RESUMEN
Fibroblast growth factors (FGFs) bind to extracellular matrices, especially heparin-like carbohydrates of heparan-sulfate proteoglycans which stabilize FGFs to protect against inactivation by heat, acid, proteolysis and oxidation. Moreover, binding of FGFs to cell surface proteoglycans promotes to form oligomers, which is essential for receptor oligomerization and activation. In the present study, we determined the solution structure of acidic FGF using a series of triple resonance multi-dimensional NMR experiments and simulated annealing calculations. Furthermore, we prepared the sample complexed with a heparin-derived hexasaccharide which is a minimum unit for aFGF binding. From the chemical shift differences between free aFGF and aFGF-heparin complex, we concluded that the major heparin binding site was located on the regions 110-131 and 17-21. The binding sites are quite similar to those observed for bFGF-heparin hexasaccharide complex, showing that both FGFs recognize heparin-oligosaccharides in a similar manner.
Asunto(s)
Factor 1 de Crecimiento de Fibroblastos/química , Heparina/metabolismo , Oligosacáridos/metabolismo , Conformación Proteica , Secuencia de Aminoácidos , Sitios de Unión , Factor 1 de Crecimiento de Fibroblastos/metabolismo , Factor 2 de Crecimiento de Fibroblastos/química , Heparina/química , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Oligosacáridos/química , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido , SolucionesRESUMEN
A library of long peptides displayed on the pIII protein of filamentous phage was used in biopanning experiments against several protein targets. We find that a large percentage of phage clones that bind specifically to a target contain peptide-encoding genes that do not have an ORF. Instead, the reading frame is either interrupted by one or more nonsuppressed stop codons, or a post-transcriptional frameshift is needed to account for the expression of the minor phage coat protein pIII. The percentage of frameshifted clones varies depending on the target. It can be as high as 90% for clones specific for soluble forms of certain cytokine receptors. Conversely, biopanning against four mAbs did not yield any frameshifted clones. Our studies focused on one clone that binds specifically to rat growth hormone binding protein (GHBP) yet does not have an ORF. A secondary peptide library containing random mutations of this sequence was constructed and panned against GHBP to optimize and correct the reading frame. In the last round (round two) of panning with this library, none of the phage clones that bound to GHBP had an ORF. However, careful analysis of these clones allowed us to design a synthetic peptide capable of binding to GHBP. The results of this study indicate that ORFs are not required to obtain gene expression of the minor coat protein of filamentous phage and suggest that some ORF- clones may have a selective advantage over the clones having ORFs.
Asunto(s)
Bacteriófagos/genética , Mutación del Sistema de Lectura/genética , Biblioteca de Péptidos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Técnicas Biosensibles , Cápside/genética , Proteínas Portadoras/genética , Clonación Molecular , Expresión Génica/genética , Hormona del Crecimiento/metabolismo , Datos de Secuencia Molecular , Oligopéptidos , Sistemas de Lectura Abierta/genética , Péptidos/química , Péptidos/inmunología , Unión Proteica/fisiología , Ratas , Análisis de Secuencia de ADNRESUMEN
Monoclonal antibody PAb1620 recognizes a conformational epitope on the transcription factor p53 and, upon binding, allosterically inhibits p53 binding to DNA. A highly diverse (1.5 x 10(10) members) phage-displayed library of peptides containing 40 random amino acids was used to identify the PAb1620 binding site on p53. Panning this library against PAb1620 resulted in three unique peptides which have statistically significant sequence identities with p53 sufficient to identify the binding site as being composed of amino acids 106-113 and 146-156. Based on these results, we propose a mechanism by which PAb1620 can allosterically inhibit p53 binding to DNA through an indirect interaction between the antibody binding site and the L1 loop (amino acids 112-124) of p53, which is a component of the DNA binding region.
Asunto(s)
Fragmentos de Péptidos/metabolismo , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Regulación Alostérica , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Bacteriófagos/genética , Secuencia de Bases , Sitios de Unión , ADN/metabolismo , Datos de Secuencia MolecularRESUMEN
The present studies were conducted to characterize the specific binding of recombinant human [125I]acidic fibroblast growth factor ([125]aFGF) to the cloned human fibroblast growth factor (FGF) receptor, flg, overexpressed on stably transfected NIH 3T3 mouse fibroblast (NFlg26) cell membranes. In the presence of 5 U/ml of heparin to block [125I]aFGF binding to membrane bound heparan sulfate proteoglycans, specific [125I]aFGF binding was optimal in the presence of 0.2 M NaCl and in a pH range of 7 to 9. [125I]aFGF labeled a single class of recognition sites with high affinity (Kd = 0.27 nM) and limited capacity (apparent maximum binding = 19.5 pmol/mg of protein). A similar estimate of ligand affinity (Kd = 0.25 nM) was determined from association and dissociation rate experiments. aFGF, basic fibroblast growth factor and several glycine-substituted point mutations of aFGF potently inhibited 0.1 nM [125I]aFGF binding. A variety of putative FGF receptor ligands including poly-L-lysines and poly-L-arginines, protamine, suramin and wheat germ agglutinin were shown to have weak or no affinity for the [125I]aFGF recognition site. Additional saturation studies, conducted in the presence of a lower (0.1 U/ml) heparin concentration, indicated that [125I] aFGF labeled both the high affinity (Kd = 0.02 nM) FGF-flg receptor and a separate class of lower affinity (Kd = 2 nM) recognition sites. Pretreatment of NFlg26 cell membranes with pertussis toxin resulted in a heparin-dependent decrease in the binding affinity (Kd values of 0.57-1.15 nM) of [125I]aFGF. Similar pretreatment with cholera toxin did not significantly affect [125I] aFGF binding. Guanine nucleotides were also found to significantly reduce 0.1 nM [125I]aFGF binding in a heparin-dependent fashion. The present data demonstrate that, in the presence of heparin, [125I]aFGF binds with high affinity to the cloned FGF-flg receptor on NFlg26 cell membranes. However, at a low heparin concentration (0.1 U/ml), [125I]aFGF binds to the FGF-flg receptor with higher affinity than was observed in the presence of 5 U/ml of heparin, and also binds a class of lower affinity recognition sites which are consistent with the labeling of cell surface heparan sulfate proteoglycans. The present data also indicate that agents which are known to interfere with receptor/G-protein coupling reduce the binding affinity of [125I]aFGF and suggest that the FGF-flg receptor may be coupled to a G-protein in addition to its intrinsic tyrosine kinase activity.
Asunto(s)
Factor 1 de Crecimiento de Fibroblastos/metabolismo , Guanosina Difosfato/farmacología , Heparina/farmacología , Toxina del Pertussis , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Factores de Virulencia de Bordetella/farmacología , Sitios de Unión/efectos de los fármacos , Unión Competitiva , Cationes/farmacología , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Proteínas Filagrina , HumanosRESUMEN
An experimental method to selectively label side-chain NH2 groups of glutamine and asparagine in proteins with 15N is proposed. This selective labeling method enables to observe only 15NH2 resonances and thus, to discriminate between 15NH and 15NH2 resonances in a 1H-detected heteronuclear correlation spectrum. This method gives results with approximately two times higher sensitivity than those obtained by elaborate pulse sequences such as DEPT-HSQC and will be useful for studying the molecular interaction involving the side chains of Asn and Gln residues.
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Proteínas/química , Factor 1 de Crecimiento de Fibroblastos/química , Humanos , Isótopos de NitrógenoRESUMEN
We previously reported the isolation of two partial cDNA clones encoding human acidic fibroblast growth factor (aFGF). The nucleotide (nt) sequence throughout the coding region and the deduced amino acid sequence were presented [Jaye et al., Science 233 (1986) 541-545]. In this report, the isolation of additional aFGF cDNA clones and their nt sequence are presented. The human aFGF gene is shown to encode at least four functional polyadenylation sites and multiple regulatory sequences within the 3'-untranslated region. The aFGF open reading frame resides approx. 3100 bp upstream from the most frequently utilized 3' processing and polyadenylation site. Several less abundant cDNA clones provide evidence of polyadenylation at three less distal sites, which are colinear with genomic DNA. Northern-blot analysis reveals three detectable mRNA species, whose sizes and intensities correlate with the length and relative abundance of cDNA clones representing them.
Asunto(s)
Factores de Crecimiento de Fibroblastos/genética , Genes , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Tronco Encefálico/metabolismo , Clonación Molecular , ADN/genética , ADN/aislamiento & purificación , Biblioteca de Genes , Humanos , Recién Nacido , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Poli A/análisis , Mapeo Restrictivo , Transcripción GenéticaRESUMEN
Several of the endothelial cell polypeptide mitogens that have been described probably play a role in blood vessel homeostasis. Two overlapping complementary DNA clones encoding human endothelial cell growth factor (ECGF) were isolated from a human brain stem complementary DNA library. Southern blot analysis suggested that there is a single copy of the ECGF gene and that it maps to human chromosome 5 at bands 5q31.3 to 33.2 A 4.8-kilobase messenger RNA was present in human brain stem messenger RNA. The complete amino acid sequence of human ECGF was deduced from the nucleic acid sequence of these clones; it encompasses all the well-characterized acidic endothelial cell polypeptide mitogens described by several laboratories. The ECGF-encoding open reading frame is flanked by translation stop codons and provides no signal peptide or internal hydrophobic domain for the secretion of ECGF. This property is shared by human interleukin-1, which is approximately 30 percent homologous to ECGF.