RESUMEN
We examined the distribution and processing of terrestrial organic material, derived from the disposal of material from a massive debris flow event following a major wildfire in a coastal California (USA) catchment in intertidal and nearshore subtidal marine sediments. Organic matter biomarkers, pyrogenic carbon and lignin phenols, were used to trace the distribution of terrestrial debris material in marine environments. In intertidal sediments located <1 km east of the debris deposition site, pyrogenic carbon values did not significantly change and lambda values, a lignin measure, decreased over time, indicating little lateral transport of the disposed material. In subtidal sediment, pyrogenic carbon and lambda values were greatest in 20 m water depths indicating transport and deposition of this material nearshore. An additional lignin measure indicative of degradation suggested terrestrial organic material degradation in subtidal sediment decreased with distance from shore. Terrestrial biomarkers demonstrated that the disposed material was not detected in the top 20 cm of intertidal sediment but was retained in subtidal sediment offshore of the disposal site. Results suggest coastal management should incorporate consideration of the effects of debris disposal activities on nearshore benthic communities and biogeochemical cycling.
Asunto(s)
Sedimentos Geológicos , Lignina , CarbonoRESUMEN
Activation of the proapoptotic receptor death receptor5 (DR5) in various cancer cells triggers programmed cell death through the extrinsic pathway. We have generated a fully human monoclonal antibody (Apomab) that induces tumor cell apoptosis through DR5 and investigated the structural features of its interaction with DR5. Biochemical studies showed that Apomab binds DR5 tightly and selectively. X-ray crystallographic analysis of the complex between the Apomab Fab fragment and the DR5 ectodomain revealed an interaction epitope that partially overlaps with both regions of the Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand binding site. Apomab induced DR5 clustering at the cell surface and stimulated a death-inducing signaling complex containing the adaptor molecule Fas-associated death domain and the apoptosis-initiating protease caspase-8. Fc crosslinking further augmented Apomab's proapoptotic activity. In vitro, Apomab triggered apoptosis in cancer cells, while sparing normal hepatocytes even upon anti-Fc crosslinking. In vivo, Apomab exerted potent antitumor activity as a single agent or in combination with chemotherapy in xenograft models, including those based on colorectal, non-small cell lung and pancreatic cancer cell lines. These results provide structural and functional insight into the interaction of Apomab with DR5 and support further investigation of this antibody for cancer therapy.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Neoplasias Experimentales/tratamiento farmacológico , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/agonistas , Animales , Anticuerpos Monoclonales/química , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Antineoplásicos/química , Antineoplásicos/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Sitios de Unión de Anticuerpos , Caspasa 8/metabolismo , Línea Celular Tumoral , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Mapeo Epitopo , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Femenino , Humanos , Ratones , Ratones Desnudos , Modelos Moleculares , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Unión Proteica , Conformación Proteica , Agregación de Receptores/efectos de los fármacos , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/química , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/inmunología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Vascular endothelial growth factor (VEGF-A) is a key cytokine in the development of normal blood vessels as well as the development of vessels in tumors and other tissues undergoing abnormal angiogenesis. Here, we review the molecular engineering of two humanized antibodies derived from a common mouse anti-VEGF antibody--bevacizumab, a full-length IgG1 approved for the treatment of specified cancer indications, and ranibizumab, an affinity-matured antibody Fab domain approved for use in age-related macular degeneration (AMD). In clinical trials and as FDA-approved therapeutics, these two anti-VEGF antibodies, bevacizumab (Avastin anti-VEGF antibody) and ranibizumab (Lucentis anti-VEGF antibody), have demonstrated therapeutic utility in blocking VEGF-induced angiogenesis.
Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Factor A de Crecimiento Endotelial Vascular/inmunología , Secuencia de Aminoácidos , Inhibidores de la Angiogénesis/biosíntesis , Inhibidores de la Angiogénesis/química , Inhibidores de la Angiogénesis/farmacología , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales Humanizados , Antineoplásicos/química , Antineoplásicos/farmacología , Bevacizumab , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Humanos , Degeneración Macular/tratamiento farmacológico , Modelos Moleculares , Datos de Secuencia Molecular , Neoplasias/irrigación sanguínea , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Fisiológica/efectos de los fármacos , Conformación Proteica , Ingeniería de Proteínas , Ranibizumab , Proteínas Recombinantes de Fusión/uso terapéutico , Transducción de Señal/efectos de los fármacos , Resultado del Tratamiento , Factor A de Crecimiento Endotelial Vascular/química , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
A family of structured peptides that bind to FcepsilonRIalpha, the alpha-chain of the high-affinity receptor for IgE, has been identified. Binding selections using FcepsilonRIalpha and polyvalent peptide-phage libraries yielded a dominant 18-residue peptide-phage clone, as well as related sequences that did not resemble fragments of IgE. Synthetic peptides based on these sequences inhibited IgE binding to its receptor, and were found by NMR analysis to adopt a stable beta-hairpin structure in solution. Optimized peptides with micromolar receptor affinity exhibited high stability in biological fluids and inhibited cellular histamine release in an in vitro bioassay of IgE activity. The structure-activity relationships of these peptides, which are less than 1% of the size of IgE, suggest an overlap between their binding site and that of IgE on FcepsilonRI. Thus, the peptides demonstrate that blocking a small epitope on this receptor chain is sufficient to block IgE activity. Such structured peptides represent a possible starting point for the design of novel antagonists, and offer the potential for testing in vivo a new approach for treating allergic disease.
Asunto(s)
Inmunoglobulina E/química , Inmunoglobulina E/metabolismo , Péptidos/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Aminoácidos/química , Animales , Bacteriófagos/química , Sitios de Unión , Unión Competitiva , Células CHO , Cricetinae , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Biblioteca de Péptidos , Unión Proteica , Conformación Proteica , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
Highly structured, peptide antagonists of the interaction between insulin-like growth factor 1 (IGF-I) and IGF binding protein 1 (IGFBP-1) have recently been discovered by phage display of naïve peptide libraries [Lowman, H. B., et al. (1998) Biochemistry 37, 8870--8878]. We now report a detailed analysis of the features of this turn-helix peptide motif that are necessary for IGFBP-1 binding and structural integrity. Further rounds of phage randomization indicate the importance of residues contributing to a hydrophobic patch on one face of the helix. Alanine-scanning substitutions confirm that the hydrophobic residues are necessary for binding. However, structural analysis by NMR spectroscopy indicates that some of these analogues are less well folded. Structured, high-affinity analogues that lack the disulfide bond were prepared by introducing a covalent constraint between side chains at positions i and i + 7 or i + 8 within the helix. Analogues based on this scaffold demonstrate that a helical conformation is present in the bound state, and that hydrophobic side chains in this helix, and residues immediately preceding it, interact with IGFBP-1. By comparison of alanine scanning data for IGF-I and the turn-helix peptide, we propose a model for common surface features of these molecules that recognize IGFBP-1.
Asunto(s)
Bacteriófago M13/metabolismo , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Péptidos/metabolismo , Alanina/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Animales , Unión Competitiva/genética , Células CHO , Secuencia Conservada , Cricetinae , Cristalografía por Rayos X , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Cinética , Imitación Molecular , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Biblioteca de Péptidos , Péptidos/síntesis química , Péptidos/genética , Unión Proteica/genética , Estructura Secundaria de Proteína , Relación Estructura-Actividad , Resonancia por Plasmón de Superficie , Propiedades de SuperficieRESUMEN
Insulin-like growth factor I (IGF-I) is a potent anabolic peptide that mediates most of its pleiotropic effects through association with the IGF type I receptor. Biological availability and plasma half-life of IGF-I are modulated by soluble binding proteins (IGFBPs), which sequester free IGF-I into high affinity complexes. Elevated levels of specific IGFBPs have been observed in several pathological conditions, resulting in inhibition of IGF-I activity. Administration of IGF-I variants that are unable to bind to the up-regulated IGFBP species could potentially counteract this effect. We engineered two IGFBP-selective variants that demonstrated 700- and 80,000-fold apparent reductions in affinity for IGFBP-1 while preserving low nanomolar affinity for IGFBP-3, the major carrier of IGF-I in plasma. Both variants displayed wild-type-like potency in cellular receptor kinase assays, stimulated human cartilage matrix synthesis, and retained their ability to associate with the acid-labile subunit in complex with IGFBP-3. Furthermore, pharmacokinetic parameters and tissue distribution of the IGF-I variants in rats differed from those of wild-type IGF-I as a function of their IGFBP affinities. These IGF-I variants may potentially be useful for treating disease conditions associated with up-regulated IGFBP-1 levels, such as chronic or acute renal and hepatic failure or uncontrolled diabetes. More generally, these results suggest that the complex biology of IGF-I may be clarified through in vivo studies of IGFBP-selective variants.
Asunto(s)
Cartílago Articular/metabolismo , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Anciano , Sustitución de Aminoácidos , Animales , Neoplasias de la Mama , Cartílago Articular/efectos de los fármacos , Femenino , Variación Genética , Humanos , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/farmacocinética , Cinética , Tasa de Depuración Metabólica , Mutagénesis Sitio-Dirigida , Técnicas de Cultivo de Órganos , Ratas , Ratas Sprague-Dawley , Receptor IGF Tipo 1/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacocinética , Especificidad por Sustrato , Sulfatos/metabolismo , Distribución Tisular , Células Tumorales CultivadasAsunto(s)
Proteínas de la Cápside , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/química , Biblioteca de Péptidos , Péptidos/química , Secuencia de Aminoácidos , Animales , Bacteriófagos/genética , Sitios de Unión , Cápside/química , Cápside/genética , Clonación Molecular/métodos , ADN de Cadena Simple/genética , Factores de Crecimiento Endotelial/química , Factores de Crecimiento Endotelial/metabolismo , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Vectores Genéticos , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Ligandos , Linfocinas/química , Linfocinas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Ácidos Nucleicos Heterodúplex , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Péptidos/metabolismo , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Engineering proteins to bind small molecules presents a challenge as daunting as drug discovery, for both hinge upon our understanding of receptor-ligand molecular recognition. However, powerful techniques from combinatorial molecular biology can be used to rapidly select artificial receptors. While traditionally researchers have relied upon antibody technologies as a source of new binding proteins, the lipocalin scaffold has recently emerged as an adaptable receptor for small molecule binding. 'Anticalins', engineered lipocalin variants, offer some advantages over traditional antibody technology and illuminate features of molecular recognition between receptors and small molecule ligands.
Asunto(s)
Proteínas Portadoras/metabolismo , Ingeniería de Proteínas , Secuencia de Aminoácidos , Animales , Anticuerpos/metabolismo , Anticuerpos/uso terapéutico , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas Portadoras/aislamiento & purificación , Proteínas Portadoras/uso terapéutico , Técnicas Químicas Combinatorias , Lipocalina 1 , Modelos Moleculares , Datos de Secuencia Molecular , Biblioteca de Péptidos , Unión Proteica , Estructura Terciaria de Proteína , Especificidad por SustratoRESUMEN
We have generated two mAbs, 6G4.2.5 and A5.12.14, that are similarly capable of neutralizing the biologic activity of wild-type IL-8. To characterize these antibodies further, their reactivity against a series of engineered IL-8 monomer and dimer variants was examined using a neutrophil degranulation assay. While 6G4.2.5 was found to block effectively the biologic activity of all variants regardless of their dimerization status, the results for A5.12.14 differed dramatically. A5.12.14 fully inhibited the agonist activity of one of the monomer variants, partially blocked the activity of another, and had no effect on the activity of two other variants. These results suggested that the binding epitope of A5.12.14 was being affected by the particular amino acid substitutions introduced into the dimer interface region of the variants to disfavor dimerization. If A5.12.14 indeed binds to the dimer interface region of IL-8, it could be predicted that this mAb would be unable to inhibit the activity of dimeric IL-8. This was confirmed in studies which showed that A5.12.14 had no demonstrable effect on the activity of a constitutively dimeric IL-8 variant. These studies represent the first example of a mAb specific for the dimerization status of IL-8.
Asunto(s)
Anticuerpos Monoclonales , Dimerización , Interleucina-8/metabolismo , Aminoácidos/química , Animales , Calcio/metabolismo , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Epítopos , Glucuronidasa/metabolismo , Humanos , Inmunoglobulina G/metabolismo , Ratones , Neutrófilos/enzimología , Unión ProteicaRESUMEN
Bronchoalveolar lavage fluid from mice with experimentally induced allergic pulmonary inflammation contains a novel 9.4 kDa cysteine-rich secreted protein, FIZZ1 (found in inflammatory zone). Murine (m) FIZZ1 is the founding member of a new gene family including two other murine genes expressed, respectively, in intestinal crypt epithelium and white adipose tissue, and two related human genes. In control mice, FIZZ1 mRNA and protein expression occur at low levels in a subset of bronchial epithelial cells and in non-neuronal cells adjacent to neurovascular bundles in the peribronchial stroma, and in the wall of the large and small bowel. During allergic pulmonary inflammation, mFIZZ1 expression markedly increases in hypertrophic, hyperplastic bronchial epithelium and appears in type II alveolar pneumocytes. In vitro, recombinant mFIZZ1 inhibits the nerve growth factor (NGF)-mediated survival of rat embryonic day 14 dorsal root ganglion (DRG) neurons and NGF-induced CGRP gene expression in adult rat DRG neurons. In vivo, FIZZ1 may modulate the function of neurons innervating the bronchial tree, thereby altering the local tissue response to allergic pulmonary inflammation.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular , Familia de Multigenes , Proteínas/genética , Hipersensibilidad Respiratoria/genética , Secuencia de Aminoácidos , Animales , Bronquios/citología , Líquido del Lavado Bronquioalveolar/química , Supervivencia Celular , Cisteína , Ganglios Espinales/citología , Humanos , Inmunohistoquímica , Hibridación in Situ , Mucosa Intestinal/citología , Ratones , Datos de Secuencia Molecular , Factor de Crecimiento Nervioso/metabolismo , Proteínas/aislamiento & purificación , Proteínas/metabolismo , Ratas , Mucosa Respiratoria/citología , Análisis de Secuencia de ADN , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
The "ELR" (Glu-Leu-Arg) tripeptide sequence near the N-terminus of interleukin-8 (IL-8) contributes a large part of the receptor binding free energy. Prior X-ray and nuclear magnetic resonance (NMR) structures of IL-8 have shown this region of the molecule to be highly mobile. We reasoned that a hydrophobic interaction between the leucine and the neighboring beta-turn might exist in the receptor binding conformation of the N-terminus. To test this hypothesis, we mutated two residues to cysteine and connected the N-terminus to the beta-turn. The mutant retains receptor binding affinity reasonably close to wild type and allows the characterization of a high-affinity conformation that may be useful in the design of small IL-8 mimics. The L5C/H33C mutant is refined to R-values of R = 20.6% and Rfree = 27.7% at 2.35 A resolution. Other receptor binding determinants reside in the "N-loop" found after "ELR" and preceding the first beta-strand. All available structures of IL-8 have been found with one of two distinct N-loop conformations. One of these is relevant for receptor binding, based on NMR results with receptor peptides. The other conformation obscures the receptor-peptide binding surface and may have an undetermined but necessarily different function.
Asunto(s)
Interleucina-8/química , Secuencias de Aminoácidos , Sustitución de Aminoácidos , Animales , Antígenos CD/química , Cristalografía por Rayos X , Humanos , Interleucina-8/genética , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Unión Proteica , Conformación Proteica , Receptores de Citocinas/química , Receptores de Interleucina/química , Receptores de Interleucina-8ARESUMEN
The use of libraries of phage-displayed human single-chain antibody fragments (scFv) has become a new, powerful tool in rapidly obtaining therapeutically useful antibodies. Here, we describe the generation of human scFv and F(ab')2 directed against the gamma-carboxyglutamic acid (Gla) domain of coagulation factor IX. A large library of human scFv, displayed either on M13 phage or expressed as soluble proteins, was screened for binding to human Gla-domain peptide (Tyr1-Lys43). Among a panel of scFv that bound to the factor IX-Gla domain, six scFv clones recognized full-length factor IX and exhibited strong inhibitory activity of factor IX in vitro. After reformatting as F(ab')2, the affinity for factor IX of three selected clones was determined: 10C12 Kd = 1.6 nmol/l, 13D1 Kd = 2.9 nmol/l, and 13H6 Kd = 0.46 nmol/l. The antibodies specifically bound to factor IX and not to other coagulation factors, as assessed by enzyme-linked immunosorbent-type and human plasma clotting assays. The complementarity determining region amino acid sequences of clones 10C12 and 13D1 only differed at a single residue, whereas 13H6 showed little homology, suggesting that 13H6 binds to a different epitope within the factor IX-Gla domain. Despite the slightly lower affinity of 10C12 F(ab')2 versus 13H6 F(ab')2, 10C12 was consistently more potent than 13H6 in prolonging the activated partial thromboplastin time (APTT), in inhibiting platelet-mediated plasma clotting, and in inhibiting factor X activation by the intrinsic Xase complex. Finally, 10C12 F(ab')2 also recognized and neutralized factor IX/factor IXa of different species, as demonstrated by the specific APTT prolongation of dog, mouse, baboon and rabbit plasma. In summary, the results validate the usefulness of scFv phage-displayed libraries to rapidly generate fully human antibodies as potential new therapeutics for thrombotic disorders.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/metabolismo , Factor IXa/inmunología , Biblioteca de Péptidos , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/farmacología , Afinidad de Anticuerpos , Reacciones Antígeno-Anticuerpo/efectos de los fármacos , Sitios de Unión de Anticuerpos , Unión Competitiva , Coagulación Sanguínea/efectos de los fármacos , Factores de Coagulación Sanguínea/metabolismo , Plaquetas/fisiología , Calcio/farmacología , Bovinos , Clonación Molecular , Endotelio Vascular/citología , Ensayo de Inmunoadsorción Enzimática , Epítopos/metabolismo , Factor IXa/antagonistas & inhibidores , Factor IXa/fisiología , Humanos , Fragmentos Fab de Inmunoglobulinas/metabolismo , Fragmentos Fab de Inmunoglobulinas/farmacología , Región Variable de Inmunoglobulina/aislamiento & purificación , Región Variable de Inmunoglobulina/metabolismo , Región Variable de Inmunoglobulina/farmacología , Magnesio/farmacología , Datos de Secuencia Molecular , Alineación de Secuencia , Especificidad de la EspecieRESUMEN
The Fab portion of a humanized antibody (Fab-12; IgG form known as rhuMAb VEGF) to vascular endothelial growth factor (VEGF) has been affinity-matured through complementarity-determining region (CDR) mutation, followed by affinity selection using monovalent phage display. After stringent binding selections at 37 degrees C, with dissociation (off-rate) selection periods of several days, high affinity variants were isolated from CDR-H1, H2, and H3 libraries. Mutations were combined to obtain cumulatively tighter-binding variants. The final variant identified here, Y0317, contained six mutations from the parental antibody. In vitro cell-based assays show that four mutations yielded an improvement of about 100-fold in potency for inhibition of VEGF-dependent cell proliferation by this variant, consistent with the equilibrium binding constant determined from kinetics experiments at 37 degrees C. Using X-ray crystallography, we determined a high-resolution structure of the complex between VEGF and the affinity-matured Fab fragment. The overall features of the binding interface seen previously with wild-type are preserved, and many contact residues are maintained in precise alignment in the superimposed structures. However, locally, we see evidence for improved contacts between antibody and antigen, and two mutations result in increased van der Waals contact and improved hydrogen bonding. Site-directed mutants confirm that the most favorable improvements as judged by examination of the complex structure, in fact, have the greatest impact on free energy of binding. In general, the final antibody has improved affinity for several VEGF variants as compared with the parental antibody; however, some contact residues on VEGF differ in their contribution to the energetics of Fab binding. The results show that small changes even in a large protein-protein binding interface can have significant effects on the energetics of interaction.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Afinidad de Anticuerpos/inmunología , Factores de Crecimiento Endotelial/inmunología , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/inmunología , Linfocinas/inmunología , Alanina/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/farmacología , Antígenos/química , Antígenos/inmunología , Sitios de Unión , División Celular/efectos de los fármacos , Cristalografía por Rayos X , Factores de Crecimiento Endotelial/antagonistas & inhibidores , Factores de Crecimiento Endotelial/química , Factores de Crecimiento Endotelial/farmacología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Epítopos/química , Epítopos/genética , Epítopos/inmunología , Humanos , Enlace de Hidrógeno , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/farmacología , Linfocinas/antagonistas & inhibidores , Linfocinas/química , Linfocinas/farmacología , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Biblioteca de Péptidos , Conformación Proteica , Termodinámica , Venas Umbilicales , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
The bioavailability of insulin-like growth factor I (IGF-I) in the serum and tissues is controlled by members of the IGF binding protein family (IGFBP). These proteins form high-affinity complexes with IGF-I and thereby either inhibit or potentiate its mitogenic and metabolic effects. Thus, understanding the IGF-IGFBP interaction at the molecular level is crucial for attempts to modulate IGF-I activity in vivo. We have systematically investigated the binding contribution of each IGF-I amino acid side chain toward IGFBP-1 and IGFBP-3, combining alanine-scanning mutagenesis and monovalent phage display. Surprisingly, most IGF-I residues could be substituted by alanines, resulting in less than 5-fold affinity losses for IGFBP-3. In contrast, binding of IGFBP-1 was more sensitive to alanine substitutions in IGF-I. The glutamate and phenylalanine at positions 3 and 49 were identified as major specificity determinants for IGFBP-1: the corresponding alanine mutations, E3A and F49A, selectively decreased IGFBP-1 binding by 34- and 100-fold, whereas IGFBP-3 affinity was not affected or reduced maximally 4-fold. No side chain specificity determinant was found for IGFBP-3. Instead, our results suggest that the N-terminal backbone region of IGF-I is important for binding to IGFBP-3. The fact that the functional binding epitopes on IGF-I are overlapping but distinct for both binding proteins may be exploited to design binding protein-specific IGF variants.
Asunto(s)
Alanina/genética , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/genética , Mutagénesis Sitio-Dirigida , Sustitución de Aminoácidos/genética , Bacteriófago M13/química , Bacteriófago M13/genética , Sitios de Unión/genética , Técnicas Biosensibles , Humanos , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/química , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/química , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/química , Factor I del Crecimiento Similar a la Insulina/metabolismo , Modelos Moleculares , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , SolubilidadRESUMEN
BACKGROUND: Interactions between CXC chemokines (e.g. interleukin-8, IL-8) and their receptors (e.g. CXCR-1) have a key role in host defense and disease by attracting and upregulating neutrophils to sites of inflammation. The transmembrane nature of the receptor impedes structure-based understanding of ligand interactions. Linear peptides based on the N-terminal, extracellular portion of the receptor CXCR-1 do bind to IL-8, however, and inhibit the binding of IL-8 to the full-length receptor. RESULTS: The NMR solution structure of the complex formed between IL-8 and one such receptor-based peptide indicates that a cleft between a loop and a beta hairpin constitute part of the receptor interaction surface on IL-8. Nine residues from the C terminus of the receptor peptide (corresponding to Pro21-Pro29 of CXCR-1) occupy the cleft in an extended fashion. Intermolecular contacts are mostly hydrophobic and sidechain mediated. CONCLUSIONS: The results offer the first details at an atomic level of the interaction between a chemokine and its receptor. Consideration of other biochemical data allow extrapolation to a model for the interaction of IL-8 with the full-length receptor. In this model, the heparin-binding residues of IL-8 are exposed, thereby allowing presentation of the chemokine from endothelial cell-surface glycosaminoglycans. This first glimpse of how IL-8 binds to its receptor provides a foundation for the structure-based design of chemokine antagonists.
Asunto(s)
Antígenos CD/química , Interleucina-8/química , Conformación Proteica , Receptores de Interleucina/química , Secuencia de Aminoácidos , Glicosaminoglicanos/metabolismo , Espectroscopía de Resonancia Magnética , Proteínas de la Membrana/química , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , Neutrófilos/metabolismo , Fragmentos de Péptidos/química , Unión Proteica , Estructura Secundaria de Proteína , Receptores de Interleucina-8ARESUMEN
BACKGROUND: Vascular endothelial growth factor (VEGF) is a highly specific angiogenic growth factor; anti-angiogenic treatment through inhibition of receptor activation by VEGF might have important therapeutic applications in diseases such as diabetic retinopathy and cancer. A neutralizing anti-VEGF antibody shown to suppress tumor growth in an in vivo murine model has been used as the basis for production of a humanized version. RESULTS: We present the crystal structure of the complex between VEGF and the Fab fragment of this humanized antibody, as well as a comprehensive alanine-scanning analysis of the contact residues on both sides of the interface. Although the VEGF residues critical for antibody binding are distinct from those important for high-affinity receptor binding, they occupy a common region on VEGF, demonstrating that the neutralizing effect of antibody binding results from steric blocking of VEGF-receptor interactions. Of the residues buried in the VEGF-Fab interface, only a small number are critical for high-affinity binding; the essential VEGF residues interact with those of the Fab fragment, generating a remarkable functional complementarity at the interface. CONCLUSIONS: Our findings suggest that the character of antigen-antibody interfaces is similar to that of other protein-protein interfaces, such as ligand-receptor interactions; in the case of VEGF, the principal difference is that the residues essential for binding to the Fab fragment are concentrated in one continuous segment of polypeptide chain, whereas those essential for binding to the receptor are distributed over four different segments and span across the dimer interface.
Asunto(s)
Complejo Antígeno-Anticuerpo/química , Complejo Antígeno-Anticuerpo/genética , Factores de Crecimiento Endotelial/química , Factores de Crecimiento Endotelial/genética , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/genética , Linfocinas/química , Linfocinas/genética , Alanina/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Complejo Antígeno-Anticuerpo/inmunología , Cristalografía por Rayos X , Análisis Mutacional de ADN , Dimerización , Factores de Crecimiento Endotelial/inmunología , Mapeo Epitopo , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Linfocinas/inmunología , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
IGF-1 (insulin-like growth factor 1) is a 70-residue protein hormone which has both metabolic and mitogenic activities mediated through IGF-1 binding to cell surface receptors. However, an unrelated class of proteins, the IGF-binding proteins (IGFBPs) also bind IGF-1 in the serum and tissues and block or modulate its activity in vivo. Therefore, inhibitors of the IGFBPs can alter the distribution between free and bound IGF-1 [Loddick, S. A., Liu, X.-J., Lu, Z.-X., Liu, C., Behan, D. P., Chalmers, D. C., Foster, A. C., Vale, W. W., Ling, N., and De Souza, E. B. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 1894-1898] and potentially affect the distribution of IGF-1 among body tissues. We report here that phage-displayed peptide libraries have yielded a peptide that binds IGFBP-1 and produces IGF-like activity at sub-micromolar concentrations. The 14-residue peptide has an extremely well-defined solution conformation that can aid in the design of smaller, orally active compounds. Interestingly, the peptide structure contains a helix, as does one region of IGF-1 previously implicated in IGFBP binding, yet displays side chains different from those of the IGF-1 helix I. Furthermore, an IGF-1 variant lacking receptor-signaling activity in vitro is shown here to produce IGF-like mitogenic and metabolic activity in vivo. These results suggest that small antagonist mimetics of protein ligands, identified by binding selection to otherwise inhibitory factors, may be useful as indirect agonists for a variety of therapeutic applications.
Asunto(s)
Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/antagonistas & inhibidores , Factor I del Crecimiento Similar a la Insulina/metabolismo , Imitación Molecular , Secuencia de Aminoácidos , Animales , Bacteriófago M13/metabolismo , Unión Competitiva/efectos de los fármacos , Insulina/farmacología , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/antagonistas & inhibidores , Factor I del Crecimiento Similar a la Insulina/química , Factor I del Crecimiento Similar a la Insulina/farmacología , Ligandos , Masculino , Modelos Moleculares , Datos de Secuencia Molecular , Biblioteca de Péptidos , Péptidos/síntesis química , Péptidos/farmacología , Unión Proteica/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Ratas Zucker , Receptor IGF Tipo 1/deficienciaRESUMEN
The characteristic CXC chemokine disulfide core of interleukin-8 (IL-8) has been rearranged in a variant replacing the 9-50 disulfide with a 9-38 disulfide. The new variant has been characterized by its binding affinity to IL-8 receptors A and B and the erythrocyte receptor DARC. This variant binds the three receptors with affinities between 500- and 2,500-fold lower than wild-type IL-8. Binding affinity results are also reported for the variant with alanine substituted for both cysteines 9 and 50. The Glu38-->Cys/Cys50-->Ala IL-8 crystallizes in space group P2(1)2(1)2(1) with cell parameters a = 46.4, b = 49.2, and c = 69.5 A, and has been refined to an R-value of 19.4% for data from 10 to 2 A resolution. Analysis of the structure confirms the new disulfide arrangement and suggests that changes at Ile10 may be the principal cause of the lowered affinities.
Asunto(s)
Antígenos CD/metabolismo , Cisteína/genética , Disulfuros/química , Interleucina-8/química , Interleucina-8/metabolismo , Receptores de Interleucina/metabolismo , Sitios de Unión/genética , Unión Competitiva , Simulación por Computador , Cristalografía por Rayos X , Eritrocitos/metabolismo , Interleucina-8/genética , Modelos Moleculares , Mutación , Neutrófilos/metabolismo , Conformación Proteica , Receptores de Interleucina-8A , Agua/químicaRESUMEN
IL-8 dimers have been observed in NMR and X-ray structures of the protein. We have engineered IL-8 monomers by mutations of residues throughout the dimer interface, which introduce hindrance determinants to dimerization. These IL-8 variants are shown by NMR to have wild-type monomer folding, but by ultracentrifugation to have a range of dimerization constants from microM to mM, as compared with a dimerization constant of about 10 microM for wild-type IL-8, under physiological salt and temperature conditions. The monomeric variants of IL-8 bind the erythrocyte chemokine receptor DARC, as well as the neutrophil IL-8 receptors CXCR1 and CXCR2 with affinities similar to that of wild-type IL-8. In addition, the monomeric variants were shown to have agonist activity, with similar potency to wild-type, in both Ca(2+)-flux assays on CXCR1 and CXCR2 transfected cells, and in chemotaxis assays on neutrophils. Thus, these variants confirm that monomeric IL-8 is functionally equivalent to wild-type in vitro assays. We have also investigated the effects of various solution conditions upon IL-8 dimer formation using analytical ultracentrifugation. At salt concentrations, temperatures, and pH conditions lower than physiological, the dimerization affinity of IL-8 is greatly enhanced. This suggests that, under some conditions, IL-8 dimer formation may occur at concentrations of IL-8 considerably lower than 10 microM, with consequences in vivo that are yet to be determined.