RESUMEN
The mammalian circadian clock at the hypothalamic suprachiasmatic nuclei (SCN) entrains biological rhythms to the 24-h cyclic environment, by encoding light-dark transitions in SCN neurons. Light pulses induce phase shifts in the clock and in circadian rhythms; photic signaling for circadian phase advances involves a nitric oxide (NO)/cyclic guanosine monophosphate (cGMP)/cGMP-dependent protein kinase (PKG) pathway, increasing the expression of Period (Per) genes. Effectors downstream of PKG remain unknown. Here we investigate the role of G-substrate (GS), a PKG substrate, in the hamster SCN. GS and phosphorylated G-substrate (p-GS) were present in a subset of SCN cells. Moreover, GS phosphorylation (p-GS/GS ratio) increased in SCN homogenates after light pulses delivered at circadian time (CT) 18 and intraperitoneal treatment with sildenafil, an inhibitor of phosphodiesterase 5 (a cGMP-specific phosphodiesterase). On the other hand, intracerebroventricular treatment with the PKG inhibitor KT5823, reduced photic phosphorylation of GS to basal levels. Since p-GS could act as a protein phosphatase 2 A (PP2A) inhibitor, we demonstrated physical interaction between p-GS and PP2A in SCN homogenates, and also a light-pulse dependent decrease of PP2A activity. Intracerebroventricular treatment with okadaic acid, a PP2A inhibitor, increased the magnitude of light-induced phase advances of locomotor rhythms. We provide evidence on the physiological phosphorylation of GS as a new downstream effector in the NO/cGMP/PKG photic pathway in the hamster SCN, including its role as a PP2A inhibitor.
Asunto(s)
Relojes Circadianos , Animales , Cricetinae , GMP Cíclico , Proteínas del Tejido Nervioso , Transducción de Señal , Núcleo SupraquiasmáticoRESUMEN
Plant growth-promoting rhizobacteria (PGPR) stimulate plant growth, but the underlying mechanism is poorly understood. In this study, we asked whether PROTEIN PHOSPHATASE 2A (PP2A), a regulatory molecular component of stress, growth, and developmental signaling networks in plants, contributes to the plant growth responses induced by the PGPR Azospirillum brasilense (wild type strain Sp245 and auxin deficient strain FAJ0009) and Pseudomonas simiae (WCS417r). The PGPR were co-cultivated with Arabidopsis wild type (WT) and PP2A (related) mutants. These plants had mutations in the PP2A catalytic subunits (C), and the PP2A activity-modulating genes LEUCINE CARBOXYL METHYL TRANSFERASE 1 (LCMT1) and PHOSPHOTYROSYL PHOSPHATASE ACTIVATOR (PTPA). When exposed to the three PGPR, WT and all mutant Arabidopsis revealed the typical phenotype of PGPR-treated plants with shortened primary root and increased lateral root density. Fresh weight of plants generally increased when the seedlings were exposed to the bacteria strains, with the exception of catalytic subunit double mutant c2c5. The positive effect on root and shoot fresh weight was especially pronounced in Arabidopsis mutants with low PP2A activity. Comparison of different mutants indicated a significant role of the PP2A catalytic subunits C2 and C5 for a positive response to PGPR.
RESUMEN
Since the original description as potent antianginal compounds, phosphodiesterase 5A inhibitors have continuously increased their possible therapeutic applications. In the heart, Sildenafil was shown to protect against an ischemic insult by decreasing cardiac Na+/H+ exchanger (NHE1) activity, action that was mediated by protein kinase G. p38 mitogen activated protein kinase (p38MAPK) activation was described in cardiac ischemia, but its precise role remains elusive. It has been shown that p38MAPK is activated by protein kinase G (PKG) in certain non-cardiac tissues, while in others modulates NHE1 activity. Current study was aimed to seek the role of p38MAPK in the Sildenafil-triggered pathway leading to NHE1 inhibition in myocardium. Rat isolated papillary muscles were used to evaluate NHE1 activity during intracellular pH recovery from an acidic load. Protein kinases phosphorylation (activation) was determined by western blot. Sustained acidosis promoted NHE1 hyperactivity by enhancing Ser703 phosphorylation, effect that was blunted by Sildenafil. p38MAPK inhibition reversed the effect of Sildenafil on NHE1. Activation of p38MAPK, by Sodium Arsenite or Anisomycin, mimicked the inhibitory effect of Sildenafil on the exchanger. Consistently, Sildenafil induced p38MAPK phosphorylation/activation during acidosis. Neither Sildenafil nor p38MAPK inhibition affected extracellular signal-regulated kinases 1/2 phosphorylation, kinases upstream NHE1. Furthermore, inhibition of NHE1 after p38MAPK activation was precluded by preventing the activation of protein phosphatase 2A with Okadaic Acid. Taken together, these results suggest that activation of p38MAPK is a necessary step to trigger the inhibitory effect of Sildenafil on cardiac NHE1 activity, thorough a mechanism that involves protein phosphatase 2A-mediated exchanger dephosphorylation.
Asunto(s)
Corazón/efectos de los fármacos , Miocardio/metabolismo , Citrato de Sildenafil/farmacología , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Acidosis/enzimología , Acidosis/metabolismo , Acidosis/patología , Animales , Activación Enzimática/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Miocardio/citología , Miocardio/patología , Fosforilación/efectos de los fármacos , Proteína Fosfatasa 2/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Wistar , Intercambiador 1 de Sodio-Hidrógeno/metabolismoRESUMEN
In a recent review published in Molecular Neurobiology, Kamat and colleagues (Mol Neurobiol. 2014 Dec;50(3):852-65) highlighted the cellular and molecular mechanisms involved in Okadaic acid (OKA)-induced neurotoxicity. In this review, the authors underline a wide range of pathological signaling pathways involved in OKA-induced neurotoxicity; however, the role of glutamate was only briefly described. We believe that the hyperactivation of the glutamatergic system is a key pathophysiological player in OKA-induced neurotoxicity and deserves serious attention. In this commentary, we propose an integrative model linking glutamate and PP2A and put forward some unanswered questions.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Ácido Glutámico/metabolismo , Proteína Fosfatasa 2/antagonistas & inhibidores , Animales , Humanos , Modelos Biológicos , Ácido Ocadaico/farmacología , Proteína Fosfatasa 2/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
In higher eukaryotes, eIF4A, eIF4E and eIF4G homologues interact to enable mRNA recruitment to the ribosome. eIF4G acts as a scaffold for these interactions and also interacts with other proteins of the translational machinery. Trypanosomatid protozoa have multiple homologues of eIF4E and eIF4G and the precise function of each remains unclear. Here, 2 previously described eIF4G homologues, EIF4G3 and EIF4G4, were further investigated. In vitro, both homologues bound EIF4AI, but with different interaction properties. Binding to distinct eIF4Es was also confirmed; EIF4G3 bound EIF4E4 while EIF4G4 bound EIF4E3, both these interactions required similar binding motifs. EIF4G3, but not EIF4G4, interacted with PABP1, a poly-A binding protein homolog. Work in vivo with Trypanosoma brucei showed that both EIF4G3 and EIF4G4 are cytoplasmic and essential for viability. Depletion of EIF4G3 caused a rapid reduction in total translation while EIF4G4 depletion led to changes in morphology but no substantial inhibition of translation. Site-directed mutagenesis was used to disrupt interactions of the eIF4Gs with either eIF4E or eIF4A, causing different levels of growth inhibition. Overall the results show that only EIF4G3, with its cap binding partner EIF4E4, plays a major role in translational initiation.
Asunto(s)
Factor 4A Eucariótico de Iniciación/genética , Factor 4E Eucariótico de Iniciación/genética , Factor 4G Eucariótico de Iniciación/genética , Leishmania major/genética , Iniciación de la Cadena Peptídica Traduccional , Proteínas Protozoarias/genética , Trypanosoma brucei brucei/genética , Secuencia de Aminoácidos , Sitios de Unión , Factor 4A Eucariótico de Iniciación/química , Factor 4A Eucariótico de Iniciación/metabolismo , Factor 4E Eucariótico de Iniciación/química , Factor 4E Eucariótico de Iniciación/metabolismo , Factor 4G Eucariótico de Iniciación/química , Factor 4G Eucariótico de Iniciación/metabolismo , Regulación de la Expresión Génica , Leishmania major/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteína I de Unión a Poli(A)/genética , Proteína I de Unión a Poli(A)/metabolismo , Unión Proteica , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Trypanosoma brucei brucei/metabolismoRESUMEN
Sleep is beneficial to learning, but the underlying mechanisms remain controversial. The synaptic homeostasis hypothesis (SHY) proposes that the cognitive function of sleep is related to a generalized rescaling of synaptic weights to intermediate levels, due to a passive downregulation of plasticity mechanisms. A competing hypothesis proposes that the active upscaling and downscaling of synaptic weights during sleep embosses memories in circuits respectively activated or deactivated during prior waking experience, leading to memory changes beyond rescaling. Both theories have empirical support but the experimental designs underlying the conflicting studies are not congruent, therefore a consensus is yet to be reached. To advance this issue, we used real-time PCR and electrophysiological recordings to assess gene expression related to synaptic plasticity in the hippocampus and primary somatosensory cortex of rats exposed to novel objects, then kept awake (WK) for 60 min and finally killed after a 30 min period rich in WK, slow-wave sleep (SWS) or rapid-eye-movement sleep (REM). Animals similarly treated but not exposed to novel objects were used as controls. We found that the mRNA levels of Arc, Egr1, Fos, Ppp2ca and Ppp2r2d were significantly increased in the hippocampus of exposed animals allowed to enter REM, in comparison with control animals. Experience-dependent changes during sleep were not significant in the hippocampus for Bdnf, Camk4, Creb1, and Nr4a1, and no differences were detected between exposed and control SWS groups for any of the genes tested. No significant changes in gene expression were detected in the primary somatosensory cortex during sleep, in contrast with previous studies using longer post-stimulation intervals (>180 min). The experience-dependent induction of multiple plasticity-related genes in the hippocampus during early REM adds experimental support to the synaptic embossing theory.
Asunto(s)
Hipocampo/fisiología , Memoria/fisiología , Plasticidad Neuronal/fisiología , Sueño REM/fisiología , Animales , Conducta Exploratoria/fisiología , Expresión Génica , Masculino , Plasticidad Neuronal/genética , Ratas , Ratas Wistar , Sueño REM/genética , Corteza Somatosensorial/fisiologíaRESUMEN
Canonical Wnt signaling is altered in most cases of colorectal cancer. Experimental evidence indicates that protein phosphatase 2A (PP2A) may play either positive or negative roles in Wnt signaling but its precise in vivo functions remain elusive. In this work, using colon cultured cell lines we showed that basal PP2A activity is markedly reduced in malignant cells compared to non-malignant counterparts. We found that whereas normal or cancer cells displaying not altered Wnt signaling express mRNAs coding for PP2A-A scaffold α and ß isoforms, cancer cells which have altered Wnt signaling do not express the Aß isoform mRNA. Remarkably, we found that the Aß protein levels are lost in all colon cancer cells, and in patients' tumor biopsies. In addition, all cancer cells exhibit higher levels of RalA activity, compared to non-malignant cells. Rescue experiments to restore Aß expression in malignant RKO cells, diminished the RalGTPase activation and cell proliferation, indicating that the Aß isoform acts as tumor suppressor in colon cancer cells. Reciprocal co-immunoprecipitation and immunofluorescence studies showed that the PP2A-C and -Aα subunits, expressed in all colon cells, interact in vivo with ß-catenin only in malignant cells. Selective inhibition of PP2A did not significantly affect cellular apoptosis but induced dose-dependent negative effects in ß-catenin-mediated transcriptional activity and in cell proliferation of malignant cells, indicating that the residual PP2A activity found in malignant cells, mediated by -C and Aα core subunits, is essential to maintain active Wnt signaling and cell proliferation in colon cancer cells.
Asunto(s)
Neoplasias del Colon/genética , Proteína Fosfatasa 2/genética , Subunidades de Proteína/genética , Vía de Señalización Wnt/genética , Línea Celular Tumoral , GTP Fosfohidrolasas/genética , Células HT29 , Humanos , Isoformas de Proteínas/genética , ARN Mensajero/genética , Transcripción Genética/genética , beta Catenina/genéticaRESUMEN
Therapies that selectively target cancer cells for death have been the center of intense research recently. One potential therapy may involve apoptin proteins, which are able to induce apoptosis in cancer cells leaving normal cells unharmed. Apoptin was originally discovered in the Chicken anemia virus (CAV); however, human gyroviruses (HGyV) have recently been found that also harbor apoptin-like proteins. Although the cancer cell specific activity of these apoptins appears to be well conserved, the precise functions and mechanisms of action are yet to be fully elucidated. Strategies for both delivering apoptin to treat tumors and disseminating the protein inside the tumor body are now being developed, and have shown promise in preclinical animal studies.