RESUMEN
The human ether-a-go-go-related gene (hERG) K+ channel conducts a rapidly activating delayed rectifier K+ current (IKr), which is essential for normal electrical activity of the heart. Precise regulation of hERG channel biogenesis is critical for serving its physiological functions, and deviations from the regulation result in human diseases. However, the mechanism underlying the precise regulation of hERG channel biogenesis remains elusive. Here, by using forward genetic screen, we found that PATR-1, the Caenorhabditis elegans homolog of the yeast DNA topoisomerase 2-associated protein PAT1, is a critical regulator for the biogenesis of UNC-103, the ERG K+ channel in C. elegans. A loss-of-function mutation in patr-1 down-regulates the expression level of UNC-103 proteins and suppresses the phenotypic defects resulted from a gain-of-function mutation in the unc-103 gene. Furthermore, downregulation of PATL1 and PATL2, the human homologs of PAT1, decreases protein levels and the current density of native hERG channels in SH-SY5Y cells and human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Knockdown of PATL1 and PATL2 elongates the duration of action potentials in hiPSC-CMs, suggesting that PATL1 and PATL2 affect the function of hERG channels and hence electrophysiological characteristics in the human heart. Further studies found that PATL1 and PATL2 interact with TFIIE, a general transcription factor required for forming the RNA polymerase II preinitiation complex, and dual-luciferase reporter assays indicated that PATL1 and PATL2 facilitate the transcription of hERG mRNAs. Together, our study discovers that evolutionarily conserved DNA topoisomerase 2-associated proteins regulate the biogenesis of hERG channels via a transcriptional mechanism.
Asunto(s)
Canales de Potasio Éter-A-Go-Go , Neuroblastoma , Animales , Humanos , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , ADN-Topoisomerasas de Tipo II/genética , ADN-Topoisomerasas de Tipo II/metabolismo , Canal de Potasio ERG1/genética , Canal de Potasio ERG1/metabolismo , Canales de Potasio Éter-A-Go-Go/genética , Canales de Potasio Éter-A-Go-Go/metabolismo , Miocitos Cardíacos/metabolismo , Neuroblastoma/metabolismo , Proteínas de Unión al ADN/metabolismoRESUMEN
Aging is a major risk factor for many human diseases, including cognitive impairment, which affects a large population of the elderly. In the past few decades, our understanding of the molecular and cellular mechanisms underlying the changes associated with aging and age-related diseases has expanded greatly, shedding light on the potential role of these changes in cognitive impairment. In this article, we review recent advances in understanding of the mechanisms underlying brain aging under normal and pathological conditions, compare their similarities and differences, discuss the causative and adaptive mechanisms of brain aging, and finally attempt to find some rules to guide us on how to promote healthy aging and prevent age-related diseases.
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Envejecimiento , Disfunción Cognitiva , Humanos , Anciano , Envejecimiento/patología , Encéfalo , Factores de RiesgoRESUMEN
The aims of the present study were to investigate the anti-inflammatory function of two flavonoids apigenin and genistein in rat intestinal epithelial (IEC-6) cells stimulated by tumor necrosis factor-alpha (TNF-α) and to clarify whether the heat treatment of the flavonoids might affect flavonoid activity. The flavonoids at lower dosage (e.g. 5 µmol/L) had no toxic effect but growth promotion on the cells. Meanwhile, the flavonoid pretreatment of the cells before TNF-α stimulation could maintain cellular morphology, decrease the production of prostaglandin E2 and two pro-inflammatory cytokines interleukin-1ß (IL-1ß) and IL-6, but increase the production of two anti-inflammatory cytokines IL-10 and transforming growth factor-ß. Additionally, the flavonoids could block off the nuclear translocation of nuclear factor-kappaB (NF-κB) p65, and suppress the expression of phosphorylated IκBα and p65 induced by TNF-α. Meanwhile, the NF-κB inhibitor BAY 11-7082 shared a similar function with the flavonoids to mediate the production of IL-6/IL-10. Furthermore, in silico analysis also declared that the flavonoids could interact with the IκBα-NF-κB complex at the binding pockets to yield the binding energies ranging from -31.7 to -34.0 kJ/mol. However, the heated flavonoids were consistently less effective than the unheated counterparts to perform these anti-inflammatory effects. It is thus proposed that both apigenin and genistein have anti-inflammatory potential to the TNF-α-stimulated IEC-6 cells by inactivating the NF-κB pathway, while heat treatment of the flavonoids caused a negative impact on these assessed anti-inflammatory effects.
RESUMEN
Flavonols possess several beneficial bioactivities in vitro and in vivo. In this study, two flavonols galangin and quercetin with or without heat treatment (100 °C for 15-30 min) were assessed for their anti-inflammatory activities in lipopolysaccharide (LPS)-stimulated rat intestinal epithelial (IEC-6) cells and whether the heat treatment caused activity changes. The flavonol dosages of 2.5-20 µmol/L had no cytotoxicity on the cells but could enhance cell viability (especially using 5 µmol/L flavonol dosage). The flavonols could decrease the production of prostaglandin E2 and three pro-inflammatory cytokines interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-α, and simultaneously promote the production of two anti-inflammatory cytokines IL-10 and transforming growth factor-ß. The Western-blot results verified that the flavonols could suppress the LPS-induced expression of TLR4 and phosphorylated IκBα and p65, while the molecular docking results also illustrated that the flavonols could bind with TLR4 and NF-κB to yield energy decreases of -(21.9-28.6) kJ/mol. Furthermore, an inhibitor BAY 11-7082 blocked the NF-κB signaling pathway by inhibiting the expression of phosphorylated IκBα/p65 and thus mediated the production of IL-6/IL-10 as the flavonols did, which confirmed the assessed anti-inflammatory effect of the flavonols. Consistently, galangin had higher anti-inflammatory activity than quercetin, while the heated flavonols (especially those with longer heat time) were less active than the unheated counterparts to exert these target anti-inflammatory effects. It is highlighted that the flavonols could antagonize the LPS-caused IEC-6 cells inflammation via suppressing TLR4/NF-κB activation, but heat treatment of the flavonols led to reduced anti-inflammatory efficacy.
Asunto(s)
Antiinflamatorios/farmacología , Células Epiteliales/efectos de los fármacos , Flavonoides/farmacología , Calor , Mucosa Intestinal/efectos de los fármacos , Quercetina/farmacología , Animales , Antiinflamatorios/química , Línea Celular , Supervivencia Celular/efectos de los fármacos , Citocinas/antagonistas & inhibidores , Citocinas/biosíntesis , Dinoprostona/antagonistas & inhibidores , Dinoprostona/biosíntesis , Células Epiteliales/metabolismo , Flavonoides/química , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/farmacología , Simulación del Acoplamiento Molecular , Estructura Molecular , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Quercetina/química , Ratas , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 4/antagonistas & inhibidores , Receptor Toll-Like 4/metabolismoRESUMEN
With the increase of life expectancy, the world's population is aging rapidly. Previous work in the field of aging greatly increases our understanding of biological mechanisms underlying longevity. Researchers have unraveled a number of longevity pathways conserved from yeast to mammals. However, recent evidence shows that mechanisms regulating the life span and those regulating age-related behavioral decline could be dissociated. The regulatory mechanisms underlying behavioral and cognitive aging is largely unknown. Previous work has described a significant age-related decline in cognitive behaviors including episodic memory, working memory, processing speed, as well as motor function deterioration and circadian dysfunction. With the advance of neuroscience and technology, more and more studies have focused on the age-related changes in structure and function of the brain. In this review, we briefly describe the deterioration of cognitive function and other behaviors in the aging process, and survey the role of age-related changes in brain structure and network, neuron morphology and function, transcriptome in brain and some conserved biological pathways on age-related cognitive and behavioral decline. Further studies on the mechanisms underpinning age-related cognitive and behavioral decline may provide clues not only for improving the quality of life for the ageing population, but also for developing intervention approaches for neurodegenerative diseases.
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Envejecimiento Cognitivo , Envejecimiento/genética , Animales , Cognición , Longevidad , Calidad de VidaRESUMEN
The enzyme cyclic GMP-AMP synthase (cGAS) senses cytosolic DNA in infected and malignant cells and catalyzes the formation of 2'3'cGMP-AMP (cGAMP), which in turn triggers interferon (IFN) production via the STING pathway. Here, we examined the contribution of anion channels to cGAMP transfer and anti-viral defense. A candidate screen revealed that inhibition of volume-regulated anion channels (VRACs) increased propagation of the DNA virus HSV-1 but not the RNA virus VSV. Chemical blockade or genetic ablation of LRRC8A/SWELL1, a VRAC subunit, resulted in defective IFN responses to HSV-1. Biochemical and electrophysiological analyses revealed that LRRC8A/LRRC8E-containing VRACs transport cGAMP and cyclic dinucleotides across the plasma membrane. Enhancing VRAC activity by hypotonic cell swelling, cisplatin, GTPγS, or the cytokines TNF or interleukin-1 increased STING-dependent IFN response to extracellular but not intracellular cGAMP. Lrrc8e-/- mice exhibited impaired IFN responses and compromised immunity to HSV-1. Our findings suggest that cell-to-cell transmission of cGAMP via LRRC8/VRAC channels is central to effective anti-viral immunity.
Asunto(s)
Fibroblastos/inmunología , Interferones/inmunología , Proteínas de la Membrana/inmunología , Nucleótidos Cíclicos/inmunología , Canales Aniónicos Dependientes del Voltaje/inmunología , Animales , Antivirales/inmunología , Antivirales/metabolismo , Efecto Espectador , Línea Celular , Células Cultivadas , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Células HeLa , Herpes Simple/inmunología , Herpes Simple/virología , Herpesvirus Humano 1/inmunología , Herpesvirus Humano 1/fisiología , Humanos , Interferones/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Nucleótidos Cíclicos/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/inmunología , Nucleotidiltransferasas/metabolismo , Canales Aniónicos Dependientes del Voltaje/metabolismoRESUMEN
It has long been assumed that lifespan and healthspan correlate strongly, yet the two can be clearly dissociated1-6. Although there has been a global increase in human life expectancy, increasing longevity is rarely accompanied by an extended healthspan4,7. Thus, understanding the origin of healthy behaviours in old people remains an important and challenging task. Here we report a conserved epigenetic mechanism underlying healthy ageing. Through genome-wide RNA-interference-based screening of genes that regulate behavioural deterioration in ageing Caenorhabditis elegans, we identify 59 genes as potential modulators of the rate of age-related behavioural deterioration. Among these modulators, we found that a neuronal epigenetic reader, BAZ-2, and a neuronal histone 3 lysine 9 methyltransferase, SET-6, accelerate behavioural deterioration in C. elegans by reducing mitochondrial function, repressing the expression of nuclear-encoded mitochondrial proteins. This mechanism is conserved in cultured mouse neurons and human cells. Examination of human databases8,9 shows that expression of the human orthologues of these C. elegans regulators, BAZ2B and EHMT1, in the frontal cortex increases with age and correlates positively with the progression of Alzheimer's disease. Furthermore, ablation of Baz2b, the mouse orthologue of BAZ-2, attenuates age-dependent body-weight gain and prevents cognitive decline in ageing mice. Thus our genome-wide RNA-interference screen in C. elegans has unravelled conserved epigenetic negative regulators of ageing, suggesting possible ways to achieve healthy ageing.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Epigénesis Genética , Envejecimiento Saludable/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Factores Generales de Transcripción/metabolismo , Envejecimiento/genética , Animales , Proteínas de Caenorhabditis elegans/genética , Cognición , Disfunción Cognitiva , N-Metiltransferasa de Histona-Lisina/deficiencia , N-Metiltransferasa de Histona-Lisina/genética , Histonas/química , Histonas/metabolismo , Humanos , Longevidad/genética , Lisina/metabolismo , Masculino , Memoria , Metilación , Ratones , Mitocondrias/metabolismo , Neuronas/metabolismo , Proteínas/genética , Interferencia de ARN , Aprendizaje Espacial , Factores Generales de Transcripción/deficiencia , Factores Generales de Transcripción/genéticaRESUMEN
Ion channels are important therapeutic targets, but the discovery of ion channel drugs remains challenging due to a lack of assays that allow high-throughput screening in the physiological context. Here we report C. elegans phenotype-based methods for screening ion channel drugs. Expression of modified human ether-a-go-go-related gene (hERG) potassium channels in C. elegans results in egg-laying and locomotive defects, which offer indicators for screening small-molecule channel modulators. Screening in worms expressing hERGA561V, which carries a trafficking-defective mutation A561V known to associate with long-QT syndrome, identifies two functional correctors Prostratin and ingenol-3,20-dibenzoate. These compounds activate PKCε signaling and consequently phosphorylate S606 at the pore region of the channel to promote hERGA561V trafficking to the plasma membrane. Importantly, the compounds correct electrophysiological abnormalities in hiPSC-derived cardiomyocytes bearing a heterozygous CRISPR/Cas9-edited hERGA561V. Thus, we have developed an in vivo high-throughput method for screening compounds that have therapeutic potential in treating channelopathies.
Asunto(s)
Canalopatías/genética , Canales de Potasio Éter-A-Go-Go/genética , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans , Canalopatías/tratamiento farmacológico , Canalopatías/metabolismo , Modelos Animales de Enfermedad , Diterpenos/farmacología , Diterpenos/uso terapéutico , Evaluación Preclínica de Medicamentos , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Canales de Potasio Éter-A-Go-Go/metabolismo , Humanos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Ésteres del Forbol/farmacología , Ésteres del Forbol/uso terapéutico , Proteína Quinasa C/metabolismo , Triterpenos/farmacología , Triterpenos/uso terapéuticoRESUMEN
The human ether-a-go-go-related gene (hERG) encodes a voltage-gated potassium channel that controls repolarization of cardiac action potentials. Accumulating evidence suggests that most disease-related hERG mutations reduce the function of the channel by disrupting protein biogenesis of the channel in the endoplasmic reticulum (ER). However, the molecular mechanism underlying the biogenesis of ERG K+ channels is largely unknown. By forward genetic screening, we identified an ER-located chaperone CNX-1, the worm homologue of mammalian chaperone Calnexin, as a critical regulator for the protein biogenesis of UNC-103, the ERG-type K+ channel in Caenorhabditis elegans Loss-of-function mutations of cnx-1 decreased the protein level and current density of the UNC-103 K+ channel and suppressed the behavioral defects caused by a gain-of-function mutation in unc-103 Moreover, CNX-1 facilitated tetrameric assembly of UNC-103 channel subunits in a liposome-assisted cell-free translation system. Further studies showed that CNX-1 act in parallel to DNJ-1, another ER-located chaperone known to regulate maturation of UNC-103 channels, on controlling the protein biogenesis of UNC-103. Importantly, Calnexin interacted with hERG proteins in the ER in HEK293T cells. Deletion of calnexin reduced the expression and current densities of endogenous hERG K+ channels in SH-SY5Y cells. Collectively, we reveal an evolutionarily conserved chaperone CNX-1/Calnexin controlling the biogenesis of ERG-type K+ channels.
Asunto(s)
Proteínas de Caenorhabditis elegans/fisiología , Proteínas de Unión al Calcio/fisiología , Retículo Endoplásmico/metabolismo , Canales de Potasio Éter-A-Go-Go/biosíntesis , Proteínas del Choque Térmico HSP40/fisiología , Canales de Potasio/biosíntesis , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas de Unión al Calcio/genética , Calnexina/metabolismo , Células HEK293 , Proteínas del Choque Térmico HSP40/genética , Humanos , RatonesRESUMEN
The rate of behavioural decline in the ageing population is remarkably variable among individuals. Despite the considerable interest in studying natural variation in ageing rate to identify factors that control healthy ageing, no such factor has yet been found. Here we report a genetic basis for variation in ageing rates in Caenorhabditis elegans. We find that C. elegans isolates show diverse lifespan and age-related declines in virility, pharyngeal pumping, and locomotion. DNA polymorphisms in a novel peptide-coding gene, named regulatory-gene-for-behavioural-ageing-1 (rgba-1), and the neuropeptide receptor gene npr-28 influence the rate of age-related decline of worm mating behaviour; these two genes might have been subjected to recent selective sweeps. Glia-derived RGBA-1 activates NPR-28 signalling, which acts in serotonergic and dopaminergic neurons to accelerate behavioural deterioration. This signalling involves the SIR-2.1-dependent activation of the mitochondrial unfolded protein response, a pathway that modulates ageing. Thus, natural variation in neuropeptide-mediated glia-neuron signalling modulates the rate of ageing in C. elegans.
Asunto(s)
Envejecimiento/genética , Envejecimiento/fisiología , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiología , Variación Genética , Neuroglía/metabolismo , Neuronas/metabolismo , Transducción de Señal/genética , Alelos , Animales , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Neuronas Dopaminérgicas/metabolismo , Femenino , Genética de Población , Locomoción/genética , Locomoción/fisiología , Longevidad/genética , Longevidad/fisiología , Masculino , Faringe/fisiología , Polimorfismo de Nucleótido Simple/genética , Receptores Acoplados a Proteínas G/metabolismo , Neuronas Serotoninérgicas/metabolismo , Conducta Sexual Animal/fisiología , Sirtuinas/metabolismo , Respuesta de Proteína Desplegada/genética , Respuesta de Proteína Desplegada/fisiologíaRESUMEN
Tetrameric assembly of channel subunits in the endoplasmic reticulum (ER) is essential for surface expression and function of K+ channels, but the molecular mechanism underlying this process remains unclear. In this study, we found through genetic screening that ER-located J-domain-containing chaperone proteins (J-proteins) are critical for the biogenesis and physiological function of ether-a-go-go-related gene (ERG) K+ channels in both Caenorhabditis elegans and human cells. Human J-proteins DNAJB12 and DNAJB14 promoted tetrameric assembly of ERG (and Kv4.2) K+ channel subunits through a heat shock protein (HSP) 70-independent mechanism, whereas a mutated DNAJB12 that did not undergo oligomerization itself failed to assemble ERG channel subunits into tetramers in vitro and in C. elegans. Overexpressing DNAJB14 significantly rescued the defective function of human ether-a-go-go-related gene (hERG) mutant channels associated with long QT syndrome (LQTS), a condition that predisposes to life-threatening arrhythmia, by stabilizing the mutated proteins. Thus, chaperone proteins are required for subunit stability and assembly of K+ channels.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Canal de Potasio ERG1/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas del Choque Térmico HSP47/metabolismo , Canales de Potasio/metabolismo , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Línea Celular Tumoral , Canal de Potasio ERG1/química , Canal de Potasio ERG1/genética , Células HEK293 , Proteínas del Choque Térmico HSP40/química , Proteínas del Choque Térmico HSP40/genética , Proteínas del Choque Térmico HSP47/química , Proteínas del Choque Térmico HSP47/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Síndrome de QT Prolongado/genética , Síndrome de QT Prolongado/metabolismo , Potenciales de la Membrana , Chaperonas Moleculares , Mutación , Miocitos Cardíacos/metabolismo , Canales de Potasio/química , Canales de Potasio/genética , Multimerización de Proteína , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Interferencia de ARN , Canales de Potasio Shal/genética , Canales de Potasio Shal/metabolismo , Factores de Tiempo , TransfecciónRESUMEN
Voltage-gated Kv4 channels control the excitability of neurons and cardiac myocytes by conducting rapidly activating-inactivating currents. The function of Kv4 channels is profoundly modulated by K(+) channel interacting protein (KChIP) soluble auxiliary subunits. However, the in vivo mechanism of the modulation is not fully understood. Here, we identified three C. elegans KChIP-like (ceKChIP) proteins, NCS-4, NCS-5, and NCS-7. All three ceKChIPs alter electrical characteristics of SHL-1, a C. elegans Kv4 channel ortholog, currents by slowing down inactivation kinetics and shifting voltage dependence of activation to more hyperpolarizing potentials. Native SHL-1 current is completely abolished in cultured myocytes of Triple KO worms in which all three ceKChIP genes are deleted. Reexpression of NCS-4 partially restored expression of functional SHL-1 channels, whereas NCS-4(efm), a NCS-4 mutant with impaired Ca(2+)-binding ability, only enhanced expression of SHL-1 proteins, but failed to transport them from the Golgi apparatus to the cell membrane in body wall muscles of Triple KO worms. Moreover, translational reporter revealed that NCS-4 assembles with SHL-1 K(+) channels in male diagonal muscles. Deletion of either ncs-4 or shl-1 significantly impairs male turning, a behavior controlled by diagonal muscles during mating. The phenotype of the ncs-4 null mutant could be rescued by reexpression of NCS-4, but not NCS-4(efm), further emphasizing the importance of Ca(2+) binding to ceKChIPs in regulating native SHL-1 channel function. Together, these data reveal an evolutionarily conserved mechanism underlying the regulation of Kv4 channels by KChIPs and unravel critical roles of ceKChIPs in regulating muscle cell excitability and animal behavior in C. elegans.
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Potenciales de Acción , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Interacción con los Canales Kv/metabolismo , Locomoción , Proteínas Sensoras del Calcio Neuronal/metabolismo , Canales de Potasio Shal/metabolismo , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiología , Proteínas de Caenorhabditis elegans/genética , Calcio/metabolismo , Células Cultivadas , Femenino , Aparato de Golgi/metabolismo , Proteínas de Interacción con los Canales Kv/genética , Masculino , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/fisiología , Proteínas Sensoras del Calcio Neuronal/genética , Unión Proteica , Transporte de Proteínas , Conducta Sexual Animal , Canales de Potasio Shal/genéticaRESUMEN
Chloride channels belong to a superfamily of ion channels that permit passive passage of anions, mainly chloride, across cell membrane. They play a variety of important physiological roles in regulation of cytosolic pH, cell volume homeostasis, organic solute transport, cell migration, cell proliferation, and differentiation. However, little is known about the functional regulation of these channels. In this study, we generated an integrated transgenic worm strain expressing green fluorescence protein (GFP) fused CLC-type chloride channel 1 (CLH-1::GFP), a voltage-gated chloride channel in Caenorhabditis elegans (C. elegans). CLH-1::GFP was expressed in some unidentified head neurons and posterior intestinal cells of C. elegans. Interacting proteins of CLH-1::GFP were purified by GFP-Trap, a novel system for efficient isolation of GFP fusion proteins and their interacting factors. Mass spectrometry (MS) analysis revealed that a total of 27 high probability interacting proteins were co-trapped with CLHp-1::GFP. Biochemical evidence showed that eukaryotic translation elongation factor 1 (EEF-1), one of these co-trapped proteins identified by MS, physically interacted with CLH-1, in consistent with GFP-Trap experiments. Further immunostaining data revealed that the protein level of CLH-1 was significantly increased upon co-expression with EEF-1. These results suggest that the combination of GFP-Trap purification with MS is an excellent tool to identify novel interacting proteins of voltage-gated chloride channels in C. elegans. Our data also show that EEF-1 is a regulator of voltage-gated chloride channel CLH-1.
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Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Canales de Cloruro/metabolismo , Factor 1 de Elongación Peptídica/metabolismo , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/genética , Proteínas Fluorescentes Verdes/química , Espectrometría de MasasRESUMEN
Aging is accompanied with behavioral and cognitive decline. Changes in the neurotransmitter level are associated with the age-related behavioral deterioration, but whether well-known longevity manipulations affect the function of neurotransmitter system in aging animals is largely unclear. Here we report that serotonin (5-HT) and dopamine (DA) level decrease with age in C. elegans. The reduction results in downregulation of the activity of neurons controlled by 5-HT/DA signaling, and deterioration of some important behaviors, including pharyngeal pumping, food-induced slowing responses, and male mating. Longevity manipulations differentially affect the age-related decline in neuronal level of 5-HT/DA. The reduction and resultant behavioral deterioration occur in long-lived worms with defective insulin signaling [daf-2(e1370), age-1(hx546)] or mitochondria function [isp-1(qm150), tpk-1(qm162)], but not in long-lived worms with dietary restriction eat-2(ad1116). A reduced expression level of dopa decarboxylase BAS-1, the shared enzyme for 5-HT/DA synthesis, is responsible for the decline in 5-HT/DA levels. RNAi assay revealed that the sustained 5-HT/DA level in neurons of aged eat-2(ad1116) worms requires PHA-4 and its effectors superoxide dismutases and catalases, suggesting the involvement of reactive oxygen species in the 5-HT/DA decline. Furthermore, we found that elevating 5-HT/DA ameliorates age-related deterioration of pharyngeal pumping, food-induced slowing responses, and male mating in both wild-type and daf-2(e1370) worms. Together, dietary restriction preserves healthy behaviors in aged worms at least partially by sustaining a high 5-HT/DA level, and elevating the 5-HT/DA level in wild-type and daf-2(e1370) worms improves their behaviors during aging.
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Envejecimiento/metabolismo , Caenorhabditis elegans/fisiología , Dopamina/metabolismo , Neuronas/metabolismo , Serotonina/metabolismo , Envejecimiento/fisiología , Animales , Conducta Animal/fisiología , Proteínas de Caenorhabditis elegans/metabolismo , Dopamina/deficiencia , Organismos Hermafroditas , Longevidad/fisiología , Masculino , Modelos Animales , Fenómenos Fisiológicos del Sistema Nervioso/fisiología , Neuronas/fisiología , Estrés Oxidativo/fisiología , Serotonina/deficiencia , Conducta Sexual Animal/fisiología , Transducción de Señal/fisiologíaRESUMEN
A wealth of evidence underscores the tight link between oxidative stress, neurodegeneration and aging. When the level of excess reactive oxygen species (ROS) increases in the cell, a phenomenon characteristic of aging, DNA is damaged, proteins are oxidized, lipids are degraded and more ROS are produced, all culminating in significant cell injury. Recently we showed that in the nematode, Caenorhabditis elegans, oxidation of K(+) channels by ROS is a major mechanism underlying the loss of neuronal function. The C. elegans results support an argument that K(+) channels controlling neuronal excitability and survival might provide a common, functionally important substrate for ROS in aging mammals. Here we discuss the implications that oxidation of K(+) channels by ROS might have for the mammalian brain during normal aging, as well as in neurodegenerative diseases such as Alzheimer's and Parkinson's. We argue that oxidation of K(+) channels by ROS is a common theme in the aging brain and suggest directions for future experimentation.
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Envejecimiento , Degeneración Nerviosa/metabolismo , Canales de Potasio/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Humanos , Oxidación-ReducciónRESUMEN
A central effort in biomedical research concerns the development of materials for sustaining and controlling cell growth. Carbon nanotube based substrates have been shown to support the growth of different kinds of cells (Hu et al 2004 Nano Lett. 4 507-11; Kalbacova et al 2006 Phys. Status Solidi b 13 243; Zanello et al 2006 Nano Lett. 6 562-7); however the underlying molecular mechanisms remain poorly defined. To address the fundamental question of mechanisms by which nanotubes promote bone mitosis and histogenesis, primary calvariae osteoblastic cells were grown on single-walled carbon nanotube thin film (SWNT) substrates. Using a combination of biochemical and optical techniques we demonstrate here that SWNT networks promote cell development through two distinct steps. Initially, SWNTs are absorbed in a process that resembles endocytosis, inducing acute toxicity. Nanotube-mediated cell destruction, however, induces a release of endogenous factors that act to boost the activity of the surviving cells by stimulating the synthesis of extracellular matrix.
Asunto(s)
Proteínas de la Matriz Extracelular/metabolismo , Matriz Extracelular/metabolismo , Nanotubos de Carbono , Osteoblastos/metabolismo , Fosfatasa Alcalina/metabolismo , Análisis de Varianza , Animales , Técnicas de Cultivo de Célula , Muerte Celular , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Cuerpos de Inclusión/metabolismo , Cuerpos de Inclusión/ultraestructura , Ratones , Microscopía Electrónica de Rastreo , Nanotubos de Carbono/toxicidad , Nanotubos de Carbono/ultraestructura , Osteoblastos/ultraestructura , RatasRESUMEN
Here, we characterize a new K(+) channel-kinase complex that operates in the metazoan Caenorhabditis elegans to control learning behaviour. This channel is composed of a pore-forming subunit, dubbed KHT-1 (73% homology to human Kv3.1), and the accessory subunit MPS-1, which shows kinase activity. Genetic, biochemical and electrophysiological evidence show that KHT-1 and MPS-1 form a complex in vitro and in native mechanosensory PLM neurons, and that KHT-1 is a substrate for the kinase activity of MPS-1. Behavioural analysis further shows that the kinase activity of MPS-1 is specifically required for habituation to repetitive mechanical stimulation. Thus, worms bearing an inactive MPS-1 variant (D178N) respond normally to touch on the body but do not habituate to repetitive mechanical stimulation such as tapping on the side of the Petri dish. Hence, the phosphorylation status of KHT-1-MPS-1 seems to be linked to distinct behavioural responses. In the non-phosphorylated state the channel is necessary for the normal function of the touch neurons. In the auto-phosphorylated state the channel acts to induce neuronal adaptation to mechanical stimulation. Taken together, these data establish a new mechanism of dynamic regulation of electrical signalling in the nervous system.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiología , Aprendizaje/fisiología , Canales de Potasio con Entrada de Voltaje/metabolismo , Canales de Potasio/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Animales , Proteínas de Caenorhabditis elegans/genética , Datos de Secuencia Molecular , Mutación Missense , Neuronas/química , Canales de Potasio/genética , Canales de Potasio con Entrada de Voltaje/genética , Unión Proteica , Homología de Secuencia de AminoácidoRESUMEN
Potassium channels are key regulators of neuronal excitability. Here we show that oxidation of the K(+) channel KVS-1 during aging causes sensory function loss in Caenorhabditis elegans and that protection of this channel from oxidation preserves neuronal function. Chemotaxis, a function controlled by KVS-1, was significantly impaired in worms exposed to oxidizing agents, but only moderately affected in worms harboring an oxidation-resistant KVS-1 mutant (C113S). In aging C113S transgenic worms, the effects of free radical accumulation were significantly attenuated compared to those in wild type. Electrophysiological analyses showed that both reactive oxygen species (ROS) accumulation during aging and acute exposure to oxidizing agents acted primarily to alter the excitability of the neurons that mediate chemotaxis. Together, these findings establish a pivotal role for ROS-mediated oxidation of voltage-gated K(+) channels in sensorial decline during aging in invertebrates.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Sistema Nervioso/metabolismo , Estrés Oxidativo/fisiología , Canales de Potasio con Entrada de Voltaje/metabolismo , Trastornos de la Sensación/metabolismo , Potenciales de Acción/fisiología , Animales , Animales Modificados Genéticamente , Células CHO , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Quimiotaxis/fisiología , Cricetinae , Cricetulus , Radicales Libres/metabolismo , Sistema Nervioso/citología , Canales de Potasio con Entrada de Voltaje/genética , Especies Reactivas de Oxígeno/metabolismo , Trastornos de la Sensación/genética , Trastornos de la Sensación/fisiopatología , Células Receptoras Sensoriales/metabolismoRESUMEN
Voltage-dependent potassium (K+) channels (Kv) regulate cell excitability by controlling the movement of K+ ions across the membrane in response to changes in the cell voltage. The Kv family, which includes A-type channels, constitute the largest group of K+ channel genes within the superfamily of Na+, Ca2+ and K+ voltage-gated channels. The name "A-type" stems from the typical profile of these currents that results form the opposing effects of fast activation and inactivation. In neuronal cells, A-type currents (I(A)), determine the interval between two consecutive action potentials during repetitive firing. In cardiac muscle, A-type currents (I(to)), control the initial repolarization of the myocardium. Structurally, A-type channels are tetramers of alpha-subunits each containing six putative transmembrane domains including a voltage-sensor. A-type channels can be modulated by means of protein-protein interactions with so-called beta-subunits that control inactivation voltage sensitivity and other properties, and by post-transcriptional modifications such as phosphorylation or oxidation. Recently a new mode of A-type regulation has been discovered in the form of a class of hybrid beta-subunits that posses their own enzymatic activity. Here, we review the biophysical and physiological properties of these multiple modes of A-type channel regulation.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Canales de Potasio con Entrada de Voltaje/metabolismo , Subunidades de Proteína/metabolismo , Potenciales de Acción/fisiología , Animales , Humanos , Potasio/metabolismo , Canales de Potasio con Entrada de Voltaje/genética , Subunidades de Proteína/genética , Procesamiento Postranscripcional del ARN/fisiologíaRESUMEN
We report on the role of K+ currents in the mechanisms regulating the proliferation of UMR 106-01 osteoblastic osteosarcoma cells. Electrophysiological analysis showed that UMR 106-01 cells produce robust K+ currents that can be pharmacologically separated into two major components: a E-4031-susceptible current, I E-4031, and a tetraethylammonium (TEA)-susceptible component, I TEA. Western blot and RT-PCR analysis showed that I E-4031 is produced by ether a go-go (eag)-related channels (ERG). Incubation of the cells with E-4031 enhanced their proliferation by 80%. Application of E-4031 in the bath solution did not induce instantaneous changes in the membrane resting potential or in the level of cytosolic calcium; however, the cells were slightly depolarized and the calcium content was significantly increased upon prolonged incubation with the compound. Taken together these findings indicate that ERG channels can impair cell proliferation. This is a novel finding that underscores new modes of regulation of mitosis by voltage-gated K+ channels and provides an unexpected insight into the current view of the mechanisms governing bone tissue proliferation.