RESUMEN
OBJECTIVE: Maintaining a constant core body temperature is essential to homeothermic vertebrate survival. Adaptive thermogenesis in brown adipose tissue and skeletal muscle is the primary mechanism of adjustment to an external stimulus such as cold exposure. Recently, several reports have revealed that the liver can play a role as a metabolic hub during adaptive thermogenesis. In this study, we suggest that the liver plays a novel role in secreting thermogenic factors in adaptive thermogenesis. Bone morphogenetic protein 9 (BMP9) is a hepatokine that regulates many biological processes, including osteogenesis, chondrogenesis, hematopoiesis, and angiogenesis. Previously, BMP9 was suggested to affect preadipocyte proliferation and differentiation. However, the conditions and mechanisms underlying hepatic expression and secretion and adipose tissue browning of BMP9 remain largely unknown. In this study, we investigated the physiological conditions for secretion and the regulatory mechanism of hepatic Bmp9 expression and the molecular mechanism by which BMP9 induces thermogenic gene program activation in adipose tissue. Here, we also present the pharmacological effects of BMP9 on a high-fat-induced obese mouse model. METHODS: To investigate the adaptive thermogenic role of BMP9 in vivo, we challenged mice with cold temperature exposure for 3 weeks and then examined the BMP9 plasma concentration and hepatic expression level. The cellular mechanism of hepatic Bmp9 expression under cold exposure was explored through promoter analysis. To identify the role of BMP9 in the differentiation of brown and beige adipocytes, we treated pluripotent stem cells and inguinal white adipose tissue (iWAT)-derived stromal-vascular (SV) cells with BMP9, and brown adipogenesis was monitored by examining thermogenic gene expression and signaling pathways. Furthermore, to evaluate the effect of BMP9 on diet-induced obesity, changes in body composition and glucose tolerance were analyzed in mice administered recombinant BMP9 (rBMP9) for 8 weeks. RESULTS: Hepatic Bmp9 expression and plasma levels in mice were significantly increased after 3 weeks of cold exposure. Bmp9 mRNA expression in the liver was regulated by transcriptional activation induced by cAMP response-element binding protein (CREB) and CREB-binding protein (CBP) on the Bmp9 promoter. Treatment with BMP9 promoted the differentiation of multipotent stem cells and iWAT-derived SV cells into beige adipocytes, as indicated by the increased expression of brown adipocyte and mitochondrial biogenesis markers. Notably, activation of the mothers against decapentaplegic homolog 1 (Smad1) and p44/p42 mitogen-activated protein kinase (MAPK) pathways was required for the induction of uncoupling protein 1 (UCP1) and peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC1α) expression in BMP9-induced differentiation of SVs into beige adipocytes. The administration of rBMP9 in vivo also induced browning markers in white adipose tissue. In high-fat diet-induced obese mice, rBMP9 administration conferred protection against obesity and enhanced glucose tolerance. CONCLUSIONS: BMP9 is a hepatokine regulated by cold-activated CREB and CBP and enhances glucose and fat metabolism by promoting the activation of the thermogenic gene program in adipocytes. These data implicate BMP9 as a potential pharmacological tool for protecting against obesity and type 2 diabetes.
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Diabetes Mellitus Tipo 2 , Factor 2 de Diferenciación de Crecimiento/metabolismo , Adipocitos Marrones/metabolismo , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Blanco/metabolismo , Animales , Frío , Diabetes Mellitus Tipo 2/metabolismo , Glucosa/metabolismo , Factor 2 de Diferenciación de Crecimiento/farmacología , Ratones , Ratones Endogámicos C57BL , Obesidad/metabolismo , Termogénesis/genética , Proteína Desacopladora 1/metabolismoRESUMEN
OBJECTIVE: To compare the spinal bone fusion properties of activin A/BMP2 chimera (AB204) with recombinant human bone morphogenetic protein (rhBMP2) using a rat posterolateral spinal fusion model. METHODS: The study was designed to compare the effects and property at different dosages of AB204 and rhBMP2 on spinal bone fusion. Sixty-one male Sprague-Dawley rats underwent posterolateral lumbar spinal fusion using one of nine treatments during the study, that is, sham; osteon only; 3.0 µg, 6.0 µg, or 10.0 µg of rhBMP2 with osteon; and 1.0 µg, 3.0 µg, 6.0 µg, or 10.0 µg of AB204 with osteon. The effects and property on spinal bone fusion was calculated at 4 and 8 weeks after treatment using the scores of physical palpation, simple radiograph, micro-computed tomography, and immunohistochemistry. RESULTS: Bone fusion scores were significantly higher for 10.0 µg AB204 and 10.0 µg rhBMP2 than for osteon only or 1.0 µg AB204. AB204 exhibited more prolonged osteoblastic activity than rhBMP2. Bone fusion properties of AB204 were similar with the properties of rhBMP2 at doses of 6.0 and 10.0 µg, but, the properties of AB204 at doses of 3.0 µg exhibited better than the properties of rhBMP2 at doses of 3.0 µg. CONCLUSION: AB204 chimeras could to be more potent for treating spinal bone fusion than rhBMP2 substitutes with increased osteoblastic activity for over a longer period.
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This study was performed for the purpose to evaluate the effect of activin A/BMP-2 chimera (AB204) on cardiovascular system and toxicological effect in beagle dogs. When administered AB204 at the dose of 0.32 mg/kg via intravenous injection in beagle dogs, there were no changes in systolic, diastolic and mean blood pressure as well as in pulse rate, in addition that there were no differences in ORS complex, PR interval, R-R interval, QT interval and QTcV interval on the electrocardiography. Also, when administered AB204 at the doses of 0.25 and 0.5 mg/kg/day via repetitive intravenous injection for 2 weeks, it did not cause any significant changes in general symptoms, weight, food intake, ophthalmologic abnormality, urine, hematology, serum biochemistry, organ weight and autopsy values. Therefore, AB204 did not affect cardiovascular functions including blood pressure, pulse rate and ECG, when administered at the dose of ≤0.32 mg/kg via single intravenous injection in male beagle dogs. When it was administered at the dose of 0.5 mg/kg repetitive intravenous injection for 2 weeks, it did not show any toxicity.
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Sistema Cardiovascular/efectos de los fármacos , Proteínas Recombinantes de Fusión/toxicidad , Animales , Biomarcadores/sangre , Presión Sanguínea/efectos de los fármacos , Sistema Cardiovascular/patología , Sistema Cardiovascular/fisiopatología , Perros , Esquema de Medicación , Frecuencia Cardíaca/efectos de los fármacos , Inyecciones Intravenosas , Masculino , Proteínas Recombinantes de Fusión/administración & dosificación , Medición de Riesgo , Factores de Tiempo , Pruebas de ToxicidadRESUMEN
Bacteria sense and respond to osmolarity through the EnvZ-OmpR two-component system. The structure of the periplasmic sensor domain of EnvZ (EnvZ-PD) is not available yet. Here, we present the crystal structure of EnvZ-PD in the presence of CHAPS detergent. The structure of EnvZ-PD shows similar folding topology to the PDC domains of PhoQ, DcuS, and CitA, but distinct orientations of helices and ß-hairpin structures. The CD and NMR spectra of EnvZ-PD in the presence of cholate, a major component of bile salts, are similar to those with CHAPS. Chemical cross-linking shows that the dimerization of EnvZ-PD is significantly inhibited by the CHAPS and cholate. Together with ß-galactosidase assay, these results suggest that bile salts may affect the EnvZ structure and function in Escherichia coli.
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Proteínas de la Membrana Bacteriana Externa/química , Colatos/farmacología , Ácidos Cólicos/farmacología , Detergentes/farmacología , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Complejos Multienzimáticos/química , Proteínas de la Membrana Bacteriana Externa/efectos de los fármacos , Dicroismo Circular , Cristalografía por Rayos X , Proteínas de Escherichia coli/efectos de los fármacos , Modelos Moleculares , Complejos Multienzimáticos/efectos de los fármacos , Dominios Proteicos/efectos de los fármacos , Pliegue de Proteína/efectos de los fármacos , Estructura Secundaria de Proteína/efectos de los fármacosRESUMEN
Several of the bone morphogenetic proteins (BMPs) have been reported to induce white as well as brown adipogenesis. Here, we characterized the adipogenic potential of AB204, a recombinant chimeric protein of activin-ßA and BMP-2, in in vitro, ex vivo and in vivo settings. BMP-2 is generally known to promote adipogenesis. When compared with BMP-2, which previously showed varying degrees of adipogenesis, AB204 displayed superior in vitro adipogenic differentiation of mouse 3 T3-L1 pre-adipocytes and human adipose-derived stem cells (hASCs). Surprisingly, implantation of hASCs, preconditioned with AB204 for as short a time as 48 h, into the subcutaneous space of athymic nude mice effectively produced fat pads, but not with BMP-2. When BMP-2 and AB204 were injected intraperitoneally, AB204 promoted dramatic systemic adipogenesis of C57BL/6 mice on a high-fat diet very effectively. The results implicate the novel clinical potential of AB204, including induction of fat tissue ex vivo or in vivo for tissue re-engineering and regenerative medicinal purposes, more than any known natural protein ligand. Copyright © 2015 John Wiley & Sons, Ltd.
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Adipogénesis/efectos de los fármacos , Tejido Adiposo/metabolismo , Proteína Morfogenética Ósea 2/farmacología , Subunidades beta de Inhibinas/farmacología , Proteínas Recombinantes de Fusión/farmacología , Células Madre/metabolismo , Células 3T3-L1 , Tejido Adiposo/citología , Animales , Grasas de la Dieta/farmacología , Humanos , Ratones , Células Madre/citologíaRESUMEN
The biological role of BMP-9 signaling in liver cancer remains dubious. To explore the potential use of BMP-9 signaling for anti-cancer therapy, we used recombinant human BMP-9, which we referred to as MB109, to study the effect on growth of fifteen hepatocellular carcinoma (HCC) cell lines. MB109 effectively inhibits the proliferation of nine HCC cells in vitro. The anti-proliferative effect was found to be induced by turning on p21 signaling, which caused survivin suppression and G0/G1 cell cycle arrest. ID3 was identified to be the mediator of the MB109-induced p21 expression. Blocking the activity of p38 MAPK diminished ID3 and p21 expression, indicating that MB109 signals through a p38 MAPK/ID3/p21 pathway to arrest cell cycle progression. Moreover, prolonged MB109 treatment suppressed the expression of five prominent liver cancer stem cell (LCSC) markers, including CD44, CD90, AFP, GPC3 and ANPEP. Xenograft model confirmed the anti-tumor and LCSC-suppression capability of MB109 in vivo. Contrary to ongoing efforts of suppressing BMP-9 signaling to inhibit angiogenesis of cancer tissue, these results demonstrate an unexpected therapeutic potential of MB109 to stimulate BMP-9 signaling for anti-cancer therapies.
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Carcinoma Hepatocelular/metabolismo , Factor 2 de Diferenciación de Crecimiento/metabolismo , Neoplasias Hepáticas/metabolismo , Células Madre Neoplásicas/metabolismo , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Modelos Animales de Enfermedad , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Factor 2 de Diferenciación de Crecimiento/genética , Factor 2 de Diferenciación de Crecimiento/farmacología , Humanos , Proteínas Inhibidoras de la Diferenciación/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Ratones , Modelos Biológicos , Proteínas de Neoplasias/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Fosforilación , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
S-Nitrosylation is well established as an important post-translational regulator in protein function and signaling. However, relatively little is known about its structural and dynamical consequences. We have investigated the effects of S-nitrosylation on the rhodanese domain of the Escherichia coli integral membrane protein YgaP by NMR, X-ray crystallography, and mass spectrometry. The results show that the active cysteine in the rhodanese domain of YgaP is subjected to two competing modifications: S-nitrosylation and S-sulfhydration, which are naturally occurring in vivo. It has been observed that in addition to inhibition of the sulfur transfer activity, S-nitrosylation of the active site residue Cys63 causes an increase in slow motion and a displacement of helix 5 due to a weakening of the interaction between the active site and the helix dipole. These findings provide an example of how nitrosative stress can exert action at the atomic level.
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Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Procesamiento Proteico-Postraduccional , Tiosulfato Azufretransferasa/química , Tiosulfato Azufretransferasa/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Cisteína/metabolismo , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Modelos Moleculares , Conformación ProteicaRESUMEN
Although BMP-9 has been reported to induce browning of white adipose tissues (WATs) and suppress high fat diet-induced obesity, detailed molecular mechanism needs to be further elucidated. We report here that administration of MB109, a recombinant derivative of human BMP-9, into obese mice enhanced gene expression of fibroblast growth factor 21 (FGF21), a metabolic regulator, and alleviates a spectrum of pathological symptoms due to high fat diet-induced obesity. In addition, periodical injection of MB109 (500µg/kg/week) reduced an amount of lipid droplets in the liver, serum levels of alanine aminotransferase (ALT), and total cholesterol. These results indicate that MB109 is also effective to treat obesity-mediated non-alcoholic fatty liver disease (NAFLD).
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Fármacos Antiobesidad/uso terapéutico , Factores de Crecimiento de Fibroblastos/genética , Factores de Diferenciación de Crecimiento/uso terapéutico , Obesidad/tratamiento farmacológico , Regulación hacia Arriba/efectos de los fármacos , Alanina Transaminasa/sangre , Animales , Fármacos Antiobesidad/administración & dosificación , Colesterol/sangre , Dieta Alta en Grasa/efectos adversos , Factor 2 de Diferenciación de Crecimiento , Factores de Diferenciación de Crecimiento/administración & dosificación , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Obesos , Obesidad/sangre , Obesidad/etiología , Obesidad/genética , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/uso terapéutico , Proteína Desacopladora 1/genéticaRESUMEN
Multiple neurodegenerative diseases are caused by the aggregation of the human α-Synuclein (α-Syn) protein. α-Syn possesses high structural plasticity and the capability of interacting with membranes. Both features are not only essential for its physiological function but also play a role in the aggregation process. Recently it has been proposed that α-Syn is able to form lipid-protein particles reminiscent of high-density lipoproteins. Here, we present a method to obtain a stable and homogeneous population of nanometer-sized particles composed of α-Syn and anionic phospholipids. These particles are called α-Syn lipoprotein (nano)particles to indicate their relationship to high-density lipoproteins formed by human apolipoproteins in vivo and of in vitro self-assembling phospholipid bilayer nanodiscs. Structural investigations of the α-Syn lipoprotein particles by circular dichroism (CD) and magic angle solid-state nuclear magnetic resonance (MAS SS-NMR) spectroscopy establish that α-Syn adopts a helical secondary structure within these particles. Based on cryo-electron microscopy (cryo-EM) and dynamic light scattering (DLS) α-Syn lipoprotein particles have a defined size with a diameter of â¼23 nm. Chemical cross-linking in combination with solution-state NMR and multiangle static light scattering (MALS) of α-Syn particles reveal a high-order protein-lipid entity composed of â¼8-10 α-Syn molecules. The close resemblance in size between cross-linked in vitro-derived α-Syn lipoprotein particles and a cross-linked species of endogenous α-Syn from SH-SY5Y human neuroblastoma cells indicates a potential functional relevance of α-Syn lipoprotein nanoparticles.
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Lipoproteínas HDL/química , Nanopartículas/química , Fosfolípidos/química , alfa-Sinucleína/química , Línea Celular Tumoral , Humanos , Resonancia Magnética Nuclear BiomolecularRESUMEN
The purpose of this study was to determine the effects of a single intravenous injection of a novel osteoinductive material, activin A/BMP-2 (AB204), to rodents on toxicity and their respiratory functions and central nervous system (CNS). A single intravenous injection of AB204 was given to Sprague-Dawley (SD) rats in doses of 0, 0.625, 2.5 and 10 mg/kg to observe the mortality rate, the general symptoms for 14 days. The experimental groups were also given 0.2, 0.4 and 0.8 mg/kg of AB204, respectively, and the respiration rate, the tidal volume and the minute volume were measured for 240 min. The experimental groups of imprinting control region (ICR) mice were given a single intravenous injection of 0.2, 0.4 and 0.8 mg/kg of AB204, respectively. Their body temperature was taken and general behaviors were observed to evaluate the effect of AB204 on the CNS for 240 min. The study on toxicity of a single intravenous injection found no death or abnormal symptoms, abnormal findings from autopsy, or abnormal body weight gain or loss in all the experimental groups. No abnormal variation associated with the test substance was observed in the respiration rate, the tidal volume, the minute volume, body temperature or the general behaviors. On the basis of these results, the approximate lethal dose of AB204 for a single intravenous injection exceeds 10 mg/kg for SD rats and a single intravenous injection of ≤0.8 mg/kg AB204 has no effect on their respiratory system for SD rat and no effect on their CNS for ICR mice.
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Sistema Nervioso Central/efectos de los fármacos , Proteínas Recombinantes de Fusión/toxicidad , Frecuencia Respiratoria/efectos de los fármacos , Volumen de Ventilación Pulmonar/efectos de los fármacos , Animales , Conducta Animal/efectos de los fármacos , Temperatura Corporal/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inyecciones Intravenosas , Ratones Endogámicos ICR , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/administración & dosificación , Pruebas de Toxicidad AgudaRESUMEN
Optical modulation of proteins provides superior spatiotemporal resolution for understanding biological processes, and photoswitches built on light-sensitive proteins have been significantly advancing neuronal and cellular studies. Small molecule photoswitches could complement protein-based switches by mitigating potential interference and affording high specificity for modulation sites. However, genetic encodability and responsiveness to nonultraviolet light, two desired properties possessed by protein photoswitches, are challenging to be engineered into small molecule photoswitches. Here we developed a small molecule photoswitch that can be genetically installed onto proteins in situ and controlled by visible light. A pentafluoro azobenzene-based photoswitchable click amino acid (F-PSCaa) was designed to isomerize in response to visible light. After genetic incorporation into proteins via the expansion of the genetic code, F-PSCaa reacts with a nearby cysteine within the protein generating an azo bridge in situ. The resultant bridge is switchable by visible light and allows conformation and binding of CaM to be regulated by such light. This photoswitch should prove valuable in optobiology for its minimal interference, site flexibility, genetic encodability, and response to the more biocompatible visible light.
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Compuestos Azo/química , Luz , Optogenética/métodos , Proteínas/química , Proteínas/genética , Aminoácidos/química , Modelos Moleculares , Conformación Proteica , EstereoisomerismoRESUMEN
The subchronic (28-days) toxicity of an Activin A/BMP-2 chimera (AB204) was assessed in rats. Sprague-Dawley rats received repetitive intravenous injection of AB204 in doses of 0, 0.25 and 0.5 mg/kg for two weeks and in doses of 0, 0.08, 0.16 and 0.32 mg/kg/day for four weeks. No animal was dead and no change caused by the AB204 was observed in general symptoms, weight variation, and food and water intake as well as blood test and autopsy findings. In conclusion, the no observed adverse effects level (NOAEL) of the AB204 on rats was determined to be 0.32 mg/kg/day.
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Activinas/administración & dosificación , Activinas/efectos adversos , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/efectos adversos , Animales , Peso Corporal/efectos de los fármacos , Ingestión de Líquidos/efectos de los fármacos , Ingestión de Alimentos/efectos de los fármacos , Femenino , Inyecciones Intravenosas/métodos , Masculino , Nivel sin Efectos Adversos Observados , Tamaño de los Órganos/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
Although stem cells from mice deficient of FGF2 have been reported to display enhanced capacity for adipogenesis, the literature using in vitro cell culture system has so far reported conflicting results on the role of FGF2 in adipogenesis. We here demonstrate that FGF2, depending on concentration, can function as either a positive or negative factor of in vitro adipogenesis by regulating activation of the ERK signaling pathway. FGF2 at concentrations lower than 2 ng/ml enhanced in vitro adipogenesis of human adipose-derived stem cells (hASCs). However, FGF2 at concentrations higher than 10 ng/ml was able to suppress adipogenesis by maintaining sustained phosphorylation of ERK and function as a dominant negative adipogenic factor toward BMP ligands. Expression levels of FGF2 in the fat tissues from high fat diet induced obese C57BL/6 mice were lower than those from normal chow diet mice, indicating that expression levels of FGF2 in the fat tissues might be in reverse correlation with the size of fat tissues. Our observation of concentration dependent biphasic effect as well as dominant negative effect of FGF2 on adipogenesis provides a mechanistic basis to understand roles of FGF2 in adipogenesis and development of fat tissues.
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Adipogénesis , Tejido Adiposo/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Obesidad/metabolismo , Adipocitos/citología , Adipocitos/metabolismo , Animales , Diferenciación Celular , Células Cultivadas , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Factor 2 de Crecimiento de Fibroblastos/genética , Humanos , Sistema de Señalización de MAP Quinasas , Masculino , Células Madre Mesenquimatosas/citología , Ratones , Ratones Endogámicos C57BL , Obesidad/etiologíaRESUMEN
TGF-ß superfamily ligands govern normal tissue development and homeostasis, and their dysfunction is a hallmark of many diseases. These ligands are also well defined both structurally and functionally. This review focuses on TGF-ß superfamily ligand engineering for therapeutic purposes, in particular for regenerative medicine and musculoskeletal disorders. We describe the key discovery that structure-guided mutation of receptor-binding epitopes, especially swapping of these epitopes between ligands, results in new ligands with unique functional properties that can be harnessed clinically. Given the promising results with prototypical engineered TGF-ß superfamily ligands, and the vast number of such molecules that remain to be produced and tested, this strategy is likely to hold great promise for the development of new biologics.
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Factor de Crecimiento Transformador beta/química , Factor de Crecimiento Transformador beta/metabolismo , Animales , Cristalografía por Rayos X , Humanos , Ligandos , Modelos Moleculares , Ingeniería de Proteínas/métodos , Estructura Secundaria de Proteína , Relación Estructura-ActividadRESUMEN
Activins and bone morphogenetic proteins (BMPs) share activin type 2 signaling receptors but utilize different type 1 receptors and Smads. We designed AB215, a potent BMP2-like Activin A/BMP2 chimera incorporating the high-affinity type 2 receptor-binding epitope of Activin A. In this study, we compare the signaling properties of AB215 and BMP2 in HEK293T cells and gonadotroph LßT2 cells in which Activin A and BMP2 synergistically induce FSHß. In HEK293T cells, AB215 is more potent than BMP2 and competitively blocks Activin A signaling, while BMP2 has a partial blocking activity. Activin A signaling is insensitive to BMP pathway antagonism in HEK293T cells but is strongly inhibited by constitutively active (CA) BMP type 1 receptors. By contrast, the potencies of AB215 and BMP2 are indistinguishable in LßT2 cells and although AB215 blocks Activin A signaling, BMP2 has no inhibitory effect. Unlike HEK293T, Activin A signaling is strongly inhibited by BMP pathway antagonism in LßT2 cells but is largely unaffected by CA BMP type 1 receptors. BMP2 increases phospho-Smad3 levels in LßT2 cells, in both the absence and the presence of Activin A treatment, and augments Activin A-induced FSHß. AB215 has the opposite effect and sharply decreases basal phospho-Smad3 levels and blocks Smad2 phosphorylation and FSHß induction resulting from Activin A treatment. These findings together demonstrate that while AB215 activates the BMP pathway, it has opposing effects to those of BMP2 on FSHß induction in LßT2 cells apparently due to its ability to block Activin A signaling.
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Activinas/farmacología , Proteína Morfogenética Ósea 2/farmacología , Hormona Folículo Estimulante de Subunidad beta/genética , Gonadotrofos/efectos de los fármacos , Proteínas Recombinantes de Fusión/farmacología , Activinas/genética , Activinas/metabolismo , Animales , Western Blotting , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 2/metabolismo , Línea Celular , Gonadotrofos/citología , Gonadotrofos/metabolismo , Células HEK293 , Humanos , Ratones , Fosforilación/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Activación Transcripcional/efectos de los fármacosRESUMEN
BACKGROUND: One in eight women will be affected by breast cancer in her lifetime. Approximately 75% of breast cancers express estrogen receptor alpha (ERα) and/or progesterone receptor and these receptors are markers for tumor dependence on estrogen. Anti-estrogenic drugs such as tamoxifen are commonly used to block estrogen-mediated signaling in breast cancer. However, many patients either do not respond to these therapies (de novo resistance) or develop resistance to them following prolonged treatment (acquired resistance). Therefore, it is imperative to continue efforts aimed at developing new efficient and safe methods of targeting ER activity in breast cancer. METHODS: AB215 is a chimeric ligand assembled from sections of Activin A and BMP2. BMP2's and AB215's inhibition of breast cancer cells growth was investigated. In vitro luciferase and MTT proliferation assays together with western blot, RT_PCR, and mRNA knockdown methods were used to determine the mechanism of inhibition of estrogen positive breast cancer cells growth by BMP2 and AB215. Additionally in vivo xenograft tumor model was used to investigate anticancer properties of AB215. RESULTS: Here we report that AB215, a chimeric ligand assembled from sections of Activin A and BMP2 with BMP2-like signaling, possesses stronger anti-proliferative effects on ERα positive breast cancer cells than BMP2. We further show that AB215 inhibits estrogen signaling by inducing expression of inhibitor of DNA binding proteins (IDs). Specifically, we demonstrate that knockdown of ID proteins attenuates the anti-estrogen effects of AB215. Remarkably, we find that AB215 is more effective than tamoxifen in suppressing tumor growth in a xenograft model. CONCLUSION: This study shows that IDs have profound role to inhibit estrogen signaling in ERα positive breast cancer cells, and that engineered TGF-beta ligands may have high therapeutic value.
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Activinas/farmacología , Proteína Morfogenética Ósea 2/farmacología , Neoplasias de la Mama/patología , Proteínas Inhibidoras de la Diferenciación/metabolismo , Proteínas Recombinantes de Fusión/farmacocinética , Transducción de Señal/efectos de los fármacos , Activinas/uso terapéutico , Proteína Morfogenética Ósea 2/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Receptor alfa de Estrógeno/metabolismo , Femenino , Humanos , Proteínas Inhibidoras de la Diferenciación/genética , Células MCF-7 , Trasplante de Neoplasias , Proteínas Recombinantes de Fusión/uso terapéutico , Tamoxifeno/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The solution NMR structure of the α-helical integral membrane protein YgaP from Escherichia coli in mixed 1,2-diheptanoyl-sn-glycerol-3-phosphocholine/1-myristoyl-2-hydroxy-sn-glycero-3-phospho-(1'-rac-glycerol) micelles is presented. In these micelles, YgaP forms a homodimer with the two transmembrane helices being the dimer interface, whereas the N-terminal cytoplasmic domain includes a rhodanese-fold in accordance to its sequence homology to the rhodanese family of sulfurtransferases. The enzymatic sulfur transfer activity of full-length YgaP as well as of the N-terminal rhodanese domain only was investigated performing a series of titrations with sodium thiosulfate and potassium cyanide monitored by NMR and EPR. The data indicate the thiosulfate concentration-dependent addition of several sulfur atoms to the catalytic Cys-63, which process can be reversed by the addition of potassium cyanide. The catalytic reaction induces thereby conformational changes within the rhodanese domain, as well as on the transmembrane α-helices of YgaP. These results provide insights into a potential mechanism of YgaP during the catalytic thiosulfate activity in vivo.
Asunto(s)
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/fisiología , Escherichia coli/química , Proteínas de la Membrana/química , Proteínas de la Membrana/fisiología , Resonancia Magnética Nuclear Biomolecular/métodos , Modelos Moleculares , Conformación Proteica , Relación Estructura-Actividad , Tiosulfato Azufretransferasa/químicaRESUMEN
Recombinant bone morphogenetic protein 2 (rhBMP2) has been used clinically to treat bone fractures in human patients. However, the high doses of rhBMP2 required for a therapeutic response can cause undesirable side effects. Here, we demonstrate that a novel Activin A/BMP2 (AB2) chimera, AB204, promotes osteogenesis and bone healing much more potently and effectively than rhBMP2. Remarkably, 1 month of AB204 treatment completely heals tibial and calvarial defects of critical size in mice at a concentration 10-fold lower than a dose of rhBMP2 that only partially heals the defect. We determine the structure of AB204 to 2.3 Å that reveals a distinct BMP2-like fold in which the Activin A sequence segments confer insensitivity to the BMP2 antagonist Noggin and an affinity for the Activin/BMP type II receptor ActRII that is 100-fold greater than that of BMP2. The structure also led to our identification of a single Activin A-derived amino acid residue, which, when mutated to the corresponding BMP2 residue, resulted in a significant increase in the affinity of AB204 for its type I receptor BMPRIa and a further enhancement in AB204's osteogenic potency. Together, these findings demonstrate that rationally designed AB2 chimeras can provide BMP2 substitutes with enhanced potency for treating non-union bone fractures.
Asunto(s)
Activinas/farmacología , Proteína Morfogenética Ósea 2/farmacología , Proteínas Recombinantes de Fusión/farmacología , Factor de Crecimiento Transformador beta/farmacología , Cicatrización de Heridas/efectos de los fármacos , Receptores de Activinas Tipo I/metabolismo , Activinas/química , Fosfatasa Alcalina/metabolismo , Animales , Proteína Morfogenética Ósea 2/química , Calcificación Fisiológica/efectos de los fármacos , Línea Celular , Humanos , Ratones , Osteogénesis/efectos de los fármacos , Mutación Puntual/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Cráneo/efectos de los fármacos , Cráneo/patología , Tibia/efectos de los fármacos , Tibia/patología , Factor de Crecimiento Transformador beta/químicaRESUMEN
About 8000 genes encode membrane proteins in the human genome. The information about their druggability will be very useful to facilitate drug discovery and development. The main problem, however, consists of limited structural and functional information about these proteins because they are difficult to produce biochemically and to study. In this paper we describe the strategy that combines Cell-free protein expression, NMR spectroscopy, and molecular DYnamics simulation (CNDY) techniques. Results of a pilot CNDY experiment provide us with a guiding light towards expedited identification of the hit compounds against a new uncharacterized membrane protein as a potentially druggable target. These hits can then be further characterized and optimized to develop the initial lead compound quicker. We illustrate such "omics" approach for drug discovery with the CNDY strategy applied to two example proteins: hypoxia-induced genes HIGD1A and HIGD1B.
Asunto(s)
Diseño de Fármacos , Proteínas de la Membrana/química , Proteínas de Neoplasias/química , Resonancia Magnética Nuclear Biomolecular/métodos , Sitios de Unión , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ligandos , Proteínas de la Membrana/metabolismo , Proteínas Mitocondriales , Simulación del Acoplamiento Molecular , Proteínas de Neoplasias/metabolismo , Conformación ProteicaRESUMEN
BACKGROUND: The development of chemical refolding of transforming growth factor-beta (TGF-ß) superfamily ligands has been instrumental to produce the recombinant proteins for biochemical studies and exploring the potential of protein therapeutics. The osteogenic human bone morphogenetic protein-2 (hBMP-2) and its Drosophila DPP homolog were the early successful cases of refolding into functional form. Despite the similarity in their three dimensional structure and amino acid sequences, several other TGF-ß superfamily ligands could not be refolded readily by the same methods. RESULTS: Here, we report a comprehensive study on the variables of a rapid-dilution refolding method, including the concentrations of protein, salt, detergent and redox agents, pH, refolding duration and the presence of aggregation suppressors and host-cell contaminants, in order to identify the optimal condition to refold human BMP-9 (hBMP-9). To produce a recombinant form of hBMP-9 in E. coli cells, a synthetic codon-optimized gene was designed to encode the mature domain of hBMP-9 (Ser320 - Arg429) directly behind the first methionine, which we herein referred to as MB109. An effective purification scheme was also developed to purify the refolded MB109 to homogeneity with a final yield of 7.8 mg from 100 mg of chromatography-purified inclusion bodies as a starting material. The chemically refolded MB109 binds to ALK1, ActRIIb and BMPRII receptors with relatively high affinity as compared to other Type I and Type II receptors based on surface plasmon resonance analysis. Smad1-dependent luciferase assay in C2C12 cells shows that the MB109 has an EC50 of 0.61 ng/mL (25 pM), which is nearly the same as hBMP-9. CONCLUSION: MB109 is prone to be refolded as non-functional dimer and higher order multimers in most of the conditions tested, but bioactive MB109 dimer can be refolded with high efficiency in a narrow window, which is strongly dependent on the pH, refolding duration, the presence of aggregation suppressors and the concentrations of protein, salt and detergent. These results add to the current understanding of producing recombinant TGF-ß superfamily ligands in the microbial E. coli system. An application of the technique to produce a large number of synthetic TGF-ß chimeras for activity screen is also discussed.