RESUMEN
Chemokines and chemokine receptors are key modulators in inflammatory diseases and malignancies. Here, we describe the identification and pharmacologic characterization of nanobodies selectively blocking CXCR2, the most promiscuous of all chemokine receptors. Two classes of selective monovalent nanobodies were identified, and detailed epitope mapping showed that these bind to distinct, nonoverlapping epitopes on the CXCR2 receptor. The N-terminal-binding or class 1 monovalent nanobodies possessed potencies in the single-digit nanomolar range but lacked complete efficacy at high agonist concentrations. In contrast, the extracellular loop-binding or class 2 monovalent nanobodies were of lower potency but were more efficacious and competitively inhibited the CXCR2-mediated functional response in both recombinant and neutrophil in vitro assays. In addition to blocking CXCR2 signaling mediated by CXCL1 (growth-related oncogene α) and CXCL8 (interleukin-8), both classes of nanobodies displayed inverse agonist behavior. Bivalent and biparatopic nanobodies were generated, respectively combining nanobodies from the same or different classes via glycine/serine linkers. Interestingly, receptor mutation and competition studies demonstrated that the biparatopic nanobodies were able to avidly bind epitopes within one or across two CXCR2 receptor molecules. Most importantly, the biparatopic nanobodies were superior over their monovalent and bivalent counterparts in terms of potency and efficacy.
Asunto(s)
Receptores de Interleucina-8B/antagonistas & inhibidores , Receptores de Interleucina-8B/metabolismo , Transducción de Señal/fisiología , Anticuerpos de Dominio Único/metabolismo , Anticuerpos de Dominio Único/farmacología , Secuencia de Aminoácidos , Animales , Sitios de Unión/fisiología , Células CHO , Camélidos del Nuevo Mundo , Cricetinae , Cricetulus , Humanos , Datos de Secuencia Molecular , Receptores de Interleucina-8B/genética , Transducción de Señal/efectos de los fármacos , Anticuerpos de Dominio Único/genéticaRESUMEN
BACKGROUND: Sialic acid-binding immunoglobulin-like lectins (Siglecs) are a family of receptors that bind sialic acid and mostly contain immunoreceptor tyrosine-based inhibitory motifs, suggesting that these molecules possess inhibitory functions. We have recently identified Siglec-8 as an eosinophil-prominent Siglec, and cross-linking of Siglec-8 on human eosinophils induces apoptosis. In this article, we address the in vivo consequences of Siglec engagement. We and others have identified mouse Siglec-F as the closest functional paralog of human Siglec-8, based on shared ligand-binding and expression pattern. We therefore hypothesized that Siglec-F engagement would affect levels and viability of eosinophils in vivo. METHODS: Wild type and hypereosinophilic mice were administered Siglec-F antibody and levels of eosinophils in peripheral blood and tissue were measured. Eosinophil apoptosis (in vivo and in vitro) was determined by binding of Annexin-V. RESULTS: Studies in IL-5 transgenic mice, displaying hypereosinophilia, show that administration of a single dose of Siglec-F antibody results in rapid reductions in quantum of eosinophils in the blood. This decrease was accompanied by reductions in tissue eosinophils. Quantum of eosinophils in blood was decreased using two separate antibodies, as well as in other mouse models (wild type mice and in a mouse model of chronic eosinophilic leukemia). Mechanistic studies demonstrated that Siglec-F antibody administration induced apoptosis of eosinophils in vivo and in vitro. CONCLUSION: These data demonstrate that activation of innate immune receptors, like Siglec-F, can significantly reduce mouse eosinophil viability. As such, targeting Siglec-8/F may be a therapeutic approach for eosinophilic disorders.
Asunto(s)
Anticuerpos/farmacología , Antígenos de Diferenciación Mielomonocítica/metabolismo , Apoptosis/inmunología , Eosinofilia/sangre , Animales , Eosinofilia/inmunología , Eosinófilos , Humanos , Ratones , Ratones Endogámicos BALB C , Lectinas Similares a la Inmunoglobulina de Unión a Ácido SiálicoRESUMEN
BACKGROUND: The multimeric DNA-dependent RNA polymerases are widespread throughout nature. The RNA polymerase of Escherichia coli, which is the most well characterized, consists of a holoenzyme with subunit stoichiometry of alpha2betabeta'sigma. The beta subunit is conserved and has been implicated in all stages of transcription. The extreme C-terminus of the beta subunit, which includes two well-conserved sequence segments, contributes to the active centre and has been proposed to act in transcriptional termination. We describe a genetic system for further characterizing the role of the extreme C-terminus of the beta subunit of E. coli RNA polymerase. This involves random, PCR (Polymerase Chain Reaction)-mediated mutagenesis of the 3' region of rpoB encoding the C-terminal 116 amino acids of beta, followed by the isolation and characterization of trans-dominant-negative mutations. RESULTS: Substitutions of conserved residues in this region were obtained that exhibited different degrees of growth inhibition in a host expressing the chromosomal-encoded wild-type form of the beta subunit. A number of different substitutions were isolated within the highly conserved sequence motif GEME (residues 1271-->1274 of the E. coli beta subunit). In addition, substitutions were obtained in the extreme C-terminal (surface-exposed) region of beta and at two residues previously proposed to be in the active site (H1237, K1242). The properties of the purified mutant holoenzymes, assessed by transcription assays in vitro, suggested a promoter blockading action. CONCLUSIONS: We have identified an important, highly conserved motif in the beta subunit, GEME (residues 1271-->1274). The nature and effect of the amino acid substitutions at the Gly residue in GEME emphasize the importance of a small, uncharged residue at this position. The in vitro properties of the most extreme trans dominant-negative mutants altered in the GEME motif (and the mutant characteristics in vivo) were similar to those of certain previously identified active-site mutants, suggesting that the altered RNA polymerases were capable of promoter binding and RNA chain initiation but were deficient in the subsequent transcriptional stage.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/genética , Escherichia coli/enzimología , Mutagénesis Insercional , Secuencia de Aminoácidos , Secuencia Conservada , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Genes Bacterianos , Datos de Secuencia Molecular , Fenotipo , Plásmidos , Reacción en Cadena de la Polimerasa/métodos , Relación Estructura-Actividad , Transcripción GenéticaRESUMEN
beta-Lactam antibiotics exert their antibacterial effects by inactivating the high-molecular-weight penicillin-binding proteins (PBPs) that are responsible for the final stages of peptidoglycan biosynthesis. The availability of the amino acid sequences of several low-molecular-weight PBPs, high-molecular-weight PBPs, and active-site serine beta-lactamases has provided evidence that these groups of enzymes have a common, but distant, evolutionary origin. This view is strongly supported by the recent finding of a similarity in the three-dimensional structures of a low-molecular-weight PBP and class A beta-lactamases. The high-molecular-weight PBPs of Escherichia coli are believed to possess an amino-terminal peptidoglycan transglycosylase domain and a carboxy-terminal penicillin-sensitive transpeptidase domain. These enzymes are inserted in the cytoplasmic membrane only at their amino termini, and water-soluble forms have been obtained that should be suitable for crystallization and X-ray analysis. Resistance to beta-lactam antibiotics mediated by alterations of PBPs has been reported in some gram-negative bacteria. In isolates of Neisseria gonorrhoeae with chromosomally mediated resistance, penicillin-resistant PBPs have arisen from the introduction of multiple amino acid substitutions within the transpeptidase domain of the enzymes.
Asunto(s)
Proteínas Bacterianas , Proteínas Portadoras/fisiología , Bacterias Gramnegativas/metabolismo , Hexosiltransferasas , Muramoilpentapéptido Carboxipeptidasa/fisiología , Peptidil Transferasas , Secuencia de Aminoácidos , Proteínas Portadoras/análisis , Datos de Secuencia Molecular , Muramoilpentapéptido Carboxipeptidasa/análisis , Proteínas de Unión a las Penicilinas , Conformación Proteica , beta-Lactamasas/análisisRESUMEN
We report the effects in vivo of four segments of coliphage T7 DNA upon expression, from an upstream promoter, of galK in plasmids of the pKO family. Three of the segments carry the known major or putative distal terminators of host-dependent T7 early transcription. The fourth carries a novel terminator and maps in the late region of T7. We report the efficiencies of termination in these regions: evidence, based on studies with the E. coli nusA1 mutation, for an involvement of the transcription factor NusA in events at the major early and novel terminators: and the nature of the latter transcription signal.
Asunto(s)
ADN Viral/genética , Galactoquinasa/genética , Genes Reguladores , Genes Virales , Genes , Fagos T/genética , Regiones Terminadoras Genéticas , Transcripción Genética , Secuencia de Bases , Escherichia coli/enzimología , Escherichia coli/genética , Conformación de Ácido Nucleico , Plásmidos , Fagos T/enzimologíaRESUMEN
We have previously presented evidence that rifampicin promotes readthrough of various transcriptional terminators in vivo. This conclusion was based on measurements of galactokinase or beta-galactosidase synthesis in Escherichia coli strains, harbouring plasmids having the terminators fused upstream of galK or lacZ respectively. The terminators tested did not include any known example of a fully rho-dependent signal. We now show that the drug does stimulate galactokinase production when galK is fused downstream of such a terminator: the first rightward terminator of lambda. We also show that the nusA1 host mutation affects neither termination nor the drug response at this site.