these data suggest that differences in the amino acid composition and shape of the indole-N-acetamide binding pocket are responsible for the resistance of the 2b polymerase to this class of inhibitors.
- Published
- 2009
12. Development of Humanized Mice for the Study of Hepatitis C Virus Infection
- Author
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Isabella Marcucci, A. Celluci, A. Eutropi, Paolo Turrini, A. Di Marco, Julio Padron, Ralph Laufer, Emanuele Marra, Giovanni Migliaccio, Giacomo Paonessa, R. Sasso, and S. Germoni
- Subjects
Carcinoma, Hepatocellular ,Hepatitis C virus ,Xenotransplantation ,medicine.medical_treatment ,Transplantation, Heterologous ,Alpha (ethology) ,Mice, Transgenic ,Viremia ,Mice, SCID ,Biology ,medicine.disease_cause ,Antiviral Agents ,Mice ,Liver disease ,Cell Line, Tumor ,medicine ,Animals ,Humans ,Serum Albumin ,Transplantation ,Liver Diseases ,Liver Neoplasms ,Antibodies, Monoclonal ,medicine.disease ,Human serum albumin ,Hepatitis C ,Urokinase-Type Plasminogen Activator ,Virology ,Disease Models, Animal ,Immunology ,Hepatocytes ,Surgery ,medicine.drug ,Adult stem cell - Abstract
The development of a small animal model for hepatitis C virus (HCV) infection is a critical issue for the development of novel anti-HCV drugs. To this aim, we have tried many different approaches for generating mice carrying humanized liver. Main efforts were focused on the transplantation of human hepatocytes into immunocompromised mice (SCID-/-, Bg-/-) carrying a genetic lethal liver disease (Alb-uPA). Survival of homozygotic animals should largely depend on early transplantation with healthy hepatocytes. In parallel to establishing a colony of Alb-uPA/SCID/Bg mice, we developed a microsurgical procedure for intrasplenic xenotransplantation of healthy hepatocytes in 1-week-old mice. So far, we generated several chimeras by xenotransplanting human hepatocytes in Alb-uPA+/+/SCID-/-/Bg-/- mice at 1 week after birth. In a first step, identification of successfully engrafted animals is possible by quantification of human serum albumin and human alpha 1 antitrypsin in mouse sera. Additional preliminary histomorphological analysis of liver sections from chimeric animals was also carried out. One of the mice was transiently infected with HCV, reaching viremia levels of approximately 10(5) genomes/mL. However, the efficiency of this system to generate chimeric mice is still very limited. We are currently exploring the use of more robust models of hepatic disease. Moreover, we have been also exploring novel strategies for the generation of chimeric mice by xenotransplanting human adult stem cells, instead of human hepatocytes, at preimmune stages of development.
- Published
- 2006
13. Potent Inhibitors of Subgenomic Hepatitis C Virus RNA Replication through Optimization of Indole-N-Acetamide Allosteric Inhibitors of the Viral NS5B Polymerase
- Author
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Marcello Di Filippo, Licia Tomei, Giovanni Migliaccio, Giacomo Paonessa, Julio Padron, Ralph Laufer, Steven Harper, Frank Narjes, Barbara Pacini, Fabio Bonelli, Salvatore Avolio, Andrea Carfi, Raffaele De Francesco, Michael Rowley, Sergio Altamura, Claudio Giuliano, and Stefania Di Marco
- Subjects
Models, Molecular ,Receptors, Steroid ,Indoles ,Hepatitis C virus ,Allosteric regulation ,Biological Availability ,Receptors, Cytoplasmic and Nuclear ,Genome, Viral ,Hepacivirus ,Viral Nonstructural Proteins ,medicine.disease_cause ,Antiviral Agents ,Virus ,Rats, Sprague-Dawley ,Structure-Activity Relationship ,chemistry.chemical_compound ,Dogs ,Allosteric Regulation ,Cell Line, Tumor ,Acetamides ,Drug Discovery ,medicine ,Animals ,Humans ,Tissue Distribution ,NS5B ,Subgenomic mRNA ,chemistry.chemical_classification ,Pregnane X receptor ,Pregnane X Receptor ,RNA ,RNA-Dependent RNA Polymerase ,Virology ,digestive system diseases ,Rats ,Enzyme ,chemistry ,RNA, Viral ,Molecular Medicine ,Half-Life - Abstract
Infections caused by hepatitis C virus (HCV) are a significant world health problem for which novel therapies are in urgent demand. Compounds that block replication of subgenomic HCV RNA in liver cells are of interest because of their demonstrated antiviral effect in the clinic. In followup to our recent report that indole-N-acetamides (e.g., 1) are potent allosteric inhibitors of the HCV NS5B polymerase enzyme, we describe here their optimization as cell-based inhibitors. The crystal structure of 1 bound to NS5B was a guide in the design of a two-dimensional compound array that highlighted that formally zwitterionic inhibitors have strong intracellular potency and that pregnane X receptor (PXR) activation (an undesired off-target activity) is linked to a structural feature of the inhibitor. Optimized analogues devoid of PXR activation (e.g., 55, EC(50) = 127 nM) retain strong cell-based efficacy under high serum conditions and show acceptable pharmacokinetics parameters in rat and dog.
- Published
- 2005
14. Human hepatocytes in mice receiving pre-immune injection with human cord blood cells
- Author
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Giacomo Paonessa, Giuseppina Bonanno, Giovanni Monego, Giovanni Zelano, Jose Manuel Gonzalez, Sandra Cicuzza, Nadia Rosenthal, Ralph Laufer, Paolo Turrini, and Julio Padron
- Subjects
Transplantation, Heterologous ,Biophysics ,CD34 ,Mice, SCID ,Biology ,HEPATOCYTES ,Biochemistry ,Injections ,Mice ,Immune system ,Antigen ,Pregnancy ,medicine ,Animals ,Humans ,Molecular Biology ,Cell Proliferation ,Settore BIO/16 - ANATOMIA UMANA ,Cell Differentiation ,Cell Biology ,Human serum albumin ,medicine.anatomical_structure ,Liver ,Hepatocyte ,Cord blood ,Immunology ,Female ,Cord Blood Stem Cell Transplantation ,Immunocompetence ,Immunostaining ,STEM CELLS ,Adult stem cell ,medicine.drug - Abstract
It is well established that certain subpopulations of human adult stem cells can generate hepatocyte-like cells when transplanted into adult immunosuppressed mice. In the present study, we wanted to explore whether xeno-transplantation of human cord blood CD34 + (hCBCD34 + ) cells during pre-immune stages of development in immunocompetent mice might also lead to human–mouse liver chimerism. Freshly isolated hCBCD34 + cells were xeno-transplanted into non-immunosuppressed mice by both intra-blastocyst and intra-fetal injections. One and four weeks after birth, immunostaining for different human-specific hepatocyte markers: human hepatocyte-specific antigen, human serum albumin, and human α-1-antitrypsin indicated the presence of human hepatocyte-like cells in the livers of transplanted animals. Detection of human albumin mRNA further corroborated the development of pre-immune human-mouse chimeras. The current report, besides providing new evidence of the potential of hCBCD34 + cells to generate human hepatocyte-like cells, suggests novel strategies for generating immunocompetent mice harboring humanized liver.
- Published
- 2004
15. Persistent Replication of Hepatitis C Virus Replicons Expressing the β-Lactamase Reporter in Subpopulations of Highly Permissive Huh7 Cells
- Author
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Adam J. Simon, Jay A. Grobler, Osvaldo A. Flores, Giacomo Paonessa, Marco F. Pagnoni, Edward M. Murray, and Eric J. Markel
- Subjects
Permissiveness ,viruses ,Hepatitis C virus ,Immunology ,Population ,Replication ,Genome, Viral ,Hepacivirus ,Biology ,Transfection ,Virus Replication ,medicine.disease_cause ,Microbiology ,beta-Lactamases ,Cell Line ,chemistry.chemical_compound ,Genes, Reporter ,Virology ,medicine ,Humans ,Replicon ,education ,Gene ,NS5B ,Genetics ,education.field_of_study ,biochemical phenomena, metabolism, and nutrition ,Viral replication ,chemistry ,Insect Science ,RNA, Viral - Abstract
Progress toward development of better therapies for the treatment of hepatitis C virus (HCV) infection has been hampered by poor understanding of HCV biology and the lack of biological assays suitable for drug screening. Here we describe a powerful HCV replication system that employs HCV replicons expressing the β-lactamase reporter ( bla replicons) and subpopulations of Huh7 cells that are more permissive (or “enhanced”) to HCV replication than naïve Huh7 cells. Enhanced cells represent a small fraction of permissive cells present among naïve Huh7 cells that is enriched during selection with replicons expressing the neomycin phosphotransferase gene ( neo replicons). The level of permissiveness of cell lines harboring neo replicons can vary greatly, and the enhanced phenotype is usually revealed upon removal of the neo replicon with inhibitors of HCV replication. Replicon removal is responsible for increased permissiveness, since this effect could be reproduced either with alpha interferon or with an HCV NS5B inhibitor. Moreover, adaptive mutations present in the replicon genome used during selection do not influence the permissiveness of the resulting enhanced-cell population, suggesting that the mechanisms governing the permissiveness of enhanced cells are independent from viral adaptation. Because the β-lactamase reporter allows simultaneous quantitation of replicon-harboring cells and reporter activity, it was possible to investigate the relationship between genome replication activity and the frequency with which transfected genomes can establish persistent replication. Our study demonstrates that differences in the replication potential of the viral genome are manifested primarily in the frequency with which persistent replication is established but modestly affect the number of replicons observed per replicon-harboring cell. Replicon copy number was found to vary over a narrow range that may be defined by a minimal number required for persistent maintenance and a maximum that is limited by the availability of essential host factors.
- Published
- 2003
16. Cell Clones Selected from the Huh7 Human Hepatoma Cell Line Support Efficient Replication of a Subgenomic GB Virus B Replicon
- Author
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Maura Pizzuti, Andrea Sbardellati, Giacomo Paonessa, Sergio Altamura, Cinzia Traboni, Amedeo De Tomassi, and Rita Graziani
- Subjects
Carcinoma, Hepatocellular ,Sequence analysis ,Hepatitis C virus ,Molecular Sequence Data ,Immunology ,Replication ,RNA-dependent RNA polymerase ,Genome, Viral ,Biology ,Transfection ,Virus Replication ,medicine.disease_cause ,Antiviral Agents ,Microbiology ,Virus ,Cell Line ,Viral Proteins ,Virology ,Tumor Cells, Cultured ,medicine ,Animals ,Humans ,Amino Acid Sequence ,Replicon ,Subgenomic mRNA ,Base Sequence ,Flaviviridae ,RNA ,Sequence Analysis, DNA ,Molecular biology ,Clone Cells ,Viral replication ,Insect Science ,Saguinus - Abstract
Tamarins ( Saguinus species) infected by GB virus B (GBV-B) have recently been proposed as an acceptable surrogate model for hepatitis C virus (HCV) infection. The availability of infectious genomic molecular clones of both viruses will permit chimeric constructs to be tested for viability in animals. Studies in cells with parental and chimeric constructs would also be very useful for both basic research and drug discovery. For this purpose, a convenient host cell type supporting replication of in vitro-transcribed GBV-B RNA should be identified. We constructed a GBV-B subgenomic selectable replicon based on the sequence of a genomic molecular clone proved to sustain infection in tamarins. The corresponding in vitro-transcribed RNA was used to transfect the Huh7 human hepatoma cell line, and intracellular replication of transfected RNA was shown to occur, even though in a small percentage of transfected cells, giving rise to antibiotic-resistant clones. Sequence analysis of GBV-B RNA from some of those clones showed no adaptive mutations with respect to the input sequence, whereas the host cells sustained higher GBV-B RNA replication than the original Huh7 cells. The enhancement of replication depending on host cell was shown to be a feature common to the majority of clones selected. The replication of GBV-B subgenomic RNA was susceptible to inhibition by known inhibitors of HCV to a level similar to that of HCV subgenomic RNA.
- Published
- 2002
17. Human interleukin-6 receptor super-antagonists with high potency and wide spectrum on multiple myeloma cells
- Author
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Elisabetta Sporeno, Gennaro Ciliberto, Armin Lahm, Carlo Toniatti, Ren Xiao Sun, Laura Ciapponi, Giacomo Paonessa, Kari Pulkki, Bernard Klein, Rocco Savino, and Andrea Cabibbo
- Subjects
medicine.medical_specialty ,biology ,medicine.medical_treatment ,Immunology ,Antagonist ,Cell Biology ,Hematology ,Glycoprotein 130 ,Biochemistry ,In vitro ,Endocrinology ,Cytokine ,Internal medicine ,biology.protein ,Cancer research ,medicine ,Cytokine receptor ,Antagonism ,Receptor ,Interleukin 6 - Abstract
Interleukin-6 (IL-6) is the major growth factor for myeloma cells and is believed to participate in the pathogenesis of chronic autoimmune diseases and postmenopausal osteoporosis. IL-6 has been recently shown to possess three topologically distinct receptor binding sites: site 1 for binding to the subunit specific chain IL-6R alpha and sites 2 and 3 for the interaction with two subunits of the signaling chain gp130. We have generated a set of IL-6 variants that behave as potent cytokine receptor super-antagonists carrying substitutions that abolish interaction with gp130 at either site 2 alone (site 2 antagonist) or at both sites 2 and 3 (site 2 + 3 antagonist). In addition, substitutions have been introduced in site 1 that lead to variable increases in binding for IL-6R alpha up to 70-fold. IL-6 super-antagonists inhibit wild-type cytokine activity with efficacy proportional to the increase in receptor binding on a variety of human call lines of different origin, and the most potent molecules display full antagonism at low molar excess to wild-type IL-6. When tested on a representative set of IL-6-dependent human myeloma cell lines, although site 2 super- antagonists were in general quite effective, only the site 2 + 3 antagonist Sant7 showed antagonism on the full spectrum of cells tested. In conclusion, IL-6 super-antagonists are a useful tool for the study of myeloma in vitro and might constitute, in particular Sant7, effective IL-6 blocking agents in vivo.
- Published
- 1996
18. Definition of a Composite Binding Site for gp130 in Human Interleukin-6
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Laura Ciapponi, Gennaro Ciliberto, Giacomo Paonessa, Rocco Savino, Armin Lahm, and Rita Graziani
- Subjects
Receptor complex ,DNA, Complementary ,Stereochemistry ,Molecular Sequence Data ,Leukemia inhibitory factor receptor ,Biology ,Biochemistry ,Antigens, CD ,Cytokine Receptor gp130 ,Tumor Cells, Cultured ,Humans ,Binding site ,Molecular Biology ,Binding Sites ,Membrane Glycoproteins ,Base Sequence ,Interleukin-6 ,Biological activity ,Cell Biology ,Glycoprotein 130 ,In vitro ,Cell biology ,Helix ,Mutagenesis, Site-Directed ,Leukemia inhibitory factor ,Signal Transduction - Abstract
The helical cytokine interleukin-6 (IL-6) assembles a multiprotein receptor complex. The starting event in the activation of intracellular signaling is the binding of the IL-6/IL-6R alpha subcomplex to two gp130 chains. The homodimerization of gp130 is triggered by two distinct and independent regions of IL-6 called sites 2 and 3. Several IL-6 antagonists have been obtained that affect signaling, but not IL-6 IL-6R alpha subcomplex formation. In this paper, we analyze in detail the impact of these antagonists on gp130 binding and dimerization and show that each signaling variant affects gp130 dimerization in vitro and that biological activity on cells decreases in precise parallel to the decrease in gp130 dimerization in vitro. All IL-6 antagonists can be classified into two groups, mapping at either site 2 or 3 in correspondence to their mode of interaction with gp130. We found that site 3 is a large region, which includes residues at the beginning of helix D spatially flanked by residues in the putative AB loop and located at one extremity of the cytokine 4-helix bundle. Interestingly, in leukemia inhibitory factor, another cytokine that signals through gp130, site 3, is topologically conserved but has evolved to bind leukemia inhibitory factor receptor.
- Published
- 1995
19. Monovalent phage display of human interleukin (hIL)-6: selection of superbinder variants from a complex molecular repertoire in the hIL-6 D-helix
- Author
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Giacomo Paonessa, Rocco Savino, Gennaro Ciliberto, Elisabetta Sporeno, Sergio Altamura, Andrea Cabibbo, and Carlo Toniatti
- Subjects
endocrine system ,Receptor complex ,Phage display ,medicine.drug_class ,Mutant ,Biology ,Monoclonal antibody ,Protein Structure, Secondary ,Structure-Activity Relationship ,Antigens, CD ,Cytokine Receptor gp130 ,Genetics ,medicine ,Humans ,Receptors, Cytokine ,Binding site ,Membrane Glycoproteins ,Interleukin-6 ,Wild type ,Antibodies, Monoclonal ,General Medicine ,Fusion protein ,Molecular biology ,Recombinant Proteins ,In vitro ,Protein Structure, Tertiary ,Bacteriophage M13 ,Protein Binding - Abstract
Phage display of proteins can be used to study ligand-receptor interaction and for the affinity-maturation of binding sites in polypeptide hormones and/or cytokines. We have expressed human interleukin-6 (hIL-6) on M13 phage in a monovalent fashion as a fusion protein with the phage coat protein, pIII. Phage-displayed hIL-6 is correctly folded, as judged by its ability to interact with conformation-specific anti-hIL-6 monoclonal antibodies (mAb) and with the hIL-6 receptor complex in vitro. We set up an experimental protocol for the efficient affinity selection of hIL-6 phage using the extracellular portion of the hIL-6 receptor alpha (hIL-6R alpha) fixed on a solid phase. This system was used to affinity-purify from a library of hIL-6 variants, in which four residues in the predicted D-helix of the cytokine were fully randomized, mutants binding hIL-6R alpha with higher efficiency than the wild type. When the best-binder variant Q175I/Q183A was combined with a previously identified superbinder S176R [Savino et al., Proc. Natl. Acad. Sci. 90 (1993) 4067-4071], a triple-substitution mutant Q175I/S176R/Q183A (hIL-6IRA) was obtained with a fivefold increased hIL-6R alpha binding and a 2.5-fold enhanced biological activity.
- Published
- 1995
20. In vitroBinding of Ciliary Neurotrophic Factor to its Receptors: Evidence for the Formation of an IL-6-type Hexameric Complex
- Author
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Anna De Serio, Giacomo Paonessa, Ralph Laufer, Gennaro Ciliberto, and Rita Graziani
- Subjects
Receptor complex ,DNA, Complementary ,Insecta ,Immunoprecipitation ,Stereochemistry ,medicine.medical_treatment ,Molecular Sequence Data ,Nerve Tissue Proteins ,Leukemia inhibitory factor receptor ,Receptors, Nerve Growth Factor ,Ciliary neurotrophic factor ,Structural Biology ,medicine ,Animals ,Amino Acid Sequence ,Ciliary Neurotrophic Factor ,Interleukin 6 ,Receptor ,Receptor, Ciliary Neurotrophic Factor ,Molecular Biology ,Cells, Cultured ,biology ,Interleukin-6 ,Receptor Aggregation ,digestive, oral, and skin physiology ,Glycoprotein 130 ,Recombinant Proteins ,Cell biology ,Cytokine ,Glycerophosphates ,biology.protein - Abstract
Ciliary neurotrophic factor (CNTF) is a cytokine sharing structural and functional similarities with interleukin-6 (IL-6) and other helical cytokines that utilize the common signalling chain gp130. While IL-6 induces gp130 dimerization, CNTF, after the initial interaction with the specific, non-signalling receptor subunit, CNTFR, induces the formation of gp130/LIF-receptor heterodimers. Through immunoprecipitation exper iments with tagged soluble receptor molecules, we recently demonstrated that IL-6 drives the formation of a hexameric receptor complex with a defined topology and composed of two IL-6, two IL-6Rα and two gp130 molecules. Here, we apply the same strategy to study the assemblyin vitroof the CNTF receptor complex. We present evidence that both the cytokine and the specific binding chain undergo dimerization in the presence of gp130. Furthermore, although gp130 and LIFR are able to bind independently to the CNTF/CNTFR sub-complex, they never form homodimers but only heterodimers. We propose that CNTF assembles a hexameric receptor complex composed of two CNTF, two CNTFR, one gp130 and one LIFR molecule, and present a model of the reciprocal interaction of these molecules based on similarities with the IL-6 hexameric complex.
- Published
- 1995
21. Interleukin-6 (IL-6) Antagonism by Soluble IL-6 Receptor⃰ Mutated in the Predicted gp130-binding Interface
- Author
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Anna Laura Salvati, Giacomo Paonessa, Gennaro Ciliberto, Armin Lahm, and Carlo Toniatti
- Subjects
Models, Molecular ,Receptor complex ,Carcinoma, Hepatocellular ,Macromolecular Substances ,Protein Conformation ,Recombinant Fusion Proteins ,Protein subunit ,Molecular Sequence Data ,Mutant ,Spodoptera ,Biology ,Biochemistry ,Chlorocebus aethiops ,Tumor Cells, Cultured ,Animals ,Humans ,Computer Simulation ,Amino Acid Sequence ,Receptor ,Melanoma ,Molecular Biology ,Cell Line, Transformed ,Binding Sites ,Sequence Homology, Amino Acid ,Interleukin-6 ,Liver Neoplasms ,Mutagenesis ,Receptors, Interleukin ,Cell Biology ,Glycoprotein 130 ,Receptors, Interleukin-6 ,Nucleopolyhedroviruses ,Solubility ,Interleukin-6 receptor ,Mutagenesis, Site-Directed ,Signal transduction ,Sequence Alignment ,Signal Transduction - Abstract
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.
- Published
- 1995
22. Production and structural characterization of amino terminally histidine tagged human oncostatin M in E. Coli
- Author
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Giampaolo Nitti, Pietro Pucci, Gaetano Barbato, Giacomo Paonessa, Elisabetta Sporeno, Rita Graziani, Sporeno, E, Barbato, G, Graziani, R, Pucci, Pietro, Nitti, G, and Paonessa, G.
- Subjects
Peptide Biosynthesis ,Circular dichroism ,Transcription, Genetic ,Molecular Sequence Data ,Immunology ,Gene Expression ,Oncostatin M ,Spectrometry, Mass, Fast Atom Bombardment ,Polymerase Chain Reaction ,Biochemistry ,Protein Structure, Secondary ,Cell Line ,Complementary DNA ,Gene expression ,Escherichia coli ,Humans ,Immunology and Allergy ,Histidine ,Amino Acid Sequence ,Cloning, Molecular ,Molecular Biology ,Chromatography, High Pressure Liquid ,Helix bundle ,Expression vector ,Base Sequence ,biology ,Edman degradation ,Chemistry ,E. coli expression ,Hematology ,Molecular biology ,Growth Inhibitors ,Recombinant Proteins ,Models, Structural ,Histidine tag ,biology.protein ,Cytokines ,Tetradecanoylphorbol Acetate ,Peptides ,Circular dicroism - Abstract
Oncostatin M is a cytokine that acts as a growth regulator on a wide variety of cells and has diverse biological activities including acute phase protein induction, LDL receptor up-regulation and cell-specific gene expression. In order to gather information about the One M structure, we established a protocol for large scale production and single step purification of this functional cytokine from bacterial cells. The cDNA of human Onc M was cloned by RT-PCR from total RNA of PMA induced U937 cells. After the addition of a six histidine tag at the N-terminus, the coding region of mature Onc M was cloned in the pT7.7 expression vector. Histidine tagged Onc M was overexpressed in bacterial cells and purified to homogeneity in one step on a metal chelating column. We found that recombinant 6xHis-OncM remains fully active in a growth inhibition assay. Structural characterization of the purified protein was performed by electrospray mass spectrometry, automated Edman degradation and peptide mapping by high-pressure liquid chromatography/fast-atom-bombardment mass spectrometry. Thermal and pH stability dependence of Onc M was assessed by circular dichroism spectroscopy; the helical content is about 50%, in agreement with the four helix bundle fold postulated for cytokines that bind haematopoietic receptors of type I.
- Published
- 1994
23. Oncostatin M binds directly to gp130 and behaves as interleukin-6 antagonist on a cell line expressing gp130 but lacking functional oncostatin M receptors
- Author
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Rita Graziani, Paolo Delmastro, Carlo Toniatti, Elisabetta Sporeno, Gennaro Ciliberto, Anna Laura Salvati, and Giacomo Paonessa
- Subjects
CHO Cells ,Oncostatin M ,Transfection ,Biochemistry ,Antigens, CD ,Cell surface receptor ,Cricetinae ,Cytokine Receptor gp130 ,Tumor Cells, Cultured ,Animals ,Humans ,Cloning, Molecular ,Receptors, Cytokine ,Interleukin 6 ,Receptor ,Molecular Biology ,Binding Sites ,Membrane Glycoproteins ,biology ,Interleukin-6 ,digestive, oral, and skin physiology ,Oncostatin M receptor ,Receptors, Oncostatin M ,Receptors, Interleukin ,Cell Biology ,Glycoprotein 130 ,Receptors, Interleukin-6 ,Recombinant Proteins ,Transmembrane protein ,Cell biology ,Kinetics ,Liver ,biology.protein ,Cytokines ,Signal transduction ,Peptides - Abstract
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.
- Published
- 1994
24. Generation of interleukin-6 receptor antagonists by molecular-modeling guided mutagenesis of residues important for gp130 activation
- Author
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Sergio Altamura, Rocco Savino, Anna Laura Salvati, Giacomo Paonessa, Gennaro Ciliberto, Armin Lahm, Elisabetta Sporeno, Carlo Toniatti, and Laura Ciapponi
- Subjects
Models, Molecular ,Receptor complex ,Molecular Sequence Data ,Interleukin 5 receptor alpha subunit ,CHO Cells ,Interleukin-17 receptor ,Biology ,Crystallography, X-Ray ,General Biochemistry, Genetics and Molecular Biology ,Interleukin 10 receptor, alpha subunit ,Antigens, CD ,Cell surface receptor ,Cricetinae ,Granulocyte Colony-Stimulating Factor ,Cytokine Receptor gp130 ,Tumor Cells, Cultured ,Animals ,Humans ,Amino Acid Sequence ,Cytokine binding ,Molecular Biology ,Genetics ,Membrane Glycoproteins ,General Immunology and Microbiology ,Interleukin-6 ,General Neuroscience ,Receptors, Interleukin ,Glycoprotein 130 ,Receptors, Interleukin-6 ,Cell biology ,Growth Hormone ,Interleukin-6 receptor ,Mutagenesis, Site-Directed ,Cattle ,Signal Transduction ,Research Article - Abstract
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.
- Published
- 1994
25. Role of scavenger receptor class B type I in hepatitis C virus entry: kinetics and molecular determinants
- Author
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Thierry Huby, Maria Teresa Catanese, Martine Moreau, Giacomo Paonessa, Alfredo Nicosia, Alessandra Vitelli, Charles M. Rice, Jonathan K. Ball, Riccardo Cortese, Helenia Ansuini, Rita Graziani, Catanese, Mt, Ansuini, H, Graziani, R, Huby, T, Moreau, M, Ball, Jk, Paonessa, G, Rice, Cm, Cortese, R, Vitelli, A, Nicosia, Alfredo, Laboratory of Virology and Infectious Disease, Rockefeller University [New York]-Center for the Study of Hepatitis C, Istituto di Ricerche di Biologia Molecolare P. Angeletti, Dyslipidémies, inflammation et athérosclérose dans les maladies métaboliques, Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM), Service d’Endocrinologie, Métabolisme et Prévention des Risques Cardio-Vasculaires [CHU Pitié-Salpêtrière], CHU Pitié-Salpêtrière [AP-HP], Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Institute of Infection, Immunity, and Inflammation, University of Nottingham, UK (UON), CEINGE, Okairos, This work was supported by funding from the European Union (grants QLK2-CT-2001-01120 and MRTN-CT-2006-035599) and PHS grant R01 AI072613. M.T.C. was supported by a Women & Science fellowship., Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Chapman, John, Service d'Endocrinologie, Métabolisme et Prévention des Maladies Cardio-vasculaires [CHU Pitié-Salpêtrière], and Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)
- Subjects
MESH: Virus Internalization ,Hepacivirus ,medicine.disease_cause ,MESH: Lipoproteins, HDL ,MESH: Antibodies, Monoclonal ,Mice ,0302 clinical medicine ,MESH: Animals ,MESH: Hepacivirus ,Cells, Cultured ,0303 health sciences ,biology ,MESH: Kinetics ,Antibodies, Monoclonal ,Scavenger Receptors, Class B ,Ligand (biochemistry) ,Hepatitis C ,3. Good health ,[SDV.MHEP.CSC] Life Sciences [q-bio]/Human health and pathology/Cardiology and cardiovascular system ,Virus-Cell Interactions ,Receptors, Virus ,030211 gastroenterology & hepatology ,Lipoproteins, HDL ,MESH: Cells, Cultured ,Hepatitis C virus ,Immunology ,Microbiology ,Virus ,03 medical and health sciences ,Viral envelope ,Species Specificity ,[SDV.MHEP.CSC]Life Sciences [q-bio]/Human health and pathology/Cardiology and cardiovascular system ,Viral entry ,Virology ,medicine ,Animals ,Humans ,MESH: Species Specificity ,Scavenger receptor ,MESH: Mice ,030304 developmental biology ,MESH: Hepatitis C ,MESH: Humans ,Virus Internalization ,biology.organism_classification ,MESH: Receptors, Virus ,MESH: Scavenger Receptors, Class B ,NS2-3 protease ,Kinetics ,Hepadnaviridae ,Insect Science - Abstract
Scavenger receptor class B type I (SR-BI) is an essential receptor for hepatitis C virus (HCV) and a cell surface high-density-lipoprotein (HDL) receptor. The mechanism of SR-BI-mediated HCV entry, however, is not clearly understood, and the specific protein determinants required for the recognition of the virus envelope are not known. HCV infection is strictly linked to lipoprotein metabolism, and HCV virions may initially interact with SR-BI through associated lipoproteins before subsequent direct interactions of the viral glycoproteins with SR-BI occur. The kinetics of inhibition of cell culture-derived HCV (HCVcc) infection with an anti-SR-BI monoclonal antibody imply that the recognition of SR-BI by HCV is an early event of the infection process. Swapping and single-substitution mutants between mouse and human SR-BI sequences showed reduced binding to the recombinant soluble E2 (sE2) envelope glycoprotein, thus suggesting that the SR-BI interaction with the HCV envelope is likely to involve species-specific protein elements. Most importantly, SR-BI mutants defective for sE2 binding, although retaining wild-type activity for receptor oligomerization and binding to the physiological ligand HDL, were impaired in their ability to fully restore HCVcc infectivity when transduced into an SR-BI-knocked-down Huh-7.5 cell line. These findings suggest a specific and direct role for the identified residues in binding HCV and mediating virus entry. Moreover, the observation that different regions of SR-BI are involved in HCV and HDL binding supports the hypothesis that new therapeutic strategies aimed at interfering with virus/SR-BI recognition are feasible.
- Published
- 2010
26. Naturally Occurring Hepatitis C Virus Subgenomic Deletion Mutants Replicate Efficiently in Huh-7 Cells and Are trans-Packaged In Vitro To Generate Infectious Defective Particles▿ †
- Author
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Rita Graziani, Raffaele De Francesco, Laura Pacini, Linda Bartholomew, and Giacomo Paonessa
- Subjects
Autonomously replicating sequence ,Immunology ,Context (language use) ,Genome, Viral ,Hepacivirus ,Biology ,medicine.disease_cause ,Virus Replication ,Microbiology ,Virus ,Cell Line ,Virology ,medicine ,Humans ,Gene ,Subgenomic mRNA ,Sequence Deletion ,chemistry.chemical_classification ,Viral Structural Proteins ,Mutation ,Virus Assembly ,Virion ,Genome Replication and Regulation of Viral Gene Expression ,Viral replication ,chemistry ,Insect Science ,RNA, Viral ,Glycoprotein - Abstract
Naturally occurring hepatitis C virus (HCV) subgenomic RNAs have been found in several HCV patients. These subgenomic deletion mutants, mostly lacking the genes encoding envelope glycoproteins, were found in both liver and serum, where their relatively high abundance suggests that they are capable of autonomous replication and can be packaged and secreted in viral particles, presumably harboring the envelope proteins from wild type virus coinfecting the same cell. We recapitulated some of these natural subgenomic deletions in the context of the isolate JFH-1 and confirmed these hypotheses in vitro. In Huh-7.5 cells, these deletion-containing genomes show robust replication and can be efficiently trans -packaged and infect naïve Huh-7.5 cells when cotransfected with the full-length wild-type J6/JFH genome. The genome structure of these natural subgenomic deletion mutants was dissected, and the maintenance of both core and NS2 regions was proven to be significant for efficient replication and trans -packaging. To further explore the requirements needed to achieve trans -complementation, we provided different combinations of structural proteins in trans . Optimal trans -complementation was obtained when fragments of the polyprotein encompassing core to p7 or E1 to NS2 were expressed. Finally, we generated a stable helper cell line, constitutively expressing the structural proteins from core to p7, which efficiently supports trans -complementation of a subgenomic deletion encompassing amino acids 284 to 732. This cell line can produce and be infected by defective particles, thus representing a powerful tool to investigate the life cycle and relevance of natural HCV subgenomic deletion mutants in vivo.
- Published
- 2009
27. High-avidity monoclonal antibodies against the human scavenger class B type I receptor efficiently block hepatitis C virus infection in the presence of high-density lipoprotein
- Author
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Thierry Huby, Alessandra Luzzago, Alessandra Vitelli, Charles M. Rice, Giacomo Paonessa, Thomas von Hahn, Riccardo Cortese, Rita Graziani, Alfredo Nicosia, Maria Teresa Catanese, Claudia Santini, Martine Moreau, Catanese, M. T., Graziani, R., von Hahn, T., Moreau, M, Huby, T, Paonessa, G, Santini, C, Luzzago, A, Rice, Cm, Cortese, R, Vitelli, A, and Nicosia, Alfredo
- Subjects
medicine.drug_class ,Hepatitis C virus ,Hepacivirus ,Immunology ,Antibody Affinity ,medicine.disease_cause ,Monoclonal antibody ,Microbiology ,Cell Line ,Viral entry ,Virology ,medicine ,Humans ,Avidity ,Infectivity ,biology ,Antibodies, Monoclonal ,Scavenger Receptors, Class B ,biology.organism_classification ,Hepatitis C ,Cholesterol ,Cell culture ,Insect Science ,biology.protein ,Pathogenesis and Immunity ,Antibody ,Lipoproteins, HDL - Abstract
The human scavenger class B type 1 receptor (SR-B1/Cla1) was identified as a putative receptor for hepatitis C virus (HCV) because it binds to soluble recombinant HCV envelope glycoprotein E2 (sE2). High-density lipoprotein (HDL), a natural SR-B1 ligand, was shown to increase the in vitro infectivity of retroviral pseudoparticles bearing HCV envelope glycoproteins and of cell culture-derived HCV (HCVcc), suggesting that SR-B1 promotes viral entry in an HDL-dependent manner. To determine whether SR-B1 participates directly in HCV infection or facilitates HCV entry through lipoprotein uptake, we generated a panel of monoclonal antibodies (MAbs) against native human SR-B1. Two of them, 3D5 and C167, bound to conformation-dependent SR-B1 determinants and inhibited the interaction of sE2 with SR-B1. These antibodies efficiently blocked HCVcc infection of Huh-7.5 hepatoma cells in a dose-dependent manner. To examine the role of HDL in SR-B1-mediated HCVcc infection, we set up conditions for HCVcc production and infection in serum-free medium. HCVcc efficiently infected Huh-7.5 cells in the absence of serum lipoproteins, and addition of HDL led to a twofold increase in infectivity. However, the HDL-induced enhancement of infection had no impact on the neutralization potency of MAb C167, despite its ability to inhibit both HDL binding to cells and SR-B1-mediated lipid transfer. Of note, MAb C167 also potently blocked Huh-7.5 infection by an HCV strain recovered from HCVcc-infected chimpanzees. These results demonstrate that SR-B1 is essential for infection with HCV produced in vitro and in vivo and suggest the possible use of anti-SR-B1 antibodies as therapeutic agents.
- Published
- 2007
28. HCV antiviral resistance: the impact of in vitro studies on the development of antiviral agents targeting the viral NS5B polymerase
- Author
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Sergio Altamura, Raffaele De Francesco, Giovanni Migliaccio, Giacomo Paonessa, and Licia Tomei
- Subjects
0301 basic medicine ,Hepatitis C virus ,030106 microbiology ,RNA-dependent RNA polymerase ,Drug resistance ,Genome, Viral ,Hepacivirus ,Viral Nonstructural Proteins ,medicine.disease_cause ,01 natural sciences ,Antiviral Agents ,Virus ,03 medical and health sciences ,chemistry.chemical_compound ,Interferon ,Drug Resistance, Viral ,medicine ,Animals ,Humans ,NS5B ,Polymerase ,biology ,Nucleoside analogue ,Molecular Structure ,virus diseases ,General Medicine ,RNA-Dependent RNA Polymerase ,Virology ,0104 chemical sciences ,010404 medicinal & biomolecular chemistry ,chemistry ,Mutation ,biology.protein ,medicine.drug - Abstract
The high prevalence of the disease caused by hepatitis C virus (HCV) and the limited efficacy of interferon-based therapies have stimulated the search for safer and more effective drugs. The development of inhibitors of the HCV NS5B RNA polymerase represents a promising strategy for identifying novel anti-HCV therapeutics. However, the high genetic diversity, mutation rate and turnover of HCV are expected to favour the emergence of drug resistance, limiting the clinical usefulness of polymerase inhibitors. Thus, the characterization of the drug-resistance profile of these antiviral agents is considered crucial for identifying the inhibitors with a higher probability of clinical success. In the absence of an efficient in vitro infection system, HCV sub-genomic replicons have been used to study viral resistance to both nucleoside and non-nucleoside NS5B inhibitors. While these studies suggest that drug-resistant viruses are likely to evolve in vivo, they provide a wealth of information that should help in the identification of inhibitors with improved and distinct resistance profiles that might be used for combination therapy.
- Published
- 2005
29. Conventional protein kinase C inhibition prevents alpha interferon-mediated hepatitis C virus replicon clearance by impairing STAT activation
- Author
-
Giacomo Paonessa, Federica Fratini, Mauro Piacentini, Cristina Evangelisti, Tonino Alonzi, Gian Maria Fimia, Giuseppe Ippolito, Marco Tripodi, and Marta Romani
- Subjects
Gene Expression Regulation, Viral ,STAT3 Transcription Factor ,Hepatitis C virus ,Immunology ,Alpha interferon ,Hepacivirus ,Biology ,medicine.disease_cause ,Microbiology ,Antiviral Agents ,Cell Line ,chemistry.chemical_compound ,Virology ,Vaccines and Antiviral Agents ,medicine ,Humans ,STAT1 ,Protein kinase A ,Protein kinase C ,Interferon alfa ,Protein Kinase C ,Interferon-alpha ,Tyrosine phosphorylation ,DNA-Binding Proteins ,STAT1 Transcription Factor ,chemistry ,Insect Science ,biology.protein ,Trans-Activators ,RNA, Viral ,Replicon ,Signal transduction ,medicine.drug - Abstract
Hepatitis C virus (HCV) has evolved complex strategies to evade host immune responses and establish chronic infection. The only treatment available for HCV infections, alpha interferon (IFN-α), is effective in a limited percentage of patients. The mechanisms by which IFN-α interferes with the HCV life cycle and the reasons for limited effectiveness of IFN-α therapy have not yet been fully elucidated. Using a cell-based HCV replication system and specific kinase inhibitors, we examined the role played by various signaling pathways in the IFN-α-mediated HCV clearance. We reported that conventional protein kinase C (cPKC) activity is important for the effectiveness of IFN-α treatment. In cells treated with a cPKC-specific inhibitor, IFN-α failed to induce an efficient HCV RNA degradation. The lack of cPKC activity leads to a broad reduction of IFN-α-stimulated gene expression due to a significant impairment of STAT1 and STAT3 tyrosine phosphorylation. Thus, modulation of cPKC function by either host or viral factors could influence the positive outcome of IFN-α-mediated antiviral therapies.
- Published
- 2004
30. In Vitro Selection and Characterization of Hepatitis C Virus Serine Protease Variants Resistant to an Active-Site Peptide Inhibitor
- Author
-
Alessandra Ceccacci, Gabriella Biasiol, Laura Pacini, Caterina Trozzi, Frank Narjes, Uwe Koch, Linda Bartholomew, Sergio Altamura, Christian Steinkühler, Giovanni Migliaccio, Raffaele De Francesco, Giacomo Paonessa, and Ester Muraglia
- Subjects
Models, Molecular ,Serine Proteinase Inhibitors ,medicine.medical_treatment ,Hepatitis C virus ,viruses ,Immunology ,Hepacivirus ,Microbial Sensitivity Tests ,Biology ,Viral Nonstructural Proteins ,medicine.disease_cause ,Microbiology ,Viral Proteins ,Virology ,Drug Resistance, Viral ,Vaccines and Antiviral Agents ,medicine ,Tumor Cells, Cultured ,Humans ,Protease inhibitor (pharmacology) ,Selection, Genetic ,Serine protease ,Protease ,Binding Sites ,Intracellular Signaling Peptides and Proteins ,Genetic Variation ,Transfection ,Molecular biology ,NS2-3 protease ,Viral replication ,Insect Science ,Mutation ,biology.protein ,Replicon ,Carrier Proteins - Abstract
The hepatitis C virus (HCV) serine protease is necessary for viral replication and represents a valid target for developing new therapies for HCV infection. Potent and selective inhibitors of this enzyme have been identified and shown to inhibit HCV replication in tissue culture. The optimization of these inhibitors for clinical development would greatly benefit from in vitro systems for the identification and the study of resistant variants. We report the use HCV subgenomic replicons to isolate and characterize mutants resistant to a protease inhibitor. Taking advantage of the replicons' ability to transduce resistance to neomycin, we selected replicons with decreased sensitivity to the inhibitor by culturing the host cells in the presence of the inhibitor and neomycin. The selected replicons replicated to the same extent as those in parental cells. Sequence analysis followed by transfection of replicons containing isolated mutations revealed that resistance was mediated by amino acid substitutions in the protease. These results were confirmed by in vitro experiments with mutant enzymes and by modeling the inhibitor in the three-dimensional structure of the protease.
- Published
- 2003
31. High circulating levels of biologically inactive IL-6/SIL-6 receptor complexes in systemic juvenile idiopathic arthritis: evidence for serum factors interfering with the binding to gp130
- Author
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M. Massa, M. B. Hansen, Cristina Meazza, P. Galle, Laura Ciapponi, Daniela Novick, Giacomo Paonessa, F De Benedetti, Gennaro Ciliberto, Alberto Martini, and Pier Franco Pignatti
- Subjects
medicine.medical_specialty ,Adolescent ,medicine.medical_treatment ,Immunology ,Arthritis ,Binding, Competitive ,law.invention ,Biological Factors ,immune system diseases ,Antigens, CD ,law ,Internal medicine ,Cytokine Receptor gp130 ,medicine ,Humans ,Immunology and Allergy ,Child ,Interleukin 6 ,Receptor ,Autoantibodies ,Membrane Glycoproteins ,biology ,Interleukin-6 ,business.industry ,Autoantibody ,Original Articles ,Glycoprotein 130 ,medicine.disease ,Receptors, Interleukin-6 ,Arthritis, Juvenile ,C-Reactive Protein ,Cytokine ,Endocrinology ,Solubility ,Child, Preschool ,biology.protein ,Recombinant DNA ,Biological Assay ,business ,Juvenile rheumatoid arthritis ,Protein Binding ,Signal Transduction - Abstract
SUMMARYWe previously demonstrated that high levels of IL-6/sIL-6R complexes are present in sera of patients with systemic juvenile idiopathic arthritis (s-JIA) and that the amount of IL-6 estimated in the IL-6/sIL-6R complexes is markedly higher than that measured by the B9 assay. Here, we show that two additional bioassays, employing human myeloma XG-1 cells and human hepatoma Hep3B cells, detected serum IL-6 levels similar to those measured by the B9 assay and approximately 10-fold lower than the IL-6 levels estimated to be present in the IL-6/sIL-6R complex. Using an assay for the measurement of the amount of circulating IL-6 complexed with the sIL-6R and available for binding to gp130 (gp130 binding activity), we show that the IL-6/gp130 binding activity is similar to that detected by the bioassays and again significantly lower than that estimated to be present in the IL-6/sIL-6R complex. Addition of recombinant human IL-6 (rhIL-6) to sera of patients or controls results in a markedly lower increase in the gp130 binding activity in patients than in controls. Moreover, sera from s-JIA patients inhibited in a dose dependent manner the gp130 binding activity assay. These results show that sera from patients with s-JIA contain a factor, or factors, that inhibit(s) the binding of the IL-6/sIL-6R complex to gp130. This inhibitory activity does not appear to be due to soluble gp130, C-reactive protein or autoantibodies to IL-6.
- Published
- 2003
32. Ciliary neurotrophic factor corrects obesity and diabetes associated with leptin deficiency and resistance
- Author
-
Patrizia Costa, Rita Graziani, Charles Rosenblum, Ralph Laufer, Anna Demartis, Lex H.T. Van der Ploeg, Isabelle Gloaguen, Riccardo Cortese, Domenico Lazzaro, Gennaro Ciliberto, Annalise Di Marco, Giacomo Paonessa, and Fang Chen
- Subjects
Blood Glucose ,Leptin ,Male ,medicine.medical_treatment ,Mice, Obese ,Ciliary neurotrophic factor ,Mice ,Neuroblastoma ,Hyperinsulinemia ,Insulin ,Receptor ,Receptor, Ciliary Neurotrophic Factor ,Neurons ,Multidisciplinary ,Leptin Deficiency ,digestive, oral, and skin physiology ,Brain ,Biological Sciences ,Recombinant Proteins ,DNA-Binding Proteins ,Cytokine ,Receptors, Leptin ,hormones, hormone substitutes, and hormone antagonists ,medicine.medical_specialty ,Nerve Tissue Proteins ,Receptors, Cell Surface ,Receptors, Nerve Growth Factor ,Biology ,Hybrid Cells ,Motor Activity ,Cell Line ,Mice, Inbred AKR ,Internal medicine ,medicine ,Animals ,Humans ,Point Mutation ,Ciliary Neurotrophic Factor ,Nerve Growth Factors ,Obesity ,Leptin receptor ,Body Weight ,Arcuate Nucleus of Hypothalamus ,Proteins ,medicine.disease ,Dietary Fats ,Grooming ,Mice, Inbred C57BL ,Endocrinology ,Nerve growth factor ,Diabetes Mellitus, Type 2 ,biology.protein ,Carrier Proteins - Abstract
Receptor subunits for the neurocytokine ciliary neurotrophic factor (CNTF) share sequence similarity with the receptor for leptin, an adipocyte-derived cytokine involved in body weight homeostasis. We report here that CNTF and leptin activate a similar pattern of STAT factors in neuronal cells, and that mRNAs for CNTF receptor subunits, similarly to the mRNA of leptin receptor, are localized in mouse hypothalamic nuclei involved in the regulation of energy balance. Systemic administration of CNTF or leptin led to rapid induction of the tis-11 primary response gene in the arcuate nucleus, suggesting that both cytokines can signal to hypothalamic satiety centers. Consistent with this idea, CNTF treatment of ob / ob mice, which lack functional leptin, was found to reduce the adiposity, hyperphagia, and hyperinsulinemia associated with leptin deficiency. Unlike leptin, CNTF also reduced obesity-related phenotypes in db / db mice, which lack functional leptin receptor, and in mice with diet-induced obesity, which are partially resistant to the actions of leptin. The identification of a cytokine-mediated anti-obesity mechanism that acts independently of the leptin system may help to develop strategies for the treatment of obesity associated with leptin resistance.
- Published
- 1997
33. Induction of interleukin-6 (IL-6) autoantibodies through vaccination with an engineered IL-6 receptor antagonist
- Author
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Giacomo Paonessa, Laura Ciapponi, Tonino Alonzi, Morten Bagge Hansen, Gennaro Ciliberto, Ariane Scoumanne, Riccardo Cortese, Morten Svenson, Klaus Bendtzen, Patrizia Costa, Rocco Savino, Domenico Maione, Ciapponi, L., Maione, D., Scoumanne, A., Costa, P., Hansen, M. . B., Svenson, M., Bendtzen, K., Alonzi, T., Paonessa, G., Cortese, Riccardo, Ciliberto, G., and Savino, R.
- Subjects
endocrine system ,medicine.drug_class ,Biomedical Engineering ,Bioengineering ,Aluminum Hydroxide ,Enzyme-Linked Immunosorbent Assay ,Mice, Transgenic ,Cross Reactions ,Monoclonal antibody ,Applied Microbiology and Biotechnology ,Antigen-Antibody Reactions ,Mice ,medicine ,Escherichia coli ,Animals ,Humans ,Receptor ,Neutralizing antibody ,Interleukin 6 ,Autoantibodies ,Binding Sites ,biology ,Interleukin-6 ,Vaccination ,Autoantibody ,Receptor antagonist ,Receptors, Interleukin-6 ,Recombinant Proteins ,Gene Expression Regulation ,Interleukin-6 receptor ,Immunology ,biology.protein ,Molecular Medicine ,Antibody ,Genetic Engineering ,Injections, Intraperitoneal ,Biotechnology - Abstract
Neutralization of cytokine activity by monoclonal antibodies or receptor antagonists is beneficial in the treatment of immune and neoplastic diseases, but the necessity for continuous parenteral delivery of these anticytokine agents poses considerable practical limitations. A viable alternative is to induce a neutralizing antibody response. Using transgenic mice with high circulating levels of human interleukin-6 (hIL-6), we show that injection of the hIL-6 receptor antagonist Sant1 (an IL-6 variant with seven amino-acid substitutions) induces a strong anti-hIL-6 antibody response. The elicited antibodies bind circulating hIL-6 with very high affinity, totally masking it, and neutralize hIL-6 bioactivity both in vitro and in vivo.
- Published
- 1997
34. Functional expression of soluble human interleukin-11 (IL-11) receptor alpha and stoichiometry of in vitro IL-11 receptor complexes with gp130
- Author
-
Gennaro Ciliberto, Giacomo Paonessa, Rita Graziani, and Petra Neddermann
- Subjects
Receptor complex ,DNA, Complementary ,Macromolecular Substances ,Leukemia inhibitory factor receptor ,Interleukin-17 receptor ,Biology ,Biochemistry ,Liver Neoplasms, Experimental ,Growth factor receptor ,Antigens, CD ,Cytokine Receptor gp130 ,Tumor Cells, Cultured ,Animals ,Humans ,Receptors, Interleukin-11 ,5-HT5A receptor ,Interleukin-11 Receptor alpha Subunit ,Cloning, Molecular ,Molecular Biology ,Common gamma chain ,Membrane Glycoproteins ,Cell Biology ,Receptors, Interleukin ,Interleukin-11 ,Molecular biology ,Solubility ,Interleukin-21 receptor ,Estrogen-related receptor gamma ,Protein Binding ,Signal Transduction - Abstract
The interleukin-6 (IL-6) family of cytokines activates signaling through the formation of either gp130 homodimers, as for IL-6, or gp130-leukemia inhibitory factor receptor (LIFR) heterodimers as for ciliary neurotrophic factor (CNTF), leukemia inhibitory factor, oncostatinM, and cardiotrophin-1. Recent in vitro studies with IL-6 and CNTF have demonstrated that higher order hexameric receptor complexes are assembled in which signaling chain dimerization is accompanied by the dimerization of both the cytokine molecule and its specific receptor alpha subunits (IL-6Ralpha or CNTFRalpha, respectively). IL-11 is a member of the IL-6 family and known to require gp130 but not LIFR for signaling. In this study we investigate the functional and biochemical composition of the IL-11 receptor complex. The human IL-11 receptor alpha-chain was cloned from a human bone marrow cDNA library. IL-11Ralpha was shown to confer IL-11 responsiveness to human hepatoma cells either by cDNA transfection or by adding a soluble form of the receptor (sIL11Ralpha) expressed in the baculovirus system to the culture medium. In vitro immunoprecipitation experiments showed that sIL11Ralpha specifically binds IL-11 and that binding is enhanced by gp130. Similarly to IL-6 and CNTF, gp130 is able to induce dimerization of the IL-11.IL-11Ralpha subcomplex, the result of which is the formation of a pentameric receptor complex. However, in contrast to the other two cytokines, IL-11 was unable to induce either gp130 homodimerization or gp130/LIFR heterodimerization. These results strongly suggest that an as yet unidentified receptor beta-chain is involved in IL-11 signaling.
- Published
- 1996
35. Identification of ciliary neurotrophic factor (CNTF) residues essential for leukemia inhibitory factor receptor binding and generation of CNTF receptor antagonists
- Author
-
Isabelle Gloaguen, K R Hudson, Giacomo Paonessa, Rita Graziani, Isabella Saggio, A. Di Marco, and Ralph Laufer
- Subjects
Models, Molecular ,Receptor complex ,Leukemia Inhibitory Factor Receptor alpha Subunit ,Receptors, OSM-LIF ,DNA Mutational Analysis ,Molecular Sequence Data ,Leukemia inhibitory factor receptor ,Nerve Tissue Proteins ,Receptors, Nerve Growth Factor ,Ciliary neurotrophic factor ,Leukemia Inhibitory Factor ,Choline O-Acetyltransferase ,gp130 ,Structure-Activity Relationship ,Antigens, CD ,Leukemia inhibitory factor receptor binding ,lifr ,Cytokine Receptor gp130 ,Humans ,Amino Acid Sequence ,Ciliary Neurotrophic Factor ,Receptors, Cytokine ,Receptor, Ciliary Neurotrophic Factor ,Cells, Cultured ,Alanine ,antagonist ,ciliary neurotrophic factor ,cntf ,gene function ,leukemia ,leukemia inhibitory factor ,Lymphokines ,Multidisciplinary ,Membrane Glycoproteins ,biology ,Dose-Response Relationship, Drug ,Haptoglobins ,Interleukin-6 ,Glycoprotein 130 ,Growth Inhibitors ,Biochemistry ,Mutation ,biology.protein ,Mutagenesis, Site-Directed ,Leukemia inhibitory factor ,Cell Division ,Protein Binding ,Signal Transduction ,Research Article - Abstract
Ciliary neurotrophic factor (CNTF) drives the sequential assembly of a receptor complex containing the ligand-specific alpha-receptor subunit (CNTFR alpha) and the signal transducers gp130 and leukemia inhibitory factor receptor-beta (LIFR). The D1 structural motif, located at the beginning of the D-helix of human CNTF, contains two amino acid residues, F152 and K155, which are conserved among all cytokines that signal through LIFR. The functional importance of these residues was assessed by alanine mutagenesis. Substitution of either F152 or K155 with alanine was found to specifically inhibit cytokine interaction with LIFR without affecting binding to CNTFR alpha or gp130. The resulting variants behaved as partial agonists with varying degrees of residual bioactivity in different cell-based assays. Simultaneous alanine substitution of both F152 and K155 totally abolished biological activity. Combining these mutations with amino acid substitutions in the D-helix, which enhance binding affinity for the CNTFR alpha, gave rise to a potent competitive CNTF receptor antagonist. This protein constitutes a new tool for studies of CNTF function in normal physiology and disease.
- Published
- 1996
36. Six different cytokines that share GP130 as a receptor subunit, induce serum amyloid A and potentiate the induction of interleukin-6 and the activation of the hypothalamus-pituitary-adrenal axis by interleukin-1
- Author
-
Manuela Cappelletti, Giacomo Paonessa, Pietro Ghezzi, Pietro Pozzi, Silvano Sacco, Charles A. Dinarello, Diane Pennica, Valeria Poli, Jean D. Sipe, Giamila Fantuzzi, Fabio Benigni, Marina Sironi, and Nikos Panayotatos
- Subjects
Male ,medicine.medical_specialty ,Hypothalamo-Hypophyseal System ,medicine.medical_treatment ,Immunology ,Pituitary-Adrenal System ,Nerve Tissue Proteins ,Oncostatin M ,Ciliary neurotrophic factor ,Biochemistry ,Leukemia Inhibitory Factor ,chemistry.chemical_compound ,Mice ,Corticosterone ,Antigens, CD ,Internal medicine ,medicine ,Cytokine Receptor gp130 ,Animals ,Serum amyloid A ,Ciliary Neurotrophic Factor ,Receptors, Cytokine ,Interleukin 6 ,Acute-Phase Reaction ,Lymphokines ,Serum Amyloid A Protein ,Membrane Glycoproteins ,biology ,Interleukin-6 ,Acute-phase protein ,Interleukin ,Drug Synergism ,Cell Biology ,Hematology ,Interleukin-11 ,Growth Inhibitors ,Recombinant Proteins ,Endocrinology ,Cytokine ,chemistry ,Liver ,biology.protein ,Cytokines ,Peptides ,Interleukin-1 ,Signal Transduction - Abstract
Ciliary neurotrophic factor (CNTF) and interleukin-6 (IL-6) potentiate the elevation of serum corticosterone induced by suboptimal doses of interleukin-1 (IL-1). CNTF also potentiates IL-1-induced serum IL-6. Here, we report that four other cytokines (leukemia inhibitory factor [LIF], oncostatin M [OSM], interleukin-11 and cardiotrophin-1) also potentiated the elevation of serum corticosterone and IL-6 levels induced by IL-1. Furthermore, all the six cytokines studied induced the acute-phase protein serum amyloid A when administered alone. Because these cytokines differ both in structure and in function, but share gp130 as a subunit of their receptors, these results indicate that signaling through gp130 mediates potentiation of IL-1 activities. The potentiation of IL-1-induced serum corticosterone levels is not a consequence of the increased serum IL-6 observed after IL-1 administration. In fact, in IL-6 deficient mice, IL-1 increased serum corticosterone to a level comparable to that observed in wild-type mice. Thus, either endogenous IL-6 does not mediate IL-1-induced corticosterone increase, or its role may be fulfilled by other cytokines. To the extent that gp130-dependent cytokines may serve this role, they may be important feedback regulators of inflammation through the activation of the hypothalamus-pituitary-adrenal axis and the potentiation of acute-phase protein synthesis.
- Published
- 1996
37. Nonradioactive receptor binding assay for ciliary neurotrophic factor
- Author
-
Ralph Laufer, A. Deserio, Isabelle Gloaguen, Isabella Saggio, Giacomo Paonessa, and Rita Graziani
- Subjects
CNTF ,cytokine ,receptor binding ,recombinant DNA ,Arginine ,Mutant ,Blotting, Western ,Molecular Sequence Data ,Biophysics ,Biotin ,Nerve Tissue Proteins ,Receptors, Nerve Growth Factor ,Ciliary neurotrophic factor ,Biochemistry ,Binding, Competitive ,Epitope ,Escherichia coli ,Animals ,Humans ,Amino Acid Sequence ,Ciliary Neurotrophic Factor ,Nerve Growth Factors ,Cloning, Molecular ,Molecular Biology ,Receptor, Ciliary Neurotrophic Factor ,biology ,Cell Biology ,Molecular biology ,Recombinant Proteins ,Rats ,Blot ,Kinetics ,Biotinylation ,biology.protein ,Electrophoresis, Polyacrylamide Gel ,Rat Protein ,Avidin - Abstract
A nonradioactive receptor binding assay for ciliary neurotrophic factor (CNTF) is described. The assay is based on the interaction between biotinylated human CNTF, soluble gp130, and soluble myc-tagged CNTF receptor captured on a microtiter plate via an antibody against the myc epitope tag. Bound cytokine is revealed by alkaline phosphatase-conjugated avidin. Purified human and rat CNTF competed with biotinylated CNTF for receptor binding, with IC50 values of 29 and 2 nM, respectively. Since the higher affinity of rat vs human CNTF has been previously shown to be conferred by the arginine residue at position 63 of the rat protein, we also tested a human CNTF mutant carrying a Q63R substitution. Secreted forms of wild-type and mutant CNTF were expressed in Escherichia coli, and the amount of cytokines in periplasmic extracts was determined by quantitative Western blotting analysis. The human CNTF mutant (Q63R, N137S) was found to compete with biotinylated CNTF for binding to soluble CNTF receptor with an eightfold higher apparent affinity than wild-type human CNTF. The present method thus faithfully reproduces the relative activities of CNTF analogs determined in other assay systems. The possibility of assaying cytokines in crude bacterial extracts makes the new technique particularly suitable for rapidly determining the receptor binding potencies of genetically engineered CNTF variants.
- Published
- 1994
38. Chimpanzee DNA profiles on trial
- Author
-
Marina Dobosz, Giacomo Paonessa, U. Mereu, G. Destro Bisol, Ernesto D'Aloja, and Vincenzo Lorenzo Pascali
- Subjects
Genetic Markers ,Genetics ,Genome ,Polymorphism, Genetic ,Multidisciplinary ,Base Sequence ,Pan troglodytes ,Fraud ,Molecular Sequence Data ,Settore MED/43 - MEDICINA LEGALE ,Biology ,Polymerase Chain Reaction ,law.invention ,DNA profiling ,law ,Genetic marker ,Animals ,Base sequence ,Pan troglodytes/genetics ,Polymerase chain reaction - Published
- 1994
39. Agonistic and antagonistic variants of ciliary neurotrophic factor (CNTF) reveal functional differences between membrane-bound and soluble CNTF α-receptor
- Author
-
Isabelle Gloaguen, Isabella Saggio, Giacomo Paonessa, Rita Graziani, Anna Demartis, Annalise Di Marco, and Ralph Laufer
- Subjects
Agonist ,Receptor complex ,medicine.medical_specialty ,Leukemia Inhibitory Factor Receptor alpha Subunit ,Receptors, OSM-LIF ,medicine.drug_class ,Protein subunit ,receptor ,Leukemia inhibitory factor receptor ,Nerve Tissue Proteins ,Receptors, Nerve Growth Factor ,Biology ,Ciliary neurotrophic factor ,Leukemia Inhibitory Factor ,Biochemistry ,Antigens, CD ,Internal medicine ,medicine ,Cytokine Receptor gp130 ,CNTF ,Humans ,Ciliary Neurotrophic Factor ,Receptors, Cytokine ,Receptor ,Receptor, Ciliary Neurotrophic Factor ,membrane ,Molecular Biology ,Lymphokines ,Membrane Glycoproteins ,Dose-Response Relationship, Drug ,Interleukin-6 ,Membrane Proteins ,Receptor Protein-Tyrosine Kinases ,Transfection ,Cell Biology ,Glycoprotein 130 ,Growth Inhibitors ,Recombinant Proteins ,Cell biology ,Endocrinology ,Models, Chemical ,Solubility ,Mutation ,biology.protein ,Biological Assay ,Protein Binding ,Signal Transduction - Abstract
Ciliary neurotrophic factor (CNTF) drives the sequential assembly of a receptor complex containing the ligand-specific alpha-receptor subunit (CNTFR) and the signal-transducing beta-subunits gp130 and leukemia inhibitory factor receptor-beta (LIFR). CNTFR can function in either membrane-bound or soluble forms. The membrane-bound form mediates the neuronal actions of CNTF, whereas the soluble form serves to confer cytokine responsiveness to non-neuronal cells expressing gp130 and LIFR. The objective of this work was to analyze whether the two receptor isoforms differ in their ability to interact functionally with CNTF and related proteins. Two new types of CNTF variants, characterized by weakened interactions with either CNTFR or both LIFR and gp130, were developed, and the biological activities of these and other mutants were determined in non-neuronal versus neuronal cells, as well as in non-neuronal cells transfected with an expression vector for CNTFR. Membrane anchoring of CNTFR was found to render the CNTF receptor complex relatively insensitive to changes in agonist affinity for either alpha- or beta-receptor subunits and to promote a more efficient interaction with a gp130-depleting antagonistic variant of CNTF. As a result of this phenomenon, which can be rationalized in terms of the multivalent nature of CNTF receptor interaction, CNTF variants display striking changes in receptor selectivity.
- Published
- 1997
40. Generation of IL-6 receptor antagonist and super-antagonist by molecular modelling and site-directed mutagenesis
- Author
-
Sergio Altamura, Armin Lahm, R. Savino, Anna Laura Salvati, Carlo Toniatti, Andrea Cabibbo, Elisabetta Sporeno, Anna Demartis, Laura Ciapponi, Giacomo Paonessa, and Gennaro Ciliberto
- Subjects
Chemistry ,Immunology ,Interleukin-6 receptor ,Antagonist ,Immunology and Allergy ,Hematology ,Site-directed mutagenesis ,Molecular Biology ,Biochemistry ,Molecular biology - Published
- 1994
41. The 11S rat seminal vesicle mRNA directs thein vitrosynthesis of two precursors of the major secretory protein IV
- Author
-
Paolo Abrescia, Giacomo Paonessa, John Guardiola, and S. Metafora
- Subjects
Male ,Messenger RNA ,Reticulocytes ,Cell-Free System ,Prostatic Secretory Proteins ,Seminal Plasma Proteins ,Proteins ,Seminal Vesicles ,Translation (biology) ,Biology ,Rats ,Molecular Weight ,Secretory protein ,Biochemistry ,Protein Biosynthesis ,Gene expression ,Genetics ,Protein biosynthesis ,Animals ,RNA, Messenger ,Rabbits ,Polyacrylamide gel electrophoresis - Abstract
The 11s mRNA extracted from the rat seminal vesicles directs the synthesis of two different precursors of the major secretory protein RSV-IV. These two precursors are not interconvertible and seemingly originate from different translational events. Sucrose gradients, polyacrylamide gel electrophoresis and positive hybridization translation experiments do not allow the separation of the two putatively different mRNAs. It is concluded that the two RSV-IV precursors either derive from two extremely similar, but physically not separable mRNA species, or from two different modes of translation of the same mRNA molecule.
- Published
- 1984
42. Polymorphism of rat seminal vesicle secretory proteins: characterization of svp-1 and svp-2 and their identification with the major secretory proteins IV and V
- Author
-
A. Maffei, John Guardiola, P. Abrescia, Giacomo Paonessa, and S. Metafora
- Subjects
Male ,Locus (genetics) ,Biology ,Rat Seminal Vesicle ,Biochemistry ,Seminal vesicle ,Genetics ,medicine ,Animals ,Allele ,Amino Acids ,Molecular Biology ,Gene ,Ecology, Evolution, Behavior and Systematics ,Gel electrophoresis ,Polymorphism, Genetic ,Seminal Plasma Proteins ,Prostatic Secretory Proteins ,Proteins ,General Medicine ,Molecular biology ,Human genetics ,Rats ,Secretory protein ,medicine.anatomical_structure ,Gene Expression Regulation - Abstract
The proteins secreted by the rat seminal vesicle can be separated into five major fractions (namely, RSV-I through V) by gel electrophoresis in denaturing conditions. Two polymorphic proteins, svp-1 and svp-2, also present in the mouse, are produced by the seminal vesicle as well, but the procedure used for their identification makes it impossible to ascertain whether they correspond to any of the major fractions mentioned above. We show here that, on the basis of molecular weight measurements and of amino acid composition determinations, svp-1 and RSV-V are indeed the same protein. We also show that svp-2 is strictly related to another major secretory protein, RSV-IV, whose amino acid composition is almost identical, but for a few amino acid residues, to that of svp-2. We thus conclude that the latter protein is a variant of RSV-IV that can be expressed only in rats homozygous for a given allele at the svp-2 locus. This paper thus brings together published information on the genetics of the loci coding for svp-1 and for svp-2 and on the molecular biology of RSV-IV and RSV-V and of their corresponding gene.
- Published
- 1984
43. Purification of a NF1-like DNA-binding protein from rat liver and cloning of the corresponding cDNA
- Author
-
Rainer Frank, Giacomo Paonessa, Riccardo Cortese, Fotini Gounari, Paonessa, G., Gounari, F., Frank, R., and Cortese, Riccardo
- Subjects
Molecular Sequence Data ,Biology ,Molecular cloning ,General Biochemistry, Genetics and Molecular Biology ,Complementary DNA ,Tissue Distribution ,Amino Acid Sequence ,RNA, Messenger ,Binding site ,Cloning, Molecular ,Molecular Biology ,Peptide sequence ,chemistry.chemical_classification ,General Immunology and Microbiology ,Base Sequence ,cDNA library ,General Neuroscience ,Nuclear Proteins ,DNA ,Y box binding protein 1 ,Molecular biology ,Amino acid ,DNA-Binding Proteins ,Open reading frame ,NFI Transcription Factors ,chemistry ,Gene Expression Regulation ,Liver ,CCAAT-Enhancer-Binding Proteins ,Immunologic Techniques ,Y-Box-Binding Protein 1 ,Research Article ,Transcription Factors - Abstract
NF1-like proteins play a role in transcription of liver-specific genes. A DNA-binding protein, recognizing half of the canonical NF1 binding site (TGGCA) present on the human albumin and retinol-binding protein genes, has been purified from rat liver. Several peptides deriving from a tryptic digest of the purified protein were sequenced and the sequence was used to synthesize specific oligonucleotides. Two overlapping cDNA clones were obtained from a rat-liver cDNA library; their sequence reveals an open reading frame coding for 505 amino acids, including all the peptides sequenced from the purified protein. The DNA-binding domain, most likely located within the first 250 amino acids, is highly homologous to the sequence of CTF/NF1 purified from HeLa cells. Northern analysis reveals several mRNA species present in different combinations in various rat tissues.
- Published
- 1988
44. Alu sequences transcription in X. laevis oocytes: nuclear-cytoplasmic partitioning and evidence for 3' end processing reactions
- Author
-
Gennaro Ciliberto, Giacomo Paonessa, and Elda Perlino
- Subjects
Cytoplasm ,Transcription, Genetic ,DNA polymerase ,Genetic Linkage ,Alu element ,RNA polymerase III ,Xenopus laevis ,Transcription (biology) ,hemic and lymphatic diseases ,Genetics ,medicine ,Animals ,Humans ,RNA Processing, Post-Transcriptional ,Gene ,POLII ,Repetitive Sequences, Nucleic Acid ,RNA TRANSCRIPTION ,Cell Nucleus ,biology ,Base Sequence ,ALU SEQUENCES ,Protein primary structure ,Orosomucoid ,Molecular biology ,Cell Compartmentation ,Cell nucleus ,medicine.anatomical_structure ,Transfer RNA ,biology.protein ,Nucleic Acid Conformation ,A1 ACID-GLYCOPROTEIN - Abstract
A large Alu-family cluster in the 5' flanking region of a human alpha 1-acid glycoprotein gene has been identified and sequenced. Individual members microinjected into X. laevis oocytes are transcribed only when canonical box A and B components of the split pol III promoter are present. Alu transcripts accumulate in the nucleus. An unusually short Alu transcript, able to assume a stable secondary structure, undergoes a 3' end processing reaction similar to the one required for tRNA 3' end maturation.
- Published
- 1985
45. Two distinct factors interact with the promoter regions of several liver-specific genes
- Author
-
R. Cortese, M. Frain, E. M. Hardon, Giacomo Paonessa, Hardon, E. M., Frain, M., Paonessa, G., and Cortese, Riccardo
- Subjects
Genes, Viral ,Mutant ,Response element ,Molecular Sequence Data ,Heterologous ,Simian virus 40 ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Mice ,Transcription (biology) ,Albumins ,Sequence Homology, Nucleic Acid ,Gene expression ,Animals ,Humans ,Promoter Regions, Genetic ,Molecular Biology ,Transcription factor ,Gene ,Apolipoproteins A ,Genetics ,Binding Sites ,General Immunology and Microbiology ,Apolipoprotein A-I ,Base Sequence ,Haptoglobins ,General Neuroscience ,Promoter ,Gene Expression Regulation ,Liver ,Organ Specificity ,Enzyme Induction ,alpha 1-Antitrypsin ,Research Article ,Transcription Factors - Abstract
A segment of the human alpha 1-antitrypsin (alpha 1AT) 5'-flanking region comprising nucleotides -137 to -37 from the start of transcription is sufficient to drive liver-specific transcription from the homologous alpha 1AT promoter and from the heterologous SV40 promoter. In this paper we characterize two proteins, LF-A1 and LF-B1, whose ability to bind wild-type and mutant alpha 1AT promoter segments correlates with the ability of these segments to activate transcription in vivo. DNase I protection and methylation interference analysis reveals that LF-A1 recognizes sequences present in the regulatory region of the human alpha 1-antitrypsin, apolipoprotein A1 and haptoglobin-related genes. These sequences share a common 5' TGG/A A/C CC 3' motif. LF-B1 binds to the palindrome 5' TGGTTAAT/ATTCACCA 3' which is present in the human alpha 1-antitrypsin gene between positions -78 and -62 from the start of transcription. LF-B1 also recognizes a related sequence present in the human albumin gene between -66 and -50. These results suggest that LF-A1 and LF-B1 are common positive trans-acting factors which are required for the expression of several genes in the hepatocyte.
- Published
- 1988
46. Activation of the signal transducer gp130 by interleukin-11 and interleukin-6 is mediated by similar molecular interactions
- Author
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Stephane Minvielle, Gerhard Müller-Newen, Heike Dahmen, Peter C. Heinrich, Yannick Jacques, Ian M. Kerr, Ursula Horsten, Giacomo Paonessa, Andrea Küster, and Gennaro Ciliberto
- Subjects
STAT3 Transcription Factor ,Recombinant Fusion Proteins ,Biology ,Biochemistry ,Binding, Competitive ,Epitope ,stat ,Cell Line ,Epitopes ,Antigens, CD ,Cytokine Receptor gp130 ,Animals ,Humans ,Receptors, Interleukin-11 ,Interleukin-11 Receptor alpha Subunit ,Interleukin 6 ,Molecular Biology ,STAT4 ,Membrane Glycoproteins ,Interleukin-6 ,digestive, oral, and skin physiology ,Cell Biology ,Receptors, Interleukin ,Protein-Tyrosine Kinases ,Glycoprotein 130 ,Interleukin-11 ,Molecular biology ,Receptors, Interleukin-6 ,Hedgehog signaling pathway ,Recombinant Proteins ,Cell biology ,DNA-Binding Proteins ,STAT1 Transcription Factor ,Mutation ,STAT protein ,biology.protein ,Trans-Activators ,Janus kinase ,Dimerization ,Cell Division ,Research Article ,Signal Transduction - Abstract
The transmembrane glycoprotein gp130 is involved in many cytokine-mediated cellular responses and acts therein as the signal transducing receptor subunit. Interleukin-6 (IL-6) and interleukin-11 (IL-11), in complex with their specific α-receptors, homodimerize gp130 and, as a consequence, activate the Janus kinase (Jak)/signal transducer and activator of transcription (STAT) signalling pathway in their target cells. So far, it is not clear whether gp130 is bound to these cytokines and their specific α-receptor subunits through identical or different epitopes. In order to study the interaction of IL-11 and IL-11R with human gp130 the soluble form of the recently cloned human IL-11R was expressed in baculovirus-infected insect cells. By a coprecipitation binding-assay it is demonstrated that IL-11 and IL-6 compete for binding to gp130. Using deletion and point mutants of gp130 it is shown that IL-11–IL-11R and IL-6–IL-6R recognize overlapping binding motifs on gp130. Moreover, using well-established Jak-deficient cell lines we demonstrate that STAT activation by IL-11 requires Jak1. Taken together, our data support the concept that IL-6 and IL-11 activate gp130 by very similar molecular mechanisms.
47. Cis- and trans-acting elements responsible for the cell-specific expression of the human alpha 1-antitrypsin gene
- Author
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V De Simone, Gennaro Ciliberto, Franco Palla, R. Cortese, L Lundberg, Giacomo Paonessa, E. M. Hardon, DE SIMONE, Vincenzo, Ciliberto, G., Hardon, E., Paonessa, G., Palla, F., Lundberg, L., and Cortese, R.
- Subjects
Transcription, Genetic ,Molecular Sequence Data ,Mutant ,Heterologous ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,Transcription (biology) ,Gene expression ,Humans ,Molecular Biology ,Gene ,Regulation of gene expression ,Reporter gene ,Base Sequence ,General Immunology and Microbiology ,General Neuroscience ,Transfection ,Molecular biology ,Gene Expression Regulation ,Genes ,alpha 1-Antitrypsin ,DNA Transposable Elements ,Research Article ,HeLa Cells ,Plasmids - Abstract
The 5' flanking region of the human alpha 1-antitrypsin (alpha 1-AT) gene contains cis-acting signals for liver-specific expression and, when fused to a reporter gene, is able to drive the expression of this gene specifically in liver cells. Here we report the results of a functional dissection of the alpha 1-AT regulatory region. The expression of the bacterial chloramphenicol-transacetylase (CAT) gene, fused to a set of alpha 1-AT 5' flanking regions shortened by progressive deletions or mutated by base pair substitutions, has been compared by transfection in HepG2 (hepatocyte) and HeLa (non-hepatocyte) human cell lines. A minimal tissue-specific element has been identified between the nucleotides -137 and -37 (from the transcriptional start site). This DNA segment activates the heterologous SV40 promoter in hepatoma cell lines but not in HeLa cells. This element contains at least two regions referred to as the A (-125/-100) and B (-84/-70) domains, both essential for transcription. There are at least two other regulatory domains located upstream of the 'minimal element'; the most active of these is located between positions -261 and -210 from the cap site. These upstream elements activate the heterologous SV40 early promoter both in hepatoma cell lines and in HeLa cells. Upon fractionation of rat liver nuclear extracts two proteins have been identified, alpha 1TF-A and alpha 1TF-B, which bind specifically to the A and B domains respectively. Transcriptionally inactive A and B domain mutants are not able to bind these proteins.
48. Specific recognition of cruciform DNA by nuclear protein HMG1
- Author
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Marco Bianchi, Monica Beltrame, Giacomo Paonessa, Bianchi, MARCO EMILIO, Beltrame, M, and Paonessa, G.
- Subjects
Transcription, Genetic ,Immunoblotting ,Molecular Sequence Data ,Biology ,Genetic recombination ,DNA-binding protein ,chemistry.chemical_compound ,Animals ,Nuclear protein ,Cloning, Molecular ,Palindromic sequence ,Immunoassay ,Multidisciplinary ,Base Sequence ,Nucleic acid sequence ,High Mobility Group Proteins ,DNA ,Peptide Fragments ,Rats ,Molecular Weight ,Biochemistry ,chemistry ,Cruciform ,Liver ,Protein Biosynthesis ,DNA supercoil ,Nucleic Acid Conformation ,Electrophoresis, Polyacrylamide Gel - Abstract
Cruciform DNA, a non-double helix form of DNA, can be generated as an intermediate in genetic recombination as well as from palindromic sequences under the effect of supercoiling. Eukaryotic cells are equipped with a DNA-binding protein that selectively recognizes cruciform DNA. Biochemical and immunological data showed that this protein is HMG1, an evolutionarily conserved, essential, and abundant component of the nucleus. The interaction with a ubiquitous protein points to a critical role for cruciform DNA conformations.
49. Two distinct and independent sites on IL-6 trigger gp 130 dimer formation and signalling
- Author
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Armin Lahm, Giacomo Paonessa, Anna Laura Salvati, Gennaro Ciliberto, Rita Graziani, A De Serio, Carlo Toniatti, R. Savino, and Laura Ciapponi
- Subjects
Models, Molecular ,Receptor complex ,Immunoprecipitation ,Protein Conformation ,Protein subunit ,Molecular Sequence Data ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Interleukin 10 receptor, alpha subunit ,Cell Line ,Protein structure ,Antigens, CD ,Cytokine Receptor gp130 ,Animals ,Humans ,Amino Acid Sequence ,Binding site ,Molecular Biology ,G alpha subunit ,Binding Sites ,Membrane Glycoproteins ,General Immunology and Microbiology ,Interleukin-6 ,General Neuroscience ,Genetic Variation ,Receptors, Interleukin ,Glycoprotein 130 ,Receptors, Interleukin-6 ,Cell biology ,Biochemistry ,Research Article ,Signal Transduction - Abstract
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.
50. Nucleotide sequence of rat liver HMG1 cDNA
- Author
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Rainer Frank, Riccardo Cortese, and Giacomo Paonessa
- Subjects
Genetics ,Base Sequence ,Molecular Sequence Data ,DNA, Recombinant ,High Mobility Group Proteins ,Protein primary structure ,Nucleic acid sequence ,DNA ,Computational biology ,Biology ,Rats ,Non-histone protein ,Liver ,Complementary DNA ,Rat liver ,Animals ,Base sequence ,Amino Acid Sequence - Published
- 1987
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