11 results on '"Ansuini, H"'
Search Results
2. Biotin-tagged cDNA expression libraries displayed on lambda phage: a new tool for the selection of natural protein ligands
- Author
-
Ansuini, H., primary
- Published
- 2002
- Full Text
- View/download PDF
3. Role of scavenger receptor class B type I in hepatitis C virus entry: kinetics and molecular determinants
- Author
-
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
4. Role of scavenger receptor class B type I in hepatitis C virus entry: kinetics and molecular determinants.
- Author
-
Catanese MT, Ansuini H, Graziani R, Huby T, Moreau M, Ball JK, Paonessa G, Rice CM, Cortese R, Vitelli A, and Nicosia A
- Subjects
- Animals, Antibodies, Monoclonal, Cells, Cultured, Hepacivirus immunology, Hepatitis C immunology, Humans, Kinetics, Lipoproteins, HDL metabolism, Mice, Scavenger Receptors, Class B metabolism, Species Specificity, Hepacivirus physiology, Receptors, Virus, Scavenger Receptors, Class B physiology, Virus Internalization
- 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
- Full Text
- View/download PDF
5. Anti-EphA2 Antibodies with Distinct In Vitro Properties Have Equal In Vivo Efficacy in Pancreatic Cancer.
- Author
-
Ansuini H, Meola A, Gunes Z, Paradisi V, Pezzanera M, Acali S, Santini C, Luzzago A, Mori F, Lazzaro D, Ciliberto G, Nicosia A, La Monica N, and Vitelli A
- Abstract
The EphA2 receptor tyrosine kinase is overexpressed in a variety of human epithelial cancers and is a determinant of malignant cellular behavior in pancreatic adenocarcinoma cells. Moreover, it is expressed in tumor endothelium and its activation promotes angiogenesis. To better clarify the therapeutic potential of monoclonal antibodies (mAbs) directed to the EphA2 receptor, we generated a large number of mAbs by differential screening of phage-Ab libraries by oligonucleotide microarray technology and implemented a strategy for the rapid identification of antibodies with the desired properties. We selected two high-affinity and highly specific EphA2 monoclonal antibodies with different in vitro properties on the human pancreatic tumor cell line MiaPaCa2. One is a potent EphA2-agonistic antibody, IgG25, that promotes receptor endocytosis and subsequent degradation, and the second is a ligand antagonist, IgG28, that blocks the binding to ephrin A1 and is cross-reactive with the mouse EphA2 receptor. We measured the effect of antibody treatment on the growth of MiaPaCa2 cells orthotopically transplanted in nude mice. Both IgG25 and IgG28 had strong antitumor and antimetastatic efficacy. In vivo treatment with IgG25 determined the reduction of the EphA2 protein levels in the tumor and the phosphorylation of FAK on Tyr576 while administration of IgG28 caused a decrease in tumor vascularization as measured by immunohistochemical analysis of CD31 in tumor sections. These data show that in a pancreatic cancer model comparable therapeutic efficacy is obtained either by promoting receptor degradation or by blocking receptor activation.
- Published
- 2009
- Full Text
- View/download PDF
6. Differential screening of phage-ab libraries by oligonucleotide microarray technology.
- Author
-
Monaci P, Luzzago A, Santini C, De Pra A, Arcuri M, Magistri F, Bellini A, Ansuini H, Ambrosio M, Ammendola V, Bigotti MG, Cirillo A, Nuzzo M, Nasti AA, Neuner P, Orsatti L, Pezzanera M, Sbardellati A, Silvestre G, Uva P, Viti V, Barbato G, Colloca S, Demartis A, De Rinaldis E, Giampaoli S, Lahm A, Palombo F, Talamo F, Vitelli A, Nicosia A, and Cortese R
- Subjects
- Animals, Antibodies, Monoclonal metabolism, Antibodies, Monoclonal pharmacokinetics, Enzyme-Linked Immunosorbent Assay, Immunoglobulin G metabolism, Membrane Proteins metabolism, Mice, Mice, Inbred BALB C, Surface Plasmon Resonance, Antibodies, Monoclonal genetics, Bacteriophages genetics, Oligonucleotide Array Sequence Analysis
- Abstract
A novel and efficient tagArray technology was developed that allows rapid identification of antibodies which bind to receptors with a specific expression profile, in the absence of biological information. This method is based on the cloning of a specific, short nucleotide sequence (tag) in the phagemid coding for each phage-displayed antibody fragment (phage-Ab) present in a library. In order to set up and validate the method we identified about 10,000 different phage-Abs binding to receptors expressed in their native form on the cell surface (10 k Membranome collection) and tagged each individual phage-Ab. The frequency of each phage-Ab in a given population can at this point be inferred by measuring the frequency of its associated tag sequence through standard DNA hybridization methods. Using tiny amounts of biological samples we identified phage-Abs binding to receptors preferentially expressed on primary tumor cells rather than on cells obtained from matched normal tissues. These antibodies inhibited cell proliferation in vitro and tumor development in vivo, thus representing therapeutic lead candidates.
- Published
- 2008
- Full Text
- View/download PDF
7. Binding of the hepatitis C virus E2 glycoprotein to CD81 is strain specific and is modulated by a complex interplay between hypervariable regions 1 and 2.
- Author
-
Roccasecca R, Ansuini H, Vitelli A, Meola A, Scarselli E, Acali S, Pezzanera M, Ercole BB, McKeating J, Yagnik A, Lahm A, Tramontano A, Cortese R, and Nicosia A
- Subjects
- Amino Acid Sequence, Cell Line, Humans, Liver metabolism, Molecular Sequence Data, Species Specificity, Tetraspanin 28, Viral Envelope Proteins chemistry, Antigens, CD metabolism, Membrane Proteins metabolism, Viral Envelope Proteins metabolism
- Abstract
The envelope glycoprotein E2 of hepatitis C virus (HCV) is the target of neutralizing antibodies and is presently being evaluated as an HCV vaccine candidate. HCV binds to human cells through the interaction of E2 with the tetraspanin CD81, a putative viral receptor component. We have analyzed four different E2 proteins from 1a and 1b viral isolates for their ability to bind to recombinant CD81 in vitro and to the native receptor displayed on the surface of Molt-4 cells. A substantial difference in binding efficiency between these E2 variants was observed, with proteins derived from 1b subtypes showing significantly lower binding than the 1a protein. To elucidate the mechanism of E2-CD81 interaction and to identify critical regions responsible for the different binding efficiencies of the E2 variants, several mutants were generated in E2 protein regions predicted by computer modeling to be exposed on the protein surface. Functional analysis of these E2 derivatives revealed that at least two distinct domains are responsible for interaction with CD81. A first segment centered around amino acid residues 613 to 618 is essential for recognition, while a second element including the two hypervariable regions (HVRs) modulates E2 receptor binding. Binding inhibition experiments with anti-HVR monoclonal antibodies confirmed this mapping and supported the hypothesis that a complex interplay between the two HVRs of E2 is responsible for modulating receptor binding, possibly through intramolecular interactions. Finally, E2 proteins from different isolates displayed a profile of binding to human hepatic cells different from that observed on Molt-4 cells or isolated recombinant CD81, indicating that additional factors are involved in viral recognition by target liver cells.
- Published
- 2003
- Full Text
- View/download PDF
8. The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus.
- Author
-
Scarselli E, Ansuini H, Cerino R, Roccasecca RM, Acali S, Filocamo G, Traboni C, Nicosia A, Cortese R, and Vitelli A
- Subjects
- Animals, Antigens, CD physiology, CHO Cells, Carcinoma, Hepatocellular, Cloning, Molecular, Cricetinae, Flow Cytometry, Humans, Leukemia, T-Cell, Liver Neoplasms, Membrane Proteins physiology, Receptors, Lipoprotein physiology, Receptors, Scavenger, Recombinant Proteins metabolism, Scavenger Receptors, Class B, Tetraspanin 28, Tumor Cells, Cultured, CD36 Antigens physiology, Hepacivirus physiology, Receptors, Immunologic, Receptors, Virus physiology
- Abstract
We discovered that the hepatitis C virus (HCV) envelope glycoprotein E2 binds to human hepatoma cell lines independently of the previously proposed HCV receptor CD81. Comparative binding studies using recombinant E2 from the most prevalent 1a and 1b genotypes revealed that E2 recognition by hepatoma cells is independent from the viral isolate, while E2-CD81 interaction is isolate specific. Binding of soluble E2 to human hepatoma cells was impaired by deletion of the hypervariable region 1 (HVR1), but the wild-type phenotype was recovered by introducing a compensatory mutation reported previously to rescue infectivity of an HVR1-deleted HCV infectious clone. We have identified the receptor responsible for E2 binding to human hepatic cells as the human scavenger receptor class B type I (SR-BI). E2-SR-BI interaction is very selective since neither mouse SR-BI nor the closely related human scavenger receptor CD36, were able to bind E2. Finally, E2 recognition by SR-BI was competed out in an isolate-specific manner both on the hepatoma cell line and on the human SR-BI-transfected cell line by an anti-HVR1 monoclonal antibody.
- Published
- 2002
- Full Text
- View/download PDF
9. Searching for DNA-protein interactions by lambda phage display.
- Author
-
Cicchini C, Ansuini H, Amicone L, Alonzi T, Nicosia A, Cortese R, Tripodi M, and Luzzago A
- Subjects
- Adenosine Triphosphatases metabolism, Animals, Bacteriophage lambda, Binding Sites, Cell Line, Chromosome Mapping, DNA-Binding Proteins genetics, Genetic Vectors, HMGA1a Protein metabolism, Hepatocyte Nuclear Factor 1, Hepatocyte Nuclear Factor 1-alpha, Hepatocyte Nuclear Factor 1-beta, Humans, Mice, Peptide Library, Peptides, Promoter Regions, Genetic, Replication Protein C, Repressor Proteins metabolism, Transcription Factors genetics, Transcription Factors metabolism, DNA metabolism, DNA-Binding Proteins metabolism, Nuclear Proteins
- Abstract
We applied phage display technology to DNA-protein interaction studies. A cDNA expression library displayed on the surface of bacteriophage lambda was generated from the highly differentiated MMH E14 murine hepatic cell line. Selection of this library using the promoter sequence of the liver-enriched transcription factor HNF1alpha gene as ligate identified DNA-binding domains specifically interacting with different regions of this regulatory sequence. One of the selected phage showed 100% identity to a DNA-binding domain shared by differentiation specific element-binding protein, vasoactive intestinal peptide receptor-repressor protein and replication factor C and was further investigated. Specific binding of the selected protein domain was confirmed in a phage-independent context. By combining ELISA and South-Western assays using the selected phage and a bacterially expressed glutathione-S-transferase protein fused to the encoded DNA-binding domain, an array of multiple adjacent DNA-binding sites sharing a common consensus motif was identified. The strategy described represents a powerful tool to identify proteins that bind to DNA regulatory elements.
- Published
- 2002
- Full Text
- View/download PDF
10. Neutrophil proteases can inactivate human PAR3 and abolish the co-receptor function of PAR3 on murine platelets.
- Author
-
Cumashi A, Ansuini H, Celli N, De Blasi A, O'Brien PJ, Brass LF, and Molino M
- Subjects
- Amino Acid Sequence, Animals, Blood Platelets drug effects, COS Cells, Cathepsin G, Cathepsins metabolism, Humans, Molecular Sequence Data, Pancreatic Elastase metabolism, Platelet Aggregation drug effects, Receptors, Thrombin antagonists & inhibitors, Thrombin pharmacology, Transfection, Type C Phospholipases drug effects, Type C Phospholipases metabolism, Neutrophils enzymology, Receptors, Thrombin metabolism, Serine Endopeptidases metabolism
- Abstract
Three members of the protease-activated receptor family, PAR1, PAR3 and PAR4, are activated when thrombin cleaves the receptor N-terminus, exposing a tethered ligand. Proteases other than thrombin can also cleave PAR family members and, depending upon whether this exposes or removes the tethered ligand, either activate or disable the receptor. For example, on human platelets PAR1 is disabled by cathepsin G, although aggregation still occurs because cathepsin G can activate PAR4. The present studies examine the interaction of cathepsin G and a second neutrophil protease, elastase, with PAR3 using two model systems: COS-7 cells transfected with human PAR3 and mouse platelets, which express PAR3 and PAR4, but not PAR1. In contrast to human platelets, cathepsin G did not aggregate murine platelets, and prevented their activation only at low thrombin concentrations. Elastase had no effect on thrombin responses in mouse platelets, but when added to COS cells expressing human PAR3, both cathepsin G and elastase prevented activation of phospholipase C by thrombin. Notably, this inhibition occurred without loss of the binding sites for two monoclonal antibodies that flank the tethered ligand on human PAR3. We therefore conclude that 1) exposure to cathepsin G disables signaling through human PAR3, and prevents murine PAR3 from serving its normal role, which is to facilitate PAR4 cleavage at low thrombin concentrations, 2) elastase disables human, but not murine, PAR3, 3) in contrast to human PAR4, mouse PAR4 will not support platelet aggregation in response to cathepsin G, and 4) the inactivation of human PAR3 by cathepsin G and elastase involves a mechanism other than amputation of the tethered ligand domain. These results extend the range of possible interactions between PAR family members and proteases, and provide further support for species-specific differences in the interaction of these receptors with proteases other than thrombin.
- Published
- 2001
11. Transcription factors ets1, NF-kappa B, and Sp1 are major determinants of the promoter activity of the human protein kinase CK2alpha gene.
- Author
-
Krehan A, Ansuini H, Bocher O, Grein S, Wirkner U, and Pyerin W
- Subjects
- Casein Kinase II, Chromatography, Affinity, Gene Expression Regulation, Enzymologic, Humans, Mutagenesis, Site-Directed, Phosphorylation, Protein Binding, Proto-Oncogene Protein c-ets-1, Proto-Oncogene Proteins c-ets, DNA-Binding Proteins genetics, NF-kappa B physiology, Promoter Regions, Genetic, Protein Serine-Threonine Kinases genetics, Protein-Tyrosine Kinases physiology, Proto-Oncogene Proteins physiology, Sp1 Transcription Factor physiology, Transcription Factors physiology
- Abstract
CK2alpha is one of two isoforms of protein kinase CK2, a highly conserved, ubiquitous, and vital phosphotransferase whose expression is kept at constant cellular levels and whose dysregulated expression has been linked to malignant diseases. The upstream sequence of the gene coding for human CK2alpha (CSNK1A1, chromosomal location 20p13) has been examined for promoter location and transcription factor interactions using reporter gene assays (luciferase; HeLa cells), site-directed mutagenesis, electrophoretic mobility shift assays, super-shifts, UV cross-linking, Western blotting, and DNA affinity chromatography. Highest promoter activity has been found in a region comprising positions -9 to 46. Factors Sp1, Ets-1, and NF-kappaB have been identified as interaction partners and, by mutation of individual sites and simultaneous mutations of two or more sites, shown to cross-talk to each other. At least two of the factors (Sp1; NF-kappaB) were susceptible to phosphorylation by CK2 holoenzyme, a tetramer composed of two CK2alpha and two regulatory CK2beta proteins, but not by individual CK2alpha. Because the phosphorylation decreases promoter binding and repeated immunoprecipitation reveals presence of "free" CK2beta in cell extracts, it is tempting to speculate that the gene product CK2alpha might readily form CK2 holoenzyme and feed back onto gene transcription. The data represent the first promoter control analysis of a mammalian CK2alpha gene and provide a hypothesis of how the constant expression level of CK2alpha may be achieved.
- Published
- 2000
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.