RESUMEN
Endothelial heterogeneity has important implications in health and disease. Molecular markers selectively expressed in the vasculature of different organs and tissues are currently being explored in targeted therapies with promising results in preclinical and clinical studies. Noteworthy is the role that combinatorial approaches such as phage display have had in identifying such markers by using phage as nanoparticles and surrogates for billions of different peptides, screening noninvasively the vascular lumen for binding sites. Here, we show that a new peptide motif that emerged from such combinatorial screening of the vasculature binds selectively to blood vessels in the brain in vivo but not to vessels in other organs. Peptides containing a conserved motif in which amino acids Phenylalanine-Arginine-Tryptophan (FRW) predominate could be visualized by transmission electron microscopy bound to the junctions between endothelial cells in all areas of the brain, including the optic nerve, but not in other barrier-containing tissues, such as intestines and testis. Remarkably, peptides containing the motif do not bind to vessels in the retina, implying an important molecular difference between these two vascular barriers. Furthermore, the peptide allows for in vivo imaging, demonstrating that new tools for studying and imaging the brain are likely to emerge from this motif.
Asunto(s)
Secuencias de Aminoácidos , Encéfalo/metabolismo , Circulación Cerebrovascular/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Ligandos , Vasos Retinianos/efectos de los fármacos , Vasos Retinianos/metabolismo , Secuencia de Aminoácidos , Animales , Encéfalo/irrigación sanguínea , Técnicas de Visualización de Superficie Celular , Endotelio Vascular/ultraestructura , Femenino , Técnica del Anticuerpo Fluorescente , Ratones , Péptidos/química , Péptidos/metabolismo , Unión ProteicaRESUMEN
The specific genes and molecules that drive physiological angiogenesis differ from those involved in pathological angiogenesis, suggesting distinct mechanisms for these seemingly related processes. Unveiling genes and pathways preferentially associated with pathologic angiogenesis is key to understanding its mechanisms, thereby facilitating development of novel approaches to managing angiogenesis-dependent diseases. To better understand these different processes, we elucidated the transcriptome of the mouse retina in the well-accepted oxygen-induced retinopathy (OIR) model of pathological angiogenesis. We identified 153 genes changed between normal and OIR retinas, which represent a molecular signature relevant to other angiogenesis-dependent processes such as cancer. These genes robustly predict the survival of breast cancer patients, which was validated in an independent 1,000-patient test cohort (40% difference in 15-year survival; p = 2.56 x 10-21). These results suggest that the OIR model reveals key genes involved in pathological angiogenesis, and these may find important applications in stratifying tumors for treatment intensification or for angiogenesis-targeted therapies.
Asunto(s)
Neoplasias de la Mama/genética , Perfilación de la Expresión Génica/métodos , Neovascularización Patológica/genética , Oxígeno/efectos adversos , Retina/química , Anciano , Animales , Neoplasias de la Mama/mortalidad , Modelos Animales de Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , Persona de Mediana Edad , Neovascularización Patológica/inducido químicamente , Neovascularización Patológica/mortalidad , Retina/efectos de los fármacos , Análisis de Secuencia de ARNRESUMEN
Sepsis, a leading cause of death worldwide, involves exacerbated proinflammatory responses and inefficient bacterial clearance. Phagocytic cells play a crucial part in the prevention of sepsis by clearing bacteria through host innate receptors. Here, we used a phage display library to identify two peptides in Escherichia coli that interact with host innate receptors. One of these peptides, encoded by the wzxE gene of E. coli K-12, was involved in the transbilayer movement of a trisaccharide-lipid intermediate in the assembly of enterobacterial common antigen. Peptide-receptor interactions induced CD16-mediated inhibitory immunoreceptor tyrosine-based activating motif signaling, blocking the production of ROS and bacterial killing. This CD16-mediated inhibitory signaling was abrogated in a WzxE(-/-) mutant of E. coli K-12, restoring the production of ROS and bacterial killing. Taken together, the two novel CD16 ligands identified negatively regulate bacterial killing and inflammation. Our findings may contribute toward the development of new immunotherapies for E. coli-mediated infectious diseases and inflammation.
Asunto(s)
Antígenos Bacterianos/inmunología , Infecciones por Escherichia coli/inmunología , Proteínas de Escherichia coli/inmunología , Proteínas de Transporte de Membrana/inmunología , Fagocitosis , Receptores de IgG/inmunología , Animales , Escherichia coli , Humanos , Ligandos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Biblioteca de Péptidos , Fagocitos/inmunología , Sepsis/prevención & control , Transducción de SeñalRESUMEN
Olfactory sensory neurons are able to detect odorants with high sensitivity and specificity. We have demonstrated that Ric-8B, a guanine nucleotide exchange factor (GEF), interacts with Galphaolf and enhances odorant receptor signaling. Here we show that Ric-8B also interacts with Ggamma13, a divergent member of the Ggamma subunit family which has been implicated in taste signal transduction, and is abundantly expressed in the cilia of olfactory sensory neurons. We show that Gbeta1 is the predominant Gbeta subunit expressed in the olfactory sensory neurons. Ric-8B and Gbeta1, like Galphaolf and Ggamma13, are enriched in the cilia of olfactory sensory neurons. We also show that Ric-8B interacts with Galphaolf in a nucleotide dependent manner, consistent with the role as a GEF. Our results constitute the first example of a GEF protein that interacts with two different olfactory G protein subunits and further implicate Ric-8B as a regulator of odorant signal transduction.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Proteínas Nucleares/metabolismo , Neuronas Receptoras Olfatorias/metabolismo , Animales , Línea Celular , Cilios/química , Cilios/metabolismo , Subunidades alfa de la Proteína de Unión al GTP/análisis , Subunidades beta de la Proteína de Unión al GTP , Factores de Intercambio de Guanina Nucleótido , Proteínas de Unión al GTP Heterotriméricas/análisis , Humanos , Ratones , Ratones Endogámicos C57BL , Proteínas Nucleares/análisis , Vías Olfatorias/química , Vías Olfatorias/metabolismo , Neuronas Receptoras Olfatorias/química , Unión Proteica/fisiología , Células Receptoras Sensoriales/química , Células Receptoras Sensoriales/metabolismo , Transducción de Señal/fisiologíaRESUMEN
The electronegative low-density lipoprotein, LDL (-), is an endogenously modified LDL subfraction with cytotoxic and proinflammatory actions on endothelial cells, monocytes, and macrophages contributing to the progression of atherosclerosis. In this study, epitopes of LDL (-) were mapped using a phage display library of peptides and monoclonal antibodies reactive to this modified lipoprotein. Two different peptide libraries (X6 and CX8C for 6- and 8-amino acid-long peptides, respectively) were used in the mapping. Among all tested peptides, two circular peptides, P1A3 and P2C7, were selected based on their high affinities for the monoclonal antibodies. Small-angle X-ray scattering analysis confirmed their structures as circular rings. P1A3 or P2C7 were quickly internalized by bone marrow-derived murine macrophages as shown by confocal microscopy. P2C7 increased the expression of TNFα, IL-1 ß and iNOS as well as the secretion of TNFα, CCL2, and nitric oxide by murine macrophages, similar to the responses induced by LDL (-), although less intense. In contrast, P1A3 did not show pro-inflammatory effects. We identified a mimetic epitope associated with LDL (-), the P2C7 circular peptide, that activates macrophages. Our data suggest that this conformational epitope represents an important danger-associated molecular pattern of LDL (-) that triggers proinflammatory responses.
Asunto(s)
Epítopos/metabolismo , Inflamación/metabolismo , Lipoproteínas LDL/metabolismo , Epítopos/sangre , Epítopos/aislamiento & purificación , Humanos , Lipoproteínas LDL/sangre , Lipoproteínas LDL/aislamiento & purificación , Macrófagos/metabolismo , Óxido Nítrico/análisisRESUMEN
Receptor tyrosine kinases (RTKs) are key molecules in numerous cellular processes, the inhibitors of which play an important role in the clinic. Among them are the vascular endothelial growth factor (VEGF) family members and their receptors (VEGFR), which are essential in the formation of new blood vessels by angiogenesis. Anti-VEGF therapy has already shown promising results in oncology and ophthalmology, but one of the challenges in the field is the design of specific small-molecule inhibitors for these receptors. We show the identification and characterization of small 6-mer peptides that target the extracellular ligand-binding domain of all three VEGF receptors. These peptides specifically prevent the binding of VEGF family members to all three receptors and downstream signaling but do not affect other angiogenic RTKs and their ligands. One of the selected peptides was also very effective at preventing pathological angiogenesis in a mouse model of retinopathy, normalizing the vasculature to levels similar to those of a normal developing retina. Collectively, our results suggest that these peptides are pan-VEGF inhibitors directed at a common binding pocket shared by all three VEGFRs. These peptides and the druggable binding site they target might be important for the development of novel and selective small-molecule, extracellular ligand-binding inhibitors of RTKs (eTKIs) for angiogenic-dependent diseases.
Asunto(s)
Inhibidores de la Angiogénesis , Células Endoteliales/metabolismo , Biblioteca de Péptidos , Receptores de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular , Inhibidores de la Angiogénesis/química , Inhibidores de la Angiogénesis/genética , Inhibidores de la Angiogénesis/farmacología , Animales , Células Endoteliales/citología , Humanos , Ratones , Dominios Proteicos , Neovascularización Retiniana/tratamiento farmacológico , Neovascularización Retiniana/metabolismo , Neovascularización Retiniana/patología , Factor A de Crecimiento Endotelial Vascular/química , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
Paracoccidioides brasiliensis and Paracoccidioides lutzii are dimorphic fungi and are the etiological agents of paracoccidioidomycosis (PCM). Adhesion is one of the most important steps in infections with Paracoccidioides and is responsible for the differences in the virulence of isolates of these fungi. Because of the importance of adhesion to the establishment of an infection, this study focused on the preliminary development of a new therapeutic strategy to inhibit adhesion by Paracoccidioides, thus inhibiting infection and preventing the disease. We used two phage display libraries to select peptides that strongly bind to the Paracoccidioides cell wall to inhibit adhesion to host cells and extracellular matrix (ECM) components (laminin, fibronectin, and type I and type IV collagen). This approach allowed us to identify four peptides that inhibited up to 64% of the adhesion of Paracoccidioides to pneumocytes in vitro and inhibited the adhesion to the ECM components by up to 57%. Encouraged by these results, we evaluated the ability of these peptides to protect Galleria mellonella from Paracoccidioides infection by treating G. mellonella larvae with the different peptides prior to infection with Paracoccidioides and observing larval survival. The results show that all of the peptides tested increased the survival of the larvae infected with P. brasiliensis by up to 64% and by up to 60% in those infected with P. lutzii. These data may open new horizons for therapeutic strategies to prevent PCM, and anti-adhesion therapy could be an important strategy.
RESUMEN
In mammals, odorants and pheromones are detected by hundreds of odorant receptors (ORs) and vomeronasal receptors (V1Rs and V2Rs) expressed by sensory neurons that are respectively located in the main olfactory epithelium and in the vomeronasal organ. Even though these two olfactory systems are functionally and anatomically separate, their sensory neurons show a common mechanism of receptor gene regulation: each neuron expresses a single receptor gene from a single allele. The mechanisms underlying OR and VR gene expression remain unclear. Here we investigated if OR and V1R genes share common sequences in their promoter regions.We conducted a comparative analysis of promoter regions of 39 mouse V1R genes and found motifs that are common to a large number of promoters. We then searched mouse OR promoter regions for motifs that resemble the ones found in the V1R promoters. We identified motifs that are present in both the V1R and OR promoter regions. Some of these motifs correspond to the known O/E like binding sites while others resemble binding sites for transcriptional repressors. We show that one of these motifs specifically interacts with proteins extracted from both nuclei from olfactory and vomeronasal neurons. Our study is the first to identify motifs that resemble binding sites for repressors in the promoters of OR and V1R genes. Analysis of these motifs and of the proteins that bind to these motifs should reveal important aspects of the mechanisms of OR/V1R gene regulation.
Asunto(s)
Regiones Promotoras Genéticas , Receptores Odorantes/genética , Órgano Vomeronasal/metabolismo , Secuencia de Aminoácidos , Animales , ADN Complementario , Expresión Génica , Ratones , Datos de Secuencia Molecular , Neuronas/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Mouse odorant receptors (ORs) are encoded by >1000 genes dispersed throughout the genome. Each olfactory neuron expresses one single OR gene, while the rest of the genes remain silent. The mechanisms underlying OR gene expression are poorly understood. Here, we investigated if OR genes share common cis-regulatory sequences in their promoter regions. We carried out a comprehensive analysis in which the upstream regions of a large number of OR genes were compared. First, using RLM-RACE, we generated cDNAs containing the complete 5'-untranslated regions (5'-UTRs) for a total number of 198 mouse OR genes. Then, we aligned these cDNA sequences to the mouse genome so that the 5' structure and transcription start sites (TSSs) of the OR genes could be precisely determined. Sequences upstream of the TSSs were retrieved and browsed for common elements. We found DNA sequence motifs that are overrepresented in the promoter regions of the OR genes. Most motifs resemble O/E-like sites and are preferentially localized within 200 bp upstream of the TSSs. Finally, we show that these motifs specifically interact with proteins extracted from nuclei prepared from the olfactory epithelium, but not from brain or liver. Our results show that the OR genes share common promoter elements. The present strategy should provide information on the role played by cis-regulatory sequences in OR gene regulation.