RESUMO
Despite significant advances in the development of therapeutic interventions targeting autoimmune diseases and chronic inflammatory conditions, lack of effective treatment still poses a high unmet need. Modulating chronically activated T cells through the blockade of the Kv1.3 potassium channel is a promising therapeutic approach; however, developing selective Kv1.3 inhibitors is still an arduous task. Phage display-based high throughput peptide library screening is a rapid and robust approach to develop promising drug candidates; however, it requires solid-phase immobilization of target proteins with their binding site preserved. Historically, the KcsA bacterial channel chimera harboring only the turret region of the human Kv1.3 channel was used for screening campaigns. Nevertheless, literature data suggest that binding to this type of chimera does not correlate well with blocking potency on the native Kv1.3 channels. Therefore, we designed and successfully produced advanced KcsA-Kv1.3, KcsA-Kv1.1, and KcsA-Kv1.2 chimeric proteins in which both the turret and part of the filter regions of the human Kv1.x channels were transferred. These T+F (turret-filter) chimeras showed superior peptide ligand-binding predictivity compared to their T-only versions in novel phage ELISA assays. Phage ELISA binding and competition results supported with electrophysiological data confirmed that the filter region of KcsA-Kv1.x is essential for establishing adequate relative affinity order among selected peptide toxins (Vm24 toxin, Hongotoxin-1, Kaliotoxin-1, Maurotoxin, Stichodactyla toxin) and consequently obtaining more reliable selectivity data. These new findings provide a better screening tool for future drug development efforts and offer insight into the target-ligand interactions of these therapeutically relevant ion channels.
Assuntos
Canal de Potássio Kv1.3 , Bloqueadores dos Canais de Potássio , Proteínas Recombinantes de Fusão , Animais , Humanos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/antagonistas & inibidores , Sítios de Ligação , Canal de Potássio Kv1.3/metabolismo , Canal de Potássio Kv1.3/antagonistas & inibidores , Canal de Potássio Kv1.3/genética , Canal de Potássio Kv1.3/química , Ligantes , Biblioteca de Peptídeos , Bloqueadores dos Canais de Potássio/química , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/metabolismo , Canais de Potássio/química , Canais de Potássio/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Linhagem CelularRESUMO
Accumulating evidence from both experimental and clinical investigations indicates a tight interaction between metabolism and circadian timekeeping; however, knowledge of the underlying mechanism is still incomplete. Metabolic compensation allows circadian oscillators to run with a constant speed at different substrate levels and, therefore, is a substantial criterion of a robust rhythm in a changing environment. Because previous data have suggested a central role of RAS2-mediated signaling in the adaptation of yeast to different nutritional environments, we examined the involvement of RAS2 in the metabolic regulation of the clock in the circadian model organism Neurospora crassa We show that, in a ras2-deficient strain, the period is longer than in the control. Moreover, unlike in the WT, in Δras2, operation of the circadian clock was affected by glucose; compared with starvation conditions, the period was longer and the oscillation of expression of the frequency (frq) gene was dampened. In constant darkness, the delayed phosphorylation of the FRQ protein and the long-lasting accumulation of FRQ in the nucleus were in accordance with the longer period and the less robust rhythm in the mutant. Although glucose did not affect the subcellular distribution of FRQ in the WT, highly elevated FRQ levels were detected in the nucleus in Δras2 RAS2 interacted with the RAS-binding domain of the adenylate cyclase in vitro, and the cAMP analogue 8-bromo-cyclic AMP partially rescued the circadian phenotype in vivo We therefore propose that RAS2 acts via a cAMP-dependent pathway and exerts significant metabolic control on the Neurospora circadian clock.
Assuntos
Relógios Circadianos , Modelos Biológicos , Neurospora crassa/metabolismo , Proteínas ras/metabolismo , Proteínas ras/análise , Proteínas ras/deficiênciaRESUMO
The recreational drug ecstasy is widely used among dance clubbers for its acute euphoric and entactogenic effects. Ecstasy exerts its acute effects by increasing the extracellular concentration of monoamines in the brain by reversing the functions of reuptake mechanisms. These elevations in extracellular monoamine concentrations result in wake promoting effects, body hyperthermia and reductions in local cerebral blood flow. However, on the long-run, ecstasy reduces serotonin concentration and density of serotonergic markers in several brain areas. Functional deficits, like sleep disturbances, anxiogenic- and aggressive behavioral responses and mood disorders also may occur. However, one of the most prominent adverse effects is related to the cognitive functions. Following ecstasy use attenuated retro- and prospective memory and defective higher order cognitive functions can be observed, especially in heavy users. Several studies indicated the involvement of the endocannabinoid system, the sleep regulating centers and the hypothalamic-pituitary-adrenal axis based on or parallel to serotonergic damage in these processes. Recent evidence, however, also showed that changes in one of the latter systems can influence the functions of each other. In this review we summarize the related literature, and propose a complex mechanism for the long-lasting cognitive deficits following heavy ecstasy use.
Assuntos
Transtornos Cognitivos/induzido quimicamente , Cognição/efeitos dos fármacos , Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , N-Metil-3,4-Metilenodioxianfetamina/efeitos adversos , Sistema Hipófise-Suprarrenal/efeitos dos fármacos , Serotonina/metabolismo , Sono/efeitos dos fármacos , Circulação Cerebrovascular/efeitos dos fármacos , Ritmo Circadiano/efeitos dos fármacos , Transtornos Cognitivos/metabolismo , Endocanabinoides/metabolismo , Febre/induzido quimicamente , Humanos , Memória/efeitos dos fármacos , N-Metil-3,4-Metilenodioxianfetamina/química , N-Metil-3,4-Metilenodioxianfetamina/farmacologia , Sono REM/efeitos dos fármacosRESUMO
The circadian clock governs rhythmic cellular functions by driving the expression of a substantial fraction of the genome and thereby significantly contributes to the adaptation to changing environmental conditions. Using the circadian model organism Neurospora crassa, we show that molecular timekeeping is robust even under severe limitation of carbon sources, however, stoichiometry, phosphorylation and subcellular distribution of the key clock components display drastic alterations. Protein kinase A, protein phosphatase 2 A and glycogen synthase kinase are involved in the molecular reorganization of the clock. RNA-seq analysis reveals that the transcriptomic response of metabolism to starvation is highly dependent on the positive clock component WC-1. Moreover, our molecular and phenotypic data indicate that a functional clock facilitates recovery from starvation. We suggest that the molecular clock is a flexible network that allows the organism to maintain rhythmic physiology and preserve fitness even under long-term nutritional stress.
Assuntos
Relógios Circadianos , Neurospora crassa , Relógios Circadianos/genética , Neurospora crassa/metabolismo , Glucose/metabolismo , Fosforilação , Transcriptoma , Ritmo Circadiano/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão GênicaRESUMO
Endoplasmic reticulum (ER) stress-dependent accumulation of incorrectly folded proteins leads to activation of the unfolded protein response. The role of the unfolded protein response (UPR) is to avoid cell damage and restore the homeostatic state by autophagy; however, excessive ER stress results in apoptosis. Here we investigated the ER stress-dependent feedback loops inside one of the UPR branches by focusing on PERK-induced ATF4 and its two targets, called CHOP and GADD34. Our goal was to qualitatively describe the dynamic behavior of the system by exploring the key regulatory motifs using both molecular and theoretical biological techniques. Using the HEK293T cell line as a model system, we confirmed that the life-or-death decision is strictly regulated. We investigated the dynamic characteristics of the crucial elements of the PERK pathway at both the RNA and protein level upon tolerable and excessive levels of ER stress. Of particular note, inhibition of GADD34 or CHOP resulted in various phenotypes upon high levels of ER stress. Our computer simulations suggest the existence of two new feedback loops inside the UPR. First, GADD34 seems to have a positive effect on ATF4 activity, while CHOP inhibits it. We claim that these newly described feedback loops ensure the fine-tuning of the ATF4-dependent stress response mechanism of the cell.
Assuntos
Fator 4 Ativador da Transcrição , Estresse do Retículo Endoplasmático , Humanos , Fator 4 Ativador da Transcrição/metabolismo , Células HEK293 , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Resposta a Proteínas não DobradasRESUMO
The recreational drug "Ecstasy" [3,4-methylenedioxymethamphetamine (MDMA)] has a well-characterised neurotoxic effect on the 5-hydroxytryptamine (5-HT) neurons in animals. Despite intensive studies, the long-term functional consequencies of the 5-HT neurodegeneration remains elusive. The aim of this study was to investigate whether any alteration of 5-hydroxytryptamine-3 (5-HT(3)) receptor functions on the sleep-wake cycle, motor activity, and quantitative EEG could be detected 6 months after a single dose of 15 mg/kg of MDMA. The selective 5-HT(3) receptor agonist m-chlorophenylbiguanide (mCPBG; 1 mg/kg, i.p.) or vehicle was administered to freely moving rats pre-treated with MDMA (15 mg/kg, i.p.) or vehicle 6 months earlier. Polysomnographic and motor activity recordings were performed. Active wake (AW), passive wake (PW), light slow wave sleep (SWS-1), deep slow wave sleep (SWS-2), and paradoxical sleep were classified. In addition, EEG power spectra were calculated for the second hour after mCPBG treatment for each stage. AW increased and SWS-1 decreased in the second hour after mCPBG treatment in control animals. mCPBG caused significant changes in the EEG power in states with cortical activation (AW, PW, paradoxical sleep). In addition, mCPBG had a biphasic effect on hippocampal theta power in AW with a decrease in 7 Hz and a stage-selective increase in the upper range (8-9 Hz). Effects of mCPBG on the time spent in AW and SWS-1 were eliminated or reduced in MDMA-treated animals. In addition, mCPBG did not increase the upper theta power of AW in rats pre-treated with MDMA. These data suggest long-term changes in 5-HT(3) receptor function after MDMA.
Assuntos
Encéfalo/efeitos dos fármacos , Ritmo Circadiano/efeitos dos fármacos , Atividade Motora/efeitos dos fármacos , N-Metil-3,4-Metilenodioxianfetamina/farmacologia , Receptores 5-HT3 de Serotonina/metabolismo , Serotoninérgicos/farmacologia , Animais , Biguanidas/farmacologia , Encéfalo/fisiologia , Ritmo Circadiano/fisiologia , Eletroencefalografia , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Masculino , Atividade Motora/fisiologia , N-Metil-3,4-Metilenodioxianfetamina/administração & dosagem , Polissonografia , Distribuição Aleatória , Ratos , Agonistas do Receptor 5-HT3 de Serotonina , Serotoninérgicos/administração & dosagem , Agonistas do Receptor de Serotonina/farmacologia , Sono/efeitos dos fármacos , Sono/fisiologia , Fases do Sono/efeitos dos fármacos , Fases do Sono/fisiologia , Ritmo Teta , Fatores de Tempo , Vigília/efeitos dos fármacos , Vigília/fisiologiaRESUMO
The possible long-term effects of the recreational drug "ecstasy" (3,4-methylenedioxymethamphetamine, MDMA) on the function of 5-hydroxytryptamine-1B (5-HT(1B)) receptor in sleep and motor control were investigated using a selective 5-HT(1B) receptor agonist, 5-propoxy-3-(1,2,3,6-tetrahydro-4-pyrinzidyl)-1H-pyrrolo([3,2-b])pyridine hydrochloride (CP-94,253; 5 mg/kg). CP-94,253 or vehicle was administered to freely moving rats pre-treated with MDMA (15 mg/kg) or vehicle 6 months earlier, and polygraphic recording for 24 h and motor activity measurements were performed. Active wake (AW), passive wake (PW), light slow wave sleep (SWS-1), deep slow wave sleep (SWS-2), paradoxical sleep (PS), and diurnal rhythm were analyzed for the whole period. In additional, the EEG power spectrum was calculated for the second hour after the acute treatment for AW, PW, SWS-1, and SWS-2. 5-HT transporter (5-HTT) immunohistochemistry was measured in brain areas related to sleep and motor control 6 months after MDMA treatment. CP-94,253 increased AW and PW, decreased SWS-2 and PS, and altered parameters of diurnal rhythm in control animals. CP-94,253 decreased the EEG power spectra at higher frequencies. The effects of CP-94,253 on AW and diurnal rhythm were reduced or eliminated in MDMA-treated animals. MDMA treatment decreased 5-HTT fibre density in posterior hypothalamus, tuberomammillary nucleus, caudate putamen and ventrolateral striatum. These data suggest that long-term changes in 5-HT(1B) receptor function occur after serotonergic damage caused by a single dose of MDMA.
Assuntos
Encéfalo/efeitos dos fármacos , Movimento/efeitos dos fármacos , N-Metil-3,4-Metilenodioxianfetamina/toxicidade , Piridinas/farmacologia , Agonistas do Receptor 5-HT1 de Serotonina , Sono/efeitos dos fármacos , Animais , Axônios/efeitos dos fármacos , Axônios/metabolismo , Axônios/patologia , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Química Encefálica/efeitos dos fármacos , Química Encefálica/fisiologia , Ritmo Circadiano/efeitos dos fármacos , Ritmo Circadiano/fisiologia , Interações Medicamentosas/fisiologia , Eletroencefalografia/efeitos dos fármacos , Masculino , Movimento/fisiologia , Neurotoxinas/toxicidade , Ratos , Serotonina/metabolismo , Serotoninérgicos/toxicidade , Proteínas da Membrana Plasmática de Transporte de Serotonina/efeitos dos fármacos , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Agonistas do Receptor de Serotonina/farmacologia , Sono/fisiologia , Sono REM/efeitos dos fármacos , Sono REM/fisiologia , Tempo , Vigília/efeitos dos fármacos , Vigília/fisiologia , Degeneração Walleriana/induzido quimicamente , Degeneração Walleriana/metabolismo , Degeneração Walleriana/fisiopatologiaRESUMO
SIGNIFICANCE: Both circadian rhythm and the production of reactive oxygen species (ROS) are fundamental features of aerobic eukaryotic cells. The circadian clock enhances the fitness of organisms by enabling them to anticipate cycling changes in the surroundings. ROS generation in the cell is often altered in response to environmental changes, but oscillations in ROS levels may also reflect endogenous metabolic fluctuations governed by the circadian clock. On the other hand, an effective regulation and timing of antioxidant mechanisms may be crucial in the defense of cellular integrity. Thus, an interaction between the circadian timekeeping machinery and ROS homeostasis or signaling in both directions may be of advantage at all phylogenetic levels. RECENT ADVANCES: The Frequency-White Collar-1 and White Collar-2 oscillator (FWO) of the filamentous fungus Neurospora crassa is well characterized at the molecular level. Several members of the ROS homeostasis were found to be controlled by the circadian clock, and ROS levels display circadian rhythm in Neurospora. On the other hand, multiple data indicate that ROS affect the molecular oscillator. CRITICAL ISSUES: Increasing evidence suggests the interplay between ROS homeostasis and oscillators that may be partially or fully independent of the FWO. In addition, ROS may be part of a complex cellular network synchronizing non-transcriptional oscillators with timekeeping machineries based on the classical transcription-translation feedback mechanism. FUTURE DIRECTIONS: Further investigations are needed to clarify how the different layers of the bidirectional interactions between ROS homeostasis and circadian regulation are interconnected.
Assuntos
Antioxidantes/metabolismo , Ritmo Circadiano/fisiologia , Proteínas de Ligação a DNA/fisiologia , Proteínas Fúngicas/fisiologia , Homeostase/fisiologia , Neurospora crassa/fisiologia , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição/fisiologiaRESUMO
Discrepancies between sleep timing on workdays and weekends, also known as social jetlag (SJL), affect the majority of the population and have been found to be associated with increased health risk and health-impairing behaviors. In this study, we explored the relationship between SJL and academic performance in a sample of undergraduates of the Semmelweis University. We assessed SJL and other sleep-related parameters with the Munich ChronoType Questionnaire (MCTQ) (n = 753). Academic performance was measured by the average grade based on weekly test results as well as scores acquired on the final test (n = 247). The average mid-sleep point on free days in the Hungarian sample fits well the regression line plotted for longitudes within the Central European Time Zone and chronotypes, confirming that sunlight has a major impact on chronotype. Multivariate analysis showed negative effect of SJL on the weekly average grade (p = 0.028, n = 247) during the lecture term with its highly regular teaching schedules, while this association disappeared in the exam period (p = 0.871, n = 247) when students had no scheduled obligations (lower SJL). We also analyzed the relationship between the time of the weekly tests and academic performance and found that students with later sleep times on free days achieved worse in the morning (p = 0.017, n = 129), while the inverse tendency was observed for the afternoon test-takers (p = 0.10, n = 118). We did not find significant association between academic performance and sleep duration or sleep debt on work days. Our data suggest that circadian misalignment can have a significant negative effect on academic performance. One possible reason for this misalignment is socially enforced sleep times.
Assuntos
Transtornos Cronobiológicos/psicologia , Ritmo Circadiano , Escolaridade , Sono , Comportamento Social , Estudantes/psicologia , Tolerância ao Trabalho Programado , Adolescente , Adulto , Transtornos Cronobiológicos/diagnóstico , Transtornos Cronobiológicos/fisiopatologia , Currículo , Avaliação Educacional , Feminino , Alemanha , Humanos , Masculino , Inquéritos e Questionários , Ensino , Fatores de Tempo , Universidades , Adulto JovemRESUMO
Reactive oxygen species (ROS) may serve as signals coupling metabolism to other cell functions. In addition to being by-products of normal metabolism, they are generated at elevated levels under environmental stress situations. We analyzed how reactive oxygen species affect the circadian clock in the model organism Neurospora crassa. In light/dark cycles, an increase in the levels of reactive oxygen species advanced the phase of both the conidiation rhythm and the expression of the clock gene frequency. Our results indicate a dominant role of the superoxide anion in the control of the phase. Elevation of superoxide production resulted in the activation of protein phosphatase 2A, a regulator of the positive element of the circadian clock. Our data indicate that even under nonstress conditions, reactive oxygen species affect circadian timekeeping. Reduction of their basal levels results in a delay of the phase in light/dark cycles and a longer period under constant conditions. We show that under entrained conditions the phase depends on the temperature and reactive oxygen species contribute to this effect. Our results suggest that the superoxide anion is an important factor controlling the circadian oscillator and is able to reset the clock most probably by activating protein phosphatase 2A, thereby modulating the activity of the White Collar complex.
Assuntos
Relógios Circadianos/genética , Ritmo Circadiano/genética , Espécies Reativas de Oxigênio/metabolismo , Superóxidos/metabolismo , Relógios Circadianos/fisiologia , Ritmo Circadiano/fisiologia , Regulação Fúngica da Expressão Gênica , Luz , Neurospora crassa/genética , Neurospora crassa/metabolismo , Neurospora crassa/fisiologia , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismoRESUMO
Serious efforts have been made to develop anxiolytics with improved clinical utility and reduced side effects. 5-Hydroxytryptamine (5-HT)(2C) receptor antagonists are potential anxiolytics; however, their effects on vigilance are not well characterized. To compare the effects of benzodiazepines and subtype-selective 5-HT(2C) receptor antagonists on anxiety, vigilance, and electroencephalogram (EEG) power density, social interaction test and polygraphic recordings were performed in male Sprague-Dawley rats after chlordiazepoxide (CDP; 4.0 mg/kg i.p.) and SB-242084 (6-chloro-5-methyl-1-[2-(2-methylpyrid-3-yloxy)-pyrid-5-yl carbamoyl] indoline) (0.1, 0.3, and 1.0 mg/kg i.p.) treatment. CDP and SB-242084 (0.3 and 1.0 mg/kg) had similar anxiolytic effects. Spectral analysis of EEG in wakefulness (W) and paradoxical sleep (PS) showed an opposite effect on activity (5-9 Hz); it decreased after CDP, whereas it increased after SB-242084 (even at 0.1 mg/kg). In addition, CDP significantly decreased slow-wave activity (0.5-4 Hz) in deep slow-wave sleep (SWS-2) and increased power at frequencies above 12 Hz mainly in W and PS. A markedly increased intermediate stage of sleep was also found after CDP treatment. At the highest dose, SB-242084 increased W and decreased SWS-2. In summary, low but potent anxiolytic doses of the subtype-selective 5-HT(2C) receptor antagonist SB-242084 did not affect vigilance states but caused an increased activity in W, raising the possibility of a cognitive-enhancing effect of the drug. In contrast, acute CDP administration, based on spectral analysis of the EEG, produced a more superficial sleep along with a decreased activity.