RESUMEN
The Myo/Nog cell lineage was discovered in the chick embryo and is also present in adult mammalian tissues. The cells are named for their expression of mRNA for the skeletal muscle specific transcription factor MyoD and bone morphogenetic protein inhibitor Noggin. A third marker for Myo/Nog cells is the cell surface molecule recognized by the G8 monoclonal antibody (mAb). G8 has been used to detect, track, isolate and kill Myo/Nog cells. In this study, we screened a membrane proteome array for the target of the G8 mAb. The array consisted of >5,000 molecules, each synthesized in their native confirmation with appropriate post-translational modifications in a single clone of HEK-293T cells. G8 mAb binding to the clone expressing brain-specific angiogenesis inhibitor 1 (BAI1) was detected by flow cytometry, re-verified by sequencing and validated by transfection with the plasmid construct for BAI1. Further validation of the G8 target was provided by enzyme-linked immunosorbent assay. The G8 epitope was identified by screening a high-throughput, site directed mutagenesis library designed to cover 95-100% of the 954 amino acids of the extracellular domain of the BAI1 protein. The G8 mAb binds within the third thrombospondin repeat of the extracellular domain of human BAI1. Immunofluorescence localization experiments revealed that G8 and a commercially available BAI1 mAb co-localize to the subpopulation of Myo/Nog cells in the skin, eyes and brain. Expression of the multi-functional BAI1 protein in Myo/Nog cells introduces new possibilities for the roles of Myo/Nog cells in normal and diseased tissues.
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Proteínas Angiogénicas/biosíntesis , Miofibroblastos/metabolismo , Receptores Acoplados a Proteínas G/biosíntesis , Sustitución de Aminoácidos , Proteínas Angiogénicas/química , Proteínas Angiogénicas/genética , Proteínas Angiogénicas/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Reacciones Antígeno-Anticuerpo , Encéfalo/citología , Proteínas Portadoras/análisis , Linaje de la Célula , Epítopos/inmunología , Proteínas del Ojo/biosíntesis , Proteínas del Ojo/química , Proteínas del Ojo/genética , Proteínas del Ojo/inmunología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Desarrollo de Músculos , Proteína MioD/análisis , Especificidad de Órganos , Conformación Proteica , Dominios Proteicos , Conejos , Ratas Sprague-Dawley , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/inmunología , Secuencias Repetitivas de Aminoácido , Piel/citología , Especificidad de la Especie , Tatuaje , Adulto JovenRESUMEN
The two related members of the vasohibin family, VASH1 and VASH2, encode human tubulin detyrosinases. Here we demonstrate that, in contrast to VASH1, which requires binding of small vasohibin binding protein (SVBP), VASH2 has autonomous tubulin detyrosinating activity. Moreover, we demonstrate that SVBP acts as a bona fide activator of both enzymes. Phylogenetic analysis of the vasohibin family revealed that regulatory diversification of VASH-mediated tubulin detyrosination coincided with early vertebrate evolution. Thus, as a model organism for functional analysis, we used Trypanosoma brucei (Tb), an evolutionarily early-branched eukaryote that possesses a single VASH and encodes a terminal tyrosine on both α- and ß-tubulin tails, both subject to removal. Remarkably, although detyrosination levels are high in the flagellum, TbVASH knockout parasites did not present any noticeable flagellar abnormalities. In contrast, we observed reduced proliferation associated with profound morphological and mitotic defects, underscoring the importance of tubulin detyrosination in cell division.
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Proteínas Angiogénicas/metabolismo , Evolución Biológica , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Trypanosoma brucei brucei/metabolismo , Tirosina/metabolismo , Proteínas Angiogénicas/química , Proteínas Angiogénicas/genética , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Cristalografía por Rayos X , Flagelos/metabolismo , Células HEK293 , Humanos , Microtúbulos/metabolismo , Mitosis , Filogenia , Conformación Proteica , Procesamiento Proteico-Postraduccional , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crecimiento & desarrollo , Tirosina/química , Tirosina/genéticaRESUMEN
Angiogenesis plays a pivotal role in tissue regeneration following bone-grafting procedures; however, nonautogenous graft materials typically lack critical angiogenic growth factors. While much research has focused on modifying grafts with angiogenic factors, controlled delivery of these molecules remains a challenge. The current study describes a method for sustained delivery of an angiogenic peptide from hydroxyapatite (HA), a common alloplast material. Specifically, VEGF-derived "QK" peptides were synthesized with polyglutamate domains containing varying numbers of glutamates. The rate of peptide release from HA inversely correlated with glutamate number, with diglutamate-QK (E2-QK) released first, followed by tetraglutamate-QK (E4-QK), and finally, heptaglutamate-QK (E7-QK). By coating HA with a mixture of these peptides, termed, PGM-QK (polyglutamate-modified mixture), sequential peptide release was achieved, enabling gradient QK delivery. To evaluate bioactivity, HA disks were coated with PGM-QK and then placed in fresh media for 6 days. Media containing the released peptides was collected at varying time intervals and placed on human umbilical vein endothelial cells (HUVECs). Cells were evaluated for activation of angiogenic signaling pathways (ERK and Akt) and cell migration. Results showed that QK peptides were continuously released over the 6-day interval, and maintained their capacity to activate HUVECs. These findings point to a new approach for gradient delivery of an angiogenic stimulus.
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Inductores de la Angiogénesis/administración & dosificación , Proteínas Angiogénicas/administración & dosificación , Sustitutos de Huesos/química , Preparaciones de Acción Retardada/química , Durapatita/química , Ácido Poliglutámico/administración & dosificación , Inductores de la Angiogénesis/química , Inductores de la Angiogénesis/farmacología , Proteínas Angiogénicas/química , Proteínas Angiogénicas/farmacología , Liberación de Fármacos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Neovascularización Fisiológica/efectos de los fármacos , Ácido Poliglutámico/química , Ácido Poliglutámico/farmacología , Factor A de Crecimiento Endotelial Vascular/administración & dosificación , Factor A de Crecimiento Endotelial Vascular/química , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
The C-terminus of α-tubulin undergoes a detyrosination/tyrosination cycle and dysregulation of this cycle is associated with cancer and other diseases. The molecular mechanisms of tubulin tyrosination are well studied, however it has remained unknown how tyrosine is cleaved from the tubulin tail. Here, we report the crystal structure of the long-sought detyrosination enzyme, the VASH2/SVBP heterodimer at 2.2 Å resolution and the structure of the tail/VASH2/SVBP complex at 2.5 Å resolution. VASH2 possesses a non-canonical Cys-His-Ser catalytic architecture for tyrosine cleavage. The dynamics of the α1- and α2- helices of VASH2 are related to the insolubility of VASH2. SVBP plays a chaperone-like role by extensively interacting with VASH2 and stabilizing these dynamic helices. A positively charged groove around the catalytic pocket and the α1- and α2- helices of VASH2 targets the tubulin tail for detyrosination. We provide insights into the mechanisms underlying the cycle of tubulin tyrosine cleavage and religation.
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Proteínas Angiogénicas/química , Proteínas Portadoras/química , Tubulina (Proteína)/química , Proteínas Angiogénicas/genética , Animales , Proteínas Portadoras/genética , Cristalografía por Rayos X , Humanos , Conformación Proteica , Conformación Proteica en Hélice alfa , Células Sf9RESUMEN
Cancer remains one of the major causes of death worldwide. Angiogenesis is crucial for the pathogenesis of various human diseases, especially solid tumors. The discovery of anti-angiogenic peptides is a promising therapeutic route for cancer treatment. Thus, reliably identifying anti-angiogenic peptides is extremely important for understanding their biophysical and biochemical properties that serve as the basis for the discovery of new anti-cancer drugs. This study aims to develop an efficient and interpretable computational model called TargetAntiAngio for predicting and characterizing anti-angiogenic peptides. TargetAntiAngio was developed using the random forest classifier in conjunction with various classes of peptide features. It was observed via an independent validation test that TargetAntiAngio can identify anti-angiogenic peptides with an average accuracy of 77.50% on an objective benchmark dataset. Comparisons demonstrated that TargetAntiAngio is superior to other existing methods. In addition, results revealed the following important characteristics of anti-angiogenic peptides: (i) disulfide bond forming Cys residues play an important role for inhibiting blood vessel proliferation; (ii) Cys located at the C-terminal domain can decrease endothelial formatting activity and suppress tumor growth; and (iii) Cyclic disulfide-rich peptides contribute to the inhibition of angiogenesis and cell migration, selectivity and stability. Finally, for the convenience of experimental scientists, the TargetAntiAngio web server was established and made freely available online.
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Inhibidores de la Angiogénesis/química , Inhibidores de la Angiogénesis/farmacología , Proteínas Angiogénicas/química , Proteínas Angiogénicas/farmacología , Biología Computacional/métodos , Diseño de Fármacos , Programas Informáticos , Secuencia de Aminoácidos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Humanos , Aprendizaje Automático , Conformación Molecular , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Reproducibilidad de los Resultados , Relación Estructura-ActividadRESUMEN
Vasohibins are tubulin tyrosine carboxypeptidases that are important in neuron physiology. We examined the crystal structures of human vasohibin 1 and 2 in complex with small vasohibin-binding protein (SVBP) in the absence and presence of different inhibitors and a C-terminal α-tubulin peptide. In combination with functional data, we propose that SVBP acts as an activator of vasohibins. An extended groove and a distinctive surface residue patch of vasohibins define the specific determinants for recognizing and cleaving the C-terminal tyrosine of α-tubulin and for binding microtubules, respectively. The vasohibin-SVBP interaction and the ability of the enzyme complex to associate with microtubules regulate axon specification of neurons. Our results define the structural basis of tubulin detyrosination by vasohibins and show the relevance of this process for neuronal development. Our findings offer a unique platform for developing drugs against human conditions with abnormal tubulin tyrosination levels, such as cancer, heart defects and possibly brain disorders.
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Proteínas Angiogénicas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Tubulina (Proteína)/metabolismo , Proteínas Angiogénicas/química , Animales , Proteínas Portadoras/química , Proteínas de Ciclo Celular/química , Células Cultivadas , Cristalografía por Rayos X , Células HEK293 , Humanos , Ratones , Modelos Moleculares , Conformación Proteica , Mapas de Interacción de Proteínas , Tubulina (Proteína)/químicaRESUMEN
The brain-specific angiogenesis inhibitor (BAI) subfamily of adhesion G protein-coupled receptors (aGPCRs) plays crucial roles in diverse cellular processes including phagocytosis, myoblast fusion, and synaptic development through the ELMO/DOCK/Rac signaling pathway, although the underlying molecular mechanism is not well understood. Here, we demonstrate that an evolutionarily conserved fragment located in the C-terminal cytoplasmic tail of BAI-aGPCRs is specifically recognized by the RBD-ARR-ELMO (RAE) supramodule of the ELMO family scaffolds. The crystal structures of ELMO2-RAE and its complex with BAI1 uncover the molecular basis of BAI/ELMO interactions. Based on the complex structure we identify aGPCR-GPR128 as another upstream receptor for the ELMO family scaffolds, most likely with a recognition mode similar to that of BAI/ELMO interactions. Finally, we map disease-causing mutations of BAI and ELMO and analyze their effects on complex formation.
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Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Angiogénicas/genética , Proteínas del Citoesqueleto/genética , Dominios y Motivos de Interacción de Proteínas/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/aislamiento & purificación , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Angiogénicas/química , Proteínas Angiogénicas/aislamiento & purificación , Proteínas Angiogénicas/metabolismo , Animales , Línea Celular , Cristalografía por Rayos X , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/aislamiento & purificación , Proteínas del Citoesqueleto/metabolismo , Células HEK293 , Humanos , Ratones , Mutagénesis , Mutación , Neoplasias/genética , Receptores Acoplados a Proteínas G/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de SecuenciaRESUMEN
Pro-angiogenic therapy provides a promising new perspective in tackling of many common and severe pathological conditions, such as central and peripheral vascular diseases. Pro-angiogenic therapy also finds interesting applications in the regenerative medicine for the treatment of chronic wounds and in tissue engineering. However, clinical studies on therapeutic angiogenesis, mainly performed by administrating growth factors, have not led to convincing results until now, mainly due to the unfavorable pharmacokinetic and to safety concerns. Thus, the research of new pro-angiogenic molecules endowed of improved pharmacological profile is strongly encouraged. This review focuses on Vascular Endothelial Growth Factor (VEGF) mimetic peptides exerting a pro-angiogenic activity, which are considered among the most promising alternatives to the VEGF based therapy. Peptides show a great potential in drug discovery, as they feature straightforward development approaches, robust and cheap synthetic methodologies for their preparation and functionalization, improved safety and efficacy profiles. Thus, pro-angiogenic peptides represent a valuable alternative to traditional drugs for biomedical applications in cardiovascular diseases and regenerative medicine.
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Proteínas Angiogénicas/uso terapéutico , Materiales Biomiméticos/uso terapéutico , Proteínas Angiogénicas/química , Animales , Materiales Biomiméticos/química , Enfermedad , Humanos , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Zinc modulates the biological function of histidine-rich glycoprotein (HRG) through binding to its His-rich region (HRR). The Zn2+-binding properties of a 35 amino-acid biologically-active peptide mimic of the HRR, HRGP330, were investigated using dissociative mass spectrometry approaches in addition to travelling-wave ion mobility mass spectrometry (TWIM-MS). Native mass spectrometry confirmed zinc binding to HRGP330; however, broadening of the 1H NMR resonances upon addition of Zn2+ ions precluded the attainment of structural information. A complementary approach employing TWIM-MS indicated that HRGP330 has a more compact structure in the presence of Zn2+ ions. Top-down MS/MS data supported a metal-binding-induced conformational change, as fewer fragments were observed for Zn2+-bound HRGP330. Zn2+-bound fragments of both N-terminal and C-terminal ends of the peptide were identified from collision-induced dissociation (CID) and electron transfer dissociation/proton transfer reaction (ETD/PTR) experiments, suggesting that multiple binding sites exist within this region of HRG. The combination of mass spectrometry and NMR approaches provides new insight into the highly dynamic interaction between zinc and this His-rich peptide.
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Proteínas Angiogénicas/metabolismo , Proteínas/metabolismo , Espectrometría de Masa por Ionización de Electrospray/métodos , Oligoelementos/metabolismo , Zinc/metabolismo , Proteínas Angiogénicas/química , Cationes Bivalentes/metabolismo , Espectroscopía de Resonancia Magnética , Unión Proteica , Conformación Proteica , Proteínas/químicaRESUMEN
In this study, we hydrolyzed rice endosperm protein (REP) with pepsin and generated 20 fractions containing multifunctional cationic peptides with varying isoelectric point (pI) values using ampholyte-free isoelectric focusing (autofocusing). Subsequently, we determined antimicrobial activities of each fraction against the pathogens Prophyromonas gingivalis, Propionibacterium acnes, Streptocossus mutans, and Candida albicans. Fractions 18, 19, and 20 had pI values greater than 12 and exhibited antimicrobial activity against P. gingivalis, P. acnes, and C. albicans, but not against S. mutans. In further experiments, we purified and identified cationic peptides from fractions 18, 19, and 20 using reversed-phase high-performance liquid chromatography and matrix-assisted laser/desorption ionization-time-of-flight mass spectroscopy. We also chemically synthesized five identified peptides (RSVSKSR, RRVIEPR, ERFQPMFRRPG, RVRQNIDNPNRADTYNPRAG, and VVRRVIEPRGLL) with pI values greater than 10.5 and evaluated antimicrobial, lipopolysaccharide (LPS)-neutralizing, and angiogenic activities. Among these synthetic peptides, only VVRRVIEPRGLL exhibited antimicrobial activity against P. gingivalis, with an IC50 value of 87µM. However, all five cationic peptides exhibited LPS-neutralizing and angiogenic activities with little or no hemolytic activity against mammalian red blood cells at functional concentrations. These present data show dual or multiple functions of the five identified cationic peptides with little or no hemolytic activity. Therefore, fractions containing cationic peptides from REP hydrolysates have the potential to be used as dietary supplements and functional ingredients in food products.
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Proteínas Angiogénicas/química , Antiinfecciosos/química , Péptidos Catiónicos Antimicrobianos/química , Endospermo/química , Oryza/química , Proteínas de Plantas/química , Proteínas Angiogénicas/farmacología , Animales , Antiinfecciosos/farmacología , Antifúngicos/farmacología , Péptidos Catiónicos Antimicrobianos/síntesis química , Bacterias/efectos de los fármacos , Hongos/efectos de los fármacos , Hemólisis/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Hidrólisis , Concentración 50 Inhibidora , LipopolisacáridosRESUMEN
In the last decade, numerous growth factors and biomaterials have been explored for the treatment of myocardial infarction (MI). While pre-clinical studies have demonstrated promising results, clinical trials have been disappointing and inconsistent, likely due to poor translatability. In the present study, we investigate a potential myocardial regenerative therapy consisting of a protein-engineered dimeric fragment of hepatocyte growth factor (HGFdf) encapsulated in a shear-thinning, self-healing, bioengineered hydrogel (SHIELD). We hypothesized that SHIELD would facilitate targeted, sustained intramyocardial delivery of HGFdf thereby attenuating myocardial injury and post-infarction remodeling. Adult male Wistar rats (n = 45) underwent sham surgery or induction of MI followed by injection of phosphate buffered saline (PBS), 10 µg HGFdf alone, SHIELD alone, or SHIELD encapsulating 10 µg HGFdf. Ventricular function, infarct size, and angiogenic response were assessed 4 weeks post-infarction. Treatment with SHIELD + HGFdf significantly reduced infarct size and increased both ejection fraction and borderzone arteriole density compared to the controls. Thus, sustained delivery of HGFdf via SHIELD limits post-infarction adverse ventricular remodeling by increasing angiogenesis and reducing fibrosis. Encapsulation of HGFdf in SHIELD improves clinical translatability by enabling minimally-invasive delivery and subsequent retention and sustained administration of this novel, potent angiogenic protein analog. Biotechnol. Bioeng. 2017;114: 2379-2389. © 2017 Wiley Periodicals, Inc.
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Preparaciones de Acción Retardada/administración & dosificación , Factor de Crecimiento de Hepatocito/administración & dosificación , Hidrogeles/química , Infarto del Miocardio/tratamiento farmacológico , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/administración & dosificación , Disfunción Ventricular Izquierda/prevención & control , Proteínas Angiogénicas/administración & dosificación , Proteínas Angiogénicas/química , Proteínas Angiogénicas/genética , Animales , Preparaciones de Acción Retardada/química , Difusión , Factor de Crecimiento de Hepatocito/análogos & derivados , Factor de Crecimiento de Hepatocito/genética , Inyecciones , Masculino , Infarto del Miocardio/diagnóstico por imagen , Infarto del Miocardio/patología , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Resistencia al Corte , Resultado del Tratamiento , Disfunción Ventricular Izquierda/diagnóstico por imagen , Disfunción Ventricular Izquierda/patología , ViscosidadRESUMEN
Vasohibins (VASH1 and VASH2) are recently identified regulators of angiogenesis and cancer cell functions. They are secreted proteins without any classical secretion signal sequences, and are thought to be secreted instead via an unconventional protein secretion (UPS) pathway in a small vasohibin-binding protein (SVBP)-dependent manner. However, the precise mechanism of SVBP-dependent UPS is poorly understood. In this study, we identified a novel UPS regulatory system in which essential domain architecture (VASH-PS) of VASHs, comprising regions VASH191-180 and VASH280-169 , regulate the cytosolic punctate structure formation in the absence of SVBP. We also demonstrate that SVBP form a complex with VASH1 through the VASH1274-282 (SIa), VASH1139-144 (SIb), and VASH1133-137 (SIc), leading to the dispersion in the cytosol and extracellular release of VASH1. The amino acid sequences of VASH-SIa and VASH-PS, containing SIb and SIc, are highly conserved among VASH family members in vertebrates, suggesting that SVBP-dependent UPS may be common within the VASH family. This novel UPS regulatory system may open up new avenues for understanding fundamental protein secretion in vertebrates.
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Proteínas Angiogénicas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Angiogénicas/química , Proteínas Angiogénicas/genética , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Citosol/química , Citosol/metabolismo , Células HeLa , Humanos , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Dominios ProteicosRESUMEN
The adhesion G protein-coupled receptors (aGPCRs) are a large yet poorly understood family of seven-transmembrane proteins. A defining characteristic of the aGPCR family is the conserved GAIN domain, which has autoproteolytic activity and can cleave the receptors near the first transmembrane domain. Several aGPCRs, including ADGRB1 (BAI1 or B1) and ADGRG1 (GPR56 or G1), have been found to exhibit significantly increased constitutive activity when truncated to mimic GAIN domain cleavage (ΔNT). Recent reports have suggested that the new N-terminal stalk, which is revealed by GAIN domain cleavage, can directly activate aGPCRs as a tethered agonist. We tested this hypothesis in studies on two distinct aGPCRs, B1 and G1, by engineering mutant receptors lacking the entire NT including the stalk (B1- and G1-SL, with "SL" indicating "stalkless"). These receptors were evaluated in a battery of signaling assays and compared with full-length wild-type and cleavage-mimicking (ΔNT) forms of the two receptors. We found that B1-SL, in multiple assays, exhibited robust signaling activity, suggesting that the membrane-proximal stalk region is not necessary for its activation. For G1, however, the results were mixed, with the SL mutant exhibiting robust activity in several signaling assays (including TGFα shedding, activation of NFAT luciferase, and ß-arrestin recruitment) but reduced activity relative to ΔNT in a distinct assay (activation of SRF luciferase). These data support a model in which the activation of certain pathways downstream of aGPCRs is stalk-dependent, whereas signaling to other pathways is stalk-independent.
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Proteínas Angiogénicas/agonistas , Modelos Moleculares , Receptores Acoplados a Proteínas G/agonistas , Transducción de Señal , Regulación Alostérica , Sustitución de Aminoácidos , Proteínas Angiogénicas/química , Proteínas Angiogénicas/genética , Proteínas Angiogénicas/metabolismo , Arrestinas/química , Arrestinas/genética , Arrestinas/metabolismo , Secuencia Conservada , Genes Reporteros , Células HEK293 , Humanos , Ligandos , Factores de Transcripción NFATC/agonistas , Factores de Transcripción NFATC/química , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Fragmentos de Péptidos/agonistas , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Mutación Puntual , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Proteolisis , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Factor de Crecimiento Transformador alfa/química , Factor de Crecimiento Transformador alfa/genética , Factor de Crecimiento Transformador alfa/metabolismo , Ubiquitinación , beta-ArrestinasRESUMEN
INTRODUCTION: Data about angiogenic factors in diabetic foot syndrome (DFS) are insufficient. Therefore, in the present study we focus on circulating endothelial progenitor cells (EPCs) and two major angiogenic factors: vascular endothelial growth factor (VEGF-A) and fibroblast growth factor (FGF-2) in patients with DFS. MATERIALS AND METHODS: We included 75 subjects: 45 patients with type 2 diabetes and 30 controls. The study group was divided into 2 subgroups: 23 patients with diabetic foot and 22 patients without diabetic complications. The concentration of VEGF-A, soluble VEGF receptor 2 (sVEGF-R2) and FGF-2 were measured in plasma samples. The number of circulating EPCs was determined in peripheral venous blood. The number of endothelial progenitor cells was measured with FACSCalibur flow cytometer using monoclonal antibodies directed against antigens specific for EPCs. RESULTS: In our study we observed significant higher levels of VEGF-A and FGF-2 and lower sVEGF-R2 concentration in patients with T2DM compared to healthy subjects. The conducted analysis showed decreased levels of VEGF-A and elevated levels of FGF-2 in patients with DM complicated DFS compared to diabetic patients without DFS. Increased circulating EPCs number was reported in patients with DFS, and the difference was almost statistically significant. CONCLUSIONS: The high concentration of VEGF-A and FGF-2, and a positive correlation between them indicate their participation in the process of angiogenesis in T2DM. Decreased sVEGF-R2 may result from inactivation of VEGF-A during complexes formation.
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Proteínas Angiogénicas/sangre , Diabetes Mellitus Tipo 2/sangre , Pie Diabético/sangre , Células Progenitoras Endoteliales/patología , Factor 2 de Crecimiento de Fibroblastos/sangre , Factor A de Crecimiento Endotelial Vascular/sangre , Receptor 2 de Factores de Crecimiento Endotelial Vascular/sangre , Anciano , Proteínas Angiogénicas/química , Biomarcadores/sangre , Recuento de Células Sanguíneas , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/patología , Pie Diabético/patología , Regulación hacia Abajo , Femenino , Citometría de Flujo , Humanos , Masculino , Persona de Mediana Edad , Reproducibilidad de los Resultados , Solubilidad , Estadística como Asunto , Regulación hacia Arriba , Receptor 2 de Factores de Crecimiento Endotelial Vascular/químicaRESUMEN
BACKGROUND: CXCL4L1 is a highly potent anti-angiogenic and anti-tumor chemokine, and its structural information is unknown. RESULTS: CXCL4L1 x-ray structure is determined, and it reveals a previously unrecognized chemokine structure adopting a novel C-terminal helix conformation. CONCLUSION: The alternative helix conformation enhances the anti-angiogenic activity of CXCL4L1 by reducing the glycosaminoglycan binding ability. SIGNIFICANCE: Chemokine C-terminal helix orientation is critical in regulating their functions. Chemokines, a subfamily of cytokines, are small, secreted proteins that mediate a variety of biological processes. Various chemokines adopt remarkable conserved tertiary structure comprising an anti-parallel ß-sheet core domain followed by a C-terminal helix that packs onto the ß-sheet. The conserved structural feature has been considered critical for chemokine function, including binding to cell surface receptor. The recently isolated variant, CXCL4L1, is a homologue of CXCL4 chemokine (or platelet factor 4) with potent anti-angiogenic activity and differed only in three amino acid residues of P58L, K66E, and L67H. In this study we show by x-ray structural determination that CXCL4L1 adopts a previously unrecognized structure at its C terminus. The orientation of the C-terminal helix protrudes into the aqueous space to expose the entire helix. The alternative helix orientation modifies the overall chemokine shape and surface properties. The L67H mutation is mainly responsible for the swing-out effect of the helix, whereas mutations of P58L and K66E only act secondarily. This is the first observation that reports an open conformation of the C-terminal helix in a chemokine. This change leads to a decrease of its glycosaminoglycan binding properties and to an enhancement of its anti-angiogenic and anti-tumor effects. This unique structure is recent in evolution and has allowed CXCL4L1 to gain novel functional properties.
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Factor Plaquetario 4/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas Angiogénicas/química , Cristalografía por Rayos X , Cistina/química , Ditiotreitol/química , Heparina/química , Humanos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Factor Plaquetario 4/genética , Factor Plaquetario 4/fisiología , Unión Proteica , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Sustancias Reductoras/químicaRESUMEN
We have identified and characterized a novel proangiogenic glycoprotein (NAP) with molecular weight of 67 kDa from synovial fluid of rheumatoid arthritis patients. Proteomic analysis of the protein revealed 29% sequence coverage with maximum identity for human retinoblastoma binding protein 2. N-terminal amino acid sequence showed no identity to recently discovered protein sequences. NAP was also identified in both normal and tumor cell lines by Western blotting. NAP is a permeability factor as verified by miles permeability assay. The proangiogenic potential of NAP was identified using shell less CAM, rat cornea and tumor on CAM assays. NAP induces expression of VEGF and Flt-1 gene as verified by promoter reporter gene analysis. Further NAP induces proliferation of endothelial cells and formation of tube like structures. NAP is also involved in migration and invasion of tumor cells. Clinical data revealed the presence of NAP in breast cancer biopsies. We have developed monoclonal antibody (mAb), and specific ELISA, which confirmed the presence of NAP in the cytosol of tumor cells. The mAb effect was evaluated with established angiogenic assays. Further, we investigated the detailed mechanism by which NAP induces angiogenesis. NAP is phosphorylated by VEGF induced activation of MAPK and JNK pathways through VEGFR2 phosphorylation. NAP involves JNK pathway predominantly with further activation of NFκB in downstream processing of VEGF activation. Together these findings establish that NAP displays angiogenic properties and promotes efficient neovascularization both in vitro and in vivo models. These observations suggest that anti-NAP-mAb can be targeted for antiangiogenic therapy of cancer.
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Proteínas Angiogénicas/metabolismo , Neovascularización Patológica , Factor A de Crecimiento Endotelial Vascular/metabolismo , Secuencia de Aminoácidos , Proteínas Angiogénicas/química , Animales , Anticuerpos Monoclonales/inmunología , Neoplasias de la Mama/irrigación sanguínea , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular , Citosol/metabolismo , Femenino , Células Endoteliales de la Vena Umbilical Humana , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Datos de Secuencia Molecular , FN-kappa B/metabolismo , Fosforilación , Regiones Promotoras Genéticas , Proteómica , Ratas , Proteína 2 de Unión a Retinoblastoma/química , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Angiogenesis, a formation of neovessels, is regulated by the local balance between angiogenesis stimulators and inhibitors. A number of such endogenous regulators of angiogenesis have been found in the body. Recently, vasohibin-1 (VASH1) was isolated as a negative feedback regulator of angiogenesis produced by endothelial cells (ECs) and subsequently vasohibin-2 (VASH2) as a homologue of VASH1. It was then explored that VASH1 is expressed in ECs to terminate angiogenesis, whereas VASH2 is expressed in cells other than ECs to promote angiogenesis in the mouse model of angiogenesis. This review will focus on the vasohibin family members, which are novel regulators of angiogenesis.
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Proteínas Angiogénicas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Neovascularización Fisiológica , Proteínas Angiogénicas/química , Proteínas Angiogénicas/genética , Proteínas Angiogénicas/aislamiento & purificación , Animales , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas Portadoras/aislamiento & purificación , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/aislamiento & purificación , Endotelio Vascular/metabolismo , Retroalimentación Fisiológica , Regulación de la Expresión Génica , Humanos , Leucocitos Mononucleares/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/aislamiento & purificación , Isoformas de Proteínas/metabolismoRESUMEN
Smaller engineered analogs of angiogenic cytokines may provide translational advantages, including enhanced stability and function, ease of synthesis, lower cost, and, most important, the potential for modulated delivery via engineered biomaterials. In order to create such a peptide, computational molecular modeling and design was employed to engineer a minimized, highly efficient polypeptide analog of the stromal cell-derived factor-1α (SDF) molecule. After removal of the large, central ß-sheet region, a designed diproline linker connected the native N-terminus (responsible for receptor activation and binding) and C-terminus (responsible for extracellular stabilization). This yielded energetic and conformational advantages resulting in a small, low-molecular-weight engineered SDF polypeptide analog (ESA) that was shown to have angiogenic activity comparable to or better than that of recombinant human SDF both in vitro and in a murine model of ischemic heart failure.
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Quimiocina CXCL12/ultraestructura , Ingeniería de Proteínas/métodos , Proteínas Angiogénicas/química , Animales , Quimiocina CXCL12/uso terapéutico , Predicción , Humanos , RatonesRESUMEN
Therapeutic angiogenesis aims at treating ischemic diseases by generating new blood vessels from existing vasculature. It relies on delivery of exogenous factors to stimulate neovasculature formation. Current strategies using genes, proteins and cells have demonstrated efficacy in animal models. However, clinical translation of any of the three approaches has proved to be challenging for various reasons. Administration of angiogenic factors is generally considered safe, according to accumulated trials, and offers off-the-shelf availability. However, many hurdles must be overcome before therapeutic angiogenesis can become a true human therapy. This article will highlight protein-based therapeutic angiogenesis, concisely review recent progress and examine critical challenges. We will discuss growth factors that have been widely utilized in promoting angiogenesis and compare their targets and functions. Lastly, since bolus injection of free proteins usually result in poor outcomes, we will focus on controlled release of proteins.
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Inductores de la Angiogénesis/administración & dosificación , Proteínas Angiogénicas/administración & dosificación , Sistemas de Liberación de Medicamentos , Isquemia/tratamiento farmacológico , Neovascularización Fisiológica/efectos de los fármacos , Inductores de la Angiogénesis/química , Proteínas Angiogénicas/biosíntesis , Proteínas Angiogénicas/química , Proteínas Angiogénicas/genética , Animales , Química Farmacéutica , Preparaciones de Acción Retardada , Portadores de Fármacos , Composición de Medicamentos , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Humanos , Isquemia/genética , Isquemia/metabolismo , Isquemia/fisiopatología , Neovascularización Fisiológica/genética , Trasplante de Células Madre , Tecnología Farmacéutica/métodosRESUMEN
Structure-activity relationship (SAR) studies are essential in the generation of peptides with enhanced activity and efficacy as therapeutic agents. In this study, we report a Structure-activity relationship study for a family of mimetic peptides derived from type IV collagen with potent anti-angiogenic properties. The Structure-activity relationship study was conducted using a number of validated in vitro assays including cell proliferation, adhesion, migration, and tubule formation. We report a critical sequence (NINNV) within this peptide series, which is required for the potent anti-angiogenic activity. Detailed amino acid substitutions resulted in peptides with superior efficacy. Specifically, substitutions with isoleucine at positions 12 and 18 along with the substitution of the methionine at position 10 with the non-natural amino acid D-alanine led to an increase in potency by two orders of magnitude over the parent peptide. Several mimetic peptides in this series exhibit a significant improvement of activity over the parent peptide. This improved in vitro activity is expected to correlate with an increase in in vivo activity leading to effective peptides for anti-angiogenic therapy for different disease applications including cancer and age-related macular degeneration.