RESUMEN
Aberrantly elevated steroid receptor coactivator-1 (SRC-1) expression and activity are strongly correlated with cancer progression and metastasis. Here we report, for the first time, the development of a proteolysis targeting chimera (PROTAC) that is composed of a selective SRC-1 binder linked to a specific ligand for UBR box, a unique class of E3 ligases recognizing N-degrons. We showed that the bifunctional molecule efficiently and selectively induced the degradation of SRC-1 in cells through the N-degron pathway. Importantly, given the ubiquitous expression of the UBR protein in most cells, PROTACs targeting the UBR box could degrade a protein of interest regardless of cell types. We also showed that the SRC-1 degrader significantly suppressed cancer cell invasion and migration in vitro and in vivo. Together, these results demonstrate that the SRC-1 degrader can be an invaluable chemical tool in the studies of SRC-1 functions. Moreover, our findings suggest PROTACs based on the N-degron pathway as a widely useful strategy to degrade disease-relevant proteins.
Asunto(s)
Coactivador 1 de Receptor Nuclear/antagonistas & inhibidores , Péptidos/farmacología , Ubiquitina-Proteína Ligasas/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos CD/metabolismo , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Biocatálisis , Cadherinas/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Humanos , Factor Estimulante de Colonias de Macrófagos/metabolismo , Ratones Endogámicos BALB C , Invasividad Neoplásica/prevención & control , Neoplasias/tratamiento farmacológico , Coactivador 1 de Receptor Nuclear/metabolismo , Péptidos/metabolismo , Péptidos/uso terapéutico , Unión Proteica , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Herein, we report a strategy for generating conformationally restricted α-helix mimetic small molecules by introducing covalent bridges that limit rotation about the central axis of α-helix mimetics. We demonstrate that the bridged α-helix mimetics have enhanced binding affinity and specificity to the target protein due to the restricted conformation as well as extra interaction of the bridge with the protein surface.
Asunto(s)
Compuestos Heterocíclicos de Anillo en Puente/química , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/química , Bibliotecas de Moléculas Pequeñas/química , Compuestos Heterocíclicos de Anillo en Puente/farmacología , Humanos , Células Jurkat , Modelos Moleculares , Conformación Molecular , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Increasing non-shivering thermogenesis (NST), which expends calories as heat rather than storing them as fat, is championed as an effective way to combat obesity and metabolic disease. Innate mechanisms constraining the capacity for NST present a fundamental limitation to this approach, yet are not well understood. Here, we provide evidence that Regulator of Calcineurin 1 (RCAN1), a feedback inhibitor of the calcium-activated protein phosphatase calcineurin (CN), acts to suppress two distinctly different mechanisms of non-shivering thermogenesis (NST): one involving the activation of UCP1 expression in white adipose tissue, the other mediated by sarcolipin (SLN) in skeletal muscle. UCP1 generates heat at the expense of reducing ATP production, whereas SLN increases ATP consumption to generate heat. Gene expression profiles demonstrate a high correlation between Rcan1 expression and metabolic syndrome. On an evolutionary timescale, in the context of limited food resources, systemic suppression of prolonged NST by RCAN1 might have been beneficial; however, in the face of caloric abundance, RCAN1-mediated suppression of these adaptive avenues of energy expenditure may now contribute to the growing epidemic of obesity.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Metabolismo , Proteínas Musculares/metabolismo , Termogénesis , Células 3T3-L1 , Adipocitos/citología , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Tejido Adiposo/metabolismo , Tejido Adiposo Beige/efectos de los fármacos , Tejido Adiposo Beige/metabolismo , Tejido Adiposo Blanco/efectos de los fármacos , Tejido Adiposo Blanco/metabolismo , Adrenérgicos/farmacología , Animales , Calcineurina/metabolismo , Proteínas de Unión al Calcio , Diferenciación Celular/efectos de los fármacos , Frío , Femenino , Resistencia a la Insulina , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Metabolismo de los Lípidos/efectos de los fármacos , Hígado/metabolismo , Masculino , Síndrome Metabólico/metabolismo , Metabolismo/efectos de los fármacos , Ratones , Ratones Noqueados , Proteínas Musculares/deficiencia , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Músculo Estriado/metabolismo , Obesidad/metabolismo , Obesidad/patología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Regiones Promotoras Genéticas/genética , Proteolípidos/genética , Proteolípidos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Termogénesis/efectos de los fármacos , Proteína Desacopladora 1/metabolismoRESUMEN
The complex formation between transcription factors (TFs) and coactivator proteins is required for transcriptional activity, and thus disruption of aberrantly activated TF/coactivator interactions could be an attractive therapeutic strategy. However, modulation of such protein-protein interactions (PPIs) has proven challenging. Here we report a cell-permeable, proteolytically stable, stapled helical peptide directly targeting nuclear receptor coactivator 1 (NCOA1), a coactivator required for the transcriptional activity of signal transducer and activator of transcription 6 (STAT6). We demonstrate that this stapled peptide disrupts the NCOA1/STAT6 complex, thereby repressing STAT6-mediated transcription. Furthermore, we solved the first crystal structure of a stapled peptide in complex with NCOA1. The stapled peptide therefore represents an invaluable chemical probe for understanding the precise role of the NCOA1/STAT6 interaction and an excellent starting point for the development of a novel class of therapeutic agents.
Asunto(s)
Coactivador 1 de Receptor Nuclear/metabolismo , Péptidos/farmacología , Mapas de Interacción de Proteínas/efectos de los fármacos , Factor de Transcripción STAT6/metabolismo , Células A549 , Secuencia de Aminoácidos , Animales , Diseño de Fármacos , Células HEK293 , Humanos , Ratones , Simulación del Acoplamiento Molecular , Coactivador 1 de Receptor Nuclear/antagonistas & inhibidores , Péptidos/química , Factor de Transcripción STAT6/antagonistas & inhibidoresRESUMEN
Many biologically active α-helical peptides adopt amphiphilic helical structures that contain hydrophobic residues on one side and hydrophilic residues on the other side. Therefore, α-helix mimetics capable of mimicking such amphiphilic helical peptides should possess higher binding affinity and specificity to target proteins. Here we describe an efficient method for generating amphiphilic α-helix mimetics. One-face α-helix mimetics having hydrophobic side chains on one side was readily converted into amphiphilic α-helix mimetics by introducing appropriate charged residues on the opposite side. We also demonstrate that such two-face amphiphilic α-helix mimetics indeed show remarkably improved binding affinity to a target protein, compared to one-face hydrophobic α-helix mimetics. We believe that generating a large combinatorial library of these amphiphilic α-helix mimetics can be valuable for rapid discovery of highly potent and specific modulators of protein-protein interactions.
Asunto(s)
Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Peptidomiméticos/química , Peptidomiméticos/farmacología , Mapas de Interacción de Proteínas/efectos de los fármacos , Secuencia de Aminoácidos , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Estructura Secundaria de Proteína , Técnicas de Síntesis en Fase Sólida/métodosRESUMEN
Skp2 is thought to have two critical roles in tumorigenesis. As part of the SCF(Skp2) ubiquitin ligase, Skp2 drives the cell cycle by mediating the degradation of cell cycle proteins. Besides the proteolytic activity, Skp2 also blocks p53-mediated apoptosis by outcompeting p53 for binding p300. Herein, we exploit the Skp2/p300 interaction as a new target for Skp2 inhibition. An affinity-based high-throughput screen of a combinatorial cyclic peptoid library identified an inhibitor that binds to Skp2 and interferes with the Skp2/p300 interaction. We show that antagonism of the Skp2/p300 interaction by the inhibitor leads to p300-mediated p53 acetylation, resulting in p53-mediated apoptosis in cancer cells, without affecting Skp2 proteolytic activity. Our results suggest that inhibition of the Skp2/p300 interaction has a great potential as a new anticancer strategy, and our Skp2 inhibitor can be developed as a chemical probe to delineate Skp2 non-proteolytic function in tumorigenesis.
Asunto(s)
Apoptosis/fisiología , Proteínas Quinasas Asociadas a Fase-S/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/fisiología , Factores de Transcripción p300-CBP/antagonistas & inhibidores , Unión Proteica , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Factores de Transcripción p300-CBP/metabolismoRESUMEN
α-Helices play a critical role in mediating many protein-protein interactions (PPIs) as recognition motifs. Therefore, there is a considerable interest in developing small molecules that can mimic helical peptide segments to modulate α-helix-mediated PPIs. Due to the relatively low aqueous solubility and synthetic difficulty of most current α-helix mimetic small molecules, one important goal in this area is to develop small molecules with favorable physicochemical properties and ease of synthesis. Here we designed phenyl-piperazine-triazine-based α-helix mimetics that possess improved water solubility and excellent synthetic accessibility. We developed a facile solid-phase synthetic route that allows for rapid creation of a large, diverse combinatorial library of α-helix mimetics. Further, we identified a selective inhibitor of the Mcl-1/BH3 interaction by screening a focused library of phenyl-piperazine-triazines, demonstrating that the scaffold is able to serve as functional mimetics of α-helical peptides. We believe that our phenyl-piperazine-triazine-based α-helix mimetics, along with the facile and divergent solid-phase synthetic method, have great potential as powerful tools for discovering potent inhibitors of given α-helix-mediated PPIs.
Asunto(s)
Benzoatos/síntesis química , Biomimética , Piperazinas/síntesis química , Triazinas/síntesis química , Benzoatos/química , Polarización de Fluorescencia , Modelos Moleculares , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/química , Piperazinas/química , Estructura Secundaria de Proteína , Técnicas de Síntesis en Fase Sólida/métodos , Triazinas/químicaRESUMEN
Pharmacological chaperones are small molecules that bind to proteins and stabilize them against thermal denaturation or proteolytic degradation, as well as assist or prevent certain protein-protein assemblies. These activities are being exploited for the development of treatments for diseases caused by protein instability and/or aberrant protein-protein interactions, such as those found in certain forms of cancers and neurodegenerative diseases. However, designing or discovering pharmacological chaperones for specific targets is challenging because of the relatively featureless protein target surfaces, the lack of suitable chemical libraries, and the shortage of efficient high-throughput screening methods. In this study, we attempted to address all these challenges by synthesizing a diverse library of small molecules that mimic protein α-helical secondary structures commonly found in protein-protein interaction surfaces. This was accompanied by establishing a facile "on-bead" high-throughput screening method that allows for rapid and efficient discovery of potential pharmacological chaperones and for identifying novel chaperones/inhibitors against a cancer-associated protein, myeloid cell leukemia 1 (MCL-1), and a Parkinson disease-associated protein, α-synuclein. Our data suggest that the compounds and methods described here will be useful tools for the development of pharmaceuticals for complex-disease targets that are traditionally deemed "undruggable."
Asunto(s)
Descubrimiento de Drogas , Chaperonas Moleculares , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Neoplasias , Enfermedad de Parkinson , alfa-Sinucleína , Humanos , Células Jurkat , Chaperonas Moleculares/síntesis química , Chaperonas Moleculares/química , Chaperonas Moleculares/farmacología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/metabolismo , alfa-Sinucleína/antagonistas & inhibidores , alfa-Sinucleína/metabolismoRESUMEN
BACKGROUND: Mechanical assist device therapy has emerged recently as an important and rapidly expanding therapy in advanced heart failure, triggering in some patients a beneficial reverse remodeling response. However, mechanisms underlying this benefit are unclear. METHODS AND RESULTS: In a model of mechanical unloading of the left ventricle, we observed progressive myocyte atrophy, autophagy, and robust activation of the transcription factor FoxO3, an established regulator of catabolic processes in other cell types. Evidence for FoxO3 activation was similarly detected in unloaded failing human myocardium. To determine the role of FoxO3 activation in cardiac muscle in vivo, we engineered transgenic mice harboring a cardiomyocyte-specific constitutively active FoxO3 mutant (caFoxO3(flox);αMHC-Mer-Cre-Mer). Expression of caFoxO3 triggered dramatic and progressive loss of cardiac mass, robust increases in cardiomyocyte autophagy, declines in mitochondrial biomass and function, and early mortality. Whereas increases in cardiomyocyte apoptosis were not apparent, we detected robust increases in Bnip3 (Bcl2/adenovirus E1B 19-kDa interacting protein 3), an established downstream target of FoxO3. To test the role of Bnip3, we crossed the caFoxO3(flox);αMHC-Mer-Cre-Mer mice with Bnip3-null animals. Remarkably, the atrophy and autophagy phenotypes were significantly blunted, yet the early mortality triggered by FoxO3 activation persisted. Rather, declines in cardiac performance were attenuated by proteasome inhibitors. Consistent with involvement of FoxO3-driven activation of the ubiquitin-proteasome system, we detected time-dependent activation of the atrogenes program and sarcomere protein breakdown. CONCLUSIONS: In aggregate, these data point to FoxO3, a protein activated by mechanical unloading, as a master regulator that governs both the autophagy-lysosomal and ubiquitin-proteasomal pathways to orchestrate cardiac muscle atrophy.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Insuficiencia Cardíaca/terapia , Corazón Auxiliar , Proteínas de la Membrana/metabolismo , Proteínas Mitocondriales/metabolismo , Miocitos Cardíacos/metabolismo , Remodelación Ventricular/fisiología , Animales , Atrofia , Autofagia , Modelos Animales de Enfermedad , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/fisiología , Trasplante de Corazón , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Ratones , Ratones Transgénicos , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/fisiología , Miocardio/metabolismo , Miocardio/patología , Miocitos Cardíacos/patología , Miocitos Cardíacos/fisiología , Complejo de la Endopetidasa Proteasomal/fisiología , Transducción de Señal , Complejos de Ubiquitina-Proteína Ligasa/fisiologíaRESUMEN
The adaptor protein Amot130 scaffolds components of the Hippo pathway to promote the inhibition of cell growth. This study describes how Amot130 through binding and activating the ubiquitin ligase AIP4/Itch achieves these effects. AIP4 is found to bind and ubiquitinate Amot130 at residue Lys-481. This both stabilizes Amot130 and promotes its residence at the plasma membrane. Furthermore, Amot130 is shown to scaffold a complex containing overexpressed AIP4 and the transcriptional co-activator Yes-associated protein (YAP). Consequently, Amot130 promotes the ubiquitination of YAP by AIP4 and prevents AIP4 from binding to large tumor suppressor 1. Amot130 is found to reduce YAP stability. Importantly, Amot130 inhibition of YAP dependent transcription is reversed by AIP4 silencing, whereas Amot130 and AIP4 expression interdependently suppress cell growth. Thus, Amot130 repurposes AIP4 from its previously described role in degrading large tumor suppressor 1 to the inhibition of YAP and cell growth.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proliferación Celular , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/metabolismo , Fosfoproteínas/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Angiomotinas , Regulación de la Expresión Génica/fisiología , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Proteínas de la Membrana/genética , Proteínas de Microfilamentos , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Estabilidad Proteica , Proteolisis , Proteínas Represoras/genética , Factores de Transcripción , Transcripción Genética/fisiología , Ubiquitina-Proteína Ligasas/genética , Proteínas Señalizadoras YAPRESUMEN
RATIONALE: Despite overwhelming evidence of the importance of circadian rhythms in cardiovascular health and disease, little is known regarding the circadian regulation of intracellular signaling pathways controlling cardiac function and remodeling. OBJECTIVE: To assess circadian changes in processes dependent on the protein phosphatase calcineurin, relative to changes in phosphorylation of cardiac proteins, in normal, hypertrophic, and failing hearts. METHODS AND RESULTS: We found evidence of large circadian oscillations in calcineurin-dependent activities in the left ventricle of healthy C57BL/6 mice. Calcineurin-dependent transcript levels and nuclear occupancy of the NFAT (nuclear factor of activated T cells) regularly fluctuated as much as 20-fold over the course of a day, peaking in the morning when mice enter a period of rest. Phosphorylation of the protein phosphatase 1 inhibitor 1 (I-1), a direct calcineurin substrate, and phospholamban, an indirect target, oscillated directly out of phase with calcineurin-dependent signaling. Using a surgical model of cardiac pressure overload, we found that although calcineurin-dependent activities were markedly elevated, the circadian pattern of activation was maintained, whereas, oscillations in phospholamban and I-1 phosphorylation were lost. Changes in the expression of fetal gene markers of heart failure did not mirror the rhythm in calcineurin/NFAT activation, suggesting that these may not be direct transcriptional target genes. Cardiac function in mice subjected to pressure overload was significantly lower in the morning than in the evening when assessed by echocardiography. CONCLUSIONS: Normal, opposing circadian oscillations in calcineurin-dependent activities and phosphorylation of proteins that regulate contractility are disrupted in heart failure.
Asunto(s)
Calcineurina/fisiología , Ritmo Circadiano/fisiología , Insuficiencia Cardíaca/metabolismo , Hemodinámica/fisiología , Proteínas/metabolismo , Transducción de Señal/fisiología , Estrés Fisiológico/fisiología , Animales , Proteínas de Unión al Calcio/metabolismo , Modelos Animales de Enfermedad , Insuficiencia Cardíaca/fisiopatología , Ventrículos Cardíacos/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Musculares/metabolismo , Factores de Transcripción NFATC/metabolismo , Fosforilación/fisiología , Proteína Fosfatasa 1/metabolismoRESUMEN
There is considerable interest in developing non-peptidic, small-molecule α-helix mimetics to disrupt α-helix-mediated protein−protein interactions. Herein, we report the design of a novel pyrrolopyrimidine-based scaffold for such α-helix mimetics with increased conformational rigidity. We also developed a facile solid-phase synthetic route that is amenable to divergent synthesis of a large library. Using a fluorescence polarization-based assay, we identified cell-permeable, dual MDMX/MDM2 inhibitors, demonstrating that the designed molecules can act as α-helix mimetics.
Asunto(s)
Materiales Biomiméticos/metabolismo , Materiales Biomiméticos/farmacología , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Pirimidinas/química , Pirroles/química , Proteína p53 Supresora de Tumor/metabolismo , Secuencia de Aminoácidos , Materiales Biomiméticos/síntesis química , Materiales Biomiméticos/química , Línea Celular Tumoral , Diseño de Fármacos , Ensayos Analíticos de Alto Rendimiento , Humanos , Modelos Moleculares , Permeabilidad , Unión Proteica/efectos de los fármacos , Estructura Secundaria de Proteína , Proteína p53 Supresora de Tumor/químicaRESUMEN
Regulator of calcineurin 1 (RCAN1) inhibits the protein phosphatase calcineurin and is required for appropriate immune responses, synaptic plasticity, vascular tone, angiogenesis, and cardiac remodeling. Expression of the RCAN1-4 isoform is under the control of the calcineurin-responsive transcription factor NFAT. Typically, NFATs act in cooperation with other transcription factors to achieve maximal activation of gene expression. In this study, we identify the CCAAT/enhancer binding protein beta (C/EBPbeta) as an NFAT binding partner that cooperates with NFAT to regulate RCAN1-4 expression. Numerous C/EBPbeta binding sites are conserved in the RCAN1-4 proximal promoter. Overexpression of C/EBPbeta increased activity of both the endogenous mouse Rcan1-4 gene and a human RCAN1-4 luciferase reporter. Binding of C/EBPbeta to multiple sites in the promoter was verified using electrophoretic mobility shift assays and chromatin immunoprecipitation. A direct interaction between C/EBPbeta and NFAT was demonstrated by co-immunoprecipitation of proteins and complex formation at NFAT-C/EBPbeta composite sites. Depletion of endogenous C/EBPbeta decreased maximal activation of RCAN1-4 expression by calcineurin, whereas inhibition of calcineurin did not alter the ability of C/EBPbeta to activate RCAN1-4 expression. Together, these findings suggest that calcineurin/NFAT activation of RCAN1-4 expression is in part dependent upon C/EBPbeta, whereas activation by C/EBPbeta is not dependent on calcineurin and may provide a calcineurin-independent pathway for regulating RCAN1-4 expression. Importantly, nuclear localization, C/EBPbeta DNA binding activity and occupancy of the Rcan1-4 promoter increased in mouse models of heart failure demonstrating in vivo activation of this pathway to regulate Rcan1-4 expression and ultimately shape the dynamics of calcineurin-dependent signaling.
Asunto(s)
Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Regulación de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Musculares/metabolismo , Factores de Transcripción NFATC/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Calcineurina/metabolismo , Proteínas de Unión al Calcio , Línea Celular , Núcleo Celular/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Transducción de Señal , Fracciones Subcelulares/metabolismoRESUMEN
BACKGROUND: Cellular hypertrophy requires coordinated regulation of progrowth and antigrowth mechanisms. In cultured neonatal cardiomyocytes, Foxo transcription factors trigger an atrophy-related gene program that counters hypertrophic growth. However, downstream molecular events are not yet well defined. METHODS AND RESULTS: Here, we report that expression of either Foxo1 or Foxo3 in cardiomyocytes attenuates calcineurin phosphatase activity and inhibits agonist-induced hypertrophic growth. Consistent with these results, Foxo proteins decrease calcineurin phosphatase activity and repress both basal and hypertrophic agonist-induced expression of MCIP1.4, a direct downstream target of the calcineurin/NFAT pathway. Furthermore, hearts from Foxo3-null mice exhibit increased MCIP1.4 abundance and a hypertrophic phenotype with normal systolic function at baseline. Together, these results suggest that Foxo proteins repress cardiac growth at least in part through inhibition of the calcineurin/NFAT pathway. Given that hypertrophic growth of the heart occurs in multiple contexts, our findings also suggest that certain hypertrophic signals are capable of overriding the antigrowth program induced by Foxo. Consistent with this, multiple hypertrophic agonists triggered inactivation of Foxo proteins in cardiomyocytes through a mechanism requiring the PI3K/Akt pathway. In addition, both Foxo1 and Foxo3 are phosphorylated and consequently inactivated in hearts undergoing hypertrophic growth induced by hemodynamic stress. CONCLUSIONS: This study suggests that inhibition of the calcineurin/NFAT signaling cascade by Foxo and release of this repressive action by the PI3K/Akt pathway are important mechanisms whereby Foxo factors govern cell growth in the heart.
Asunto(s)
Cardiomegalia/fisiopatología , Factores de Transcripción Forkhead/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Angiotensina II/farmacología , Animales , Calcineurina/fisiología , Proteínas de Unión al Calcio , Cardiomegalia/genética , Cardiomegalia/patología , Células Cultivadas , Proteína Forkhead Box O1 , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/genética , Regulación de la Expresión Génica/fisiología , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Proteínas del Tejido Nervioso/genética , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Monoéster Fosfórico Hidrolasas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Allelic exclusion ensures that individual B lymphocytes produce only one kind of antibody molecule. Previous studies have shown that allelic exclusion of the mouse Igkappa locus occurs by the combination of monoallelic silencing and a low level of monoallelic activation for rearrangement combined with a negative feedback loop blocking additional functional rearrangements. Using yeast artificial chromosome-based single-copy isotransgenic mice, we have identified a cis-acting element that negatively regulates rearrangement in this locus, specifically in B cells. The element, termed Sis, resides in the V-J intervening sequence. Sis specifies the targeting of Igkappa transgenes in pre-B and B cells to centromeric heterochromatin and associates with Ikaros, a repressor protein that also colocalizes with centromeric heterochromatin. Significantly, these are hallmarks of silenced endogenous germline Igkappa genes in B cells. These results lead us to propose that Sis participates in the monoallelic silencing aspect of allelic exclusion regulation.
Asunto(s)
Heterocromatina/genética , Factor de Transcripción Ikaros/genética , Cadenas kappa de Inmunoglobulina/genética , Recombinación Genética/genética , Elementos Reguladores de la Transcripción/genética , Animales , Reordenamiento Génico de Cadena Ligera de Linfocito B/genética , Silenciador del Gen , Genes de Inmunoglobulinas , Imagenología Tridimensional , Hibridación Fluorescente in Situ , Ratones , Ratones Transgénicos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Skeletal muscles are a mosaic of slow and fast twitch myofibers. During embryogenesis, patterns of fiber type composition are initiated that change postnatally to meet physiological demand. To examine the role of the protein phosphatase calcineurin in the initiation and maintenance of muscle fiber types, we used a "Flox-ON" approach to obtain muscle-specific overexpression of the modulatory calcineurin-interacting protein 1 (MCIP1/DSCR1), an inhibitor of calcineurin. Myo-Cre transgenic mice with early skeletal muscle-specific expression of Cre recombinase were used to activate the Flox-MCIP1 transgene. Contractile components unique to type 1 slow fibers were absent from skeletal muscle of adult Myo-Cre/Flox-MCIP1 mice, whereas oxidative capacity, myoglobin content, and mitochondrial abundance were unaltered. The soleus muscles of Myo-Cre/Flox-MCIP1 mice fatigued more rapidly than the wild type as a consequence of the replacement of the slow myosin heavy chain MyHC-1 with a fast isoform, MyHC-2A. MyHC-1 expression in Myo-Cre/Flox-MCIP1 embryos and early neonates was normal. These results demonstrate that developmental patterning of slow fibers is independent of calcineurin, while the maintenance of the slow-fiber phenotype in the adult requires calcineurin activity.
Asunto(s)
Calcineurina/fisiología , Fibras Musculares de Contracción Lenta/enzimología , Músculo Esquelético/enzimología , Animales , Proteínas Contráctiles/metabolismo , Proteínas de Unión al ADN , Desarrollo Embrionario/fisiología , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Transgénicos , Mitocondrias/enzimología , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/embriología , Mioglobina/metabolismoRESUMEN
The deletion of the top3(+) gene leads to defective nuclear division and lethality in Schizosaccharo myces pombe. This lethality is suppressed by concomitant loss of rqh1(+), the RecQ helicase. Despite extensive investigation, topoisomerase III function and its relationship with RecQ helicase remain poorly understood. We generated top3 temperature-sensitive (top3-ts) mutants and found these to be defective in nuclear division and cytokinesis and to be sensitive to DNA-damaging agents. A temperature shift of top3-ts cells to 37 degrees C, or treatment with hydroxyurea at the permissive temperature, caused an increase in 'cut' (cell untimely torn) cells and elevated rates of minichromosome loss. The viability of top3-ts cells was decreased by a temperature shift during S-phase when compared with a similar treatment in other cell cycle stages. Furthermore, the top3-ts mutant was not sensitive to M-phase specific drugs. These results indicate that topoisomerase III may play an important role in DNA metabolism during DNA replication to ensure proper chromosome segregation. Our data are consistent with Top3 acting downstream of Rqh1 to process the toxic DNA structure produced by Rqh1.