RESUMEN
The cellular implications of the interaction between Pannexin-1 (Panx1) channel and P2X7 receptor (P2X7R) have not been fully elucidated. Evidence suggests that ATP, released through Panx1, activates P2X7R, which in turn promotes further activation of Panx1. In a previous study, we reported that the C-terminus of Panx1 (Panx1-CT) attenuates P2X7R-mediated Ca2+ influx and cell death. One of the distinctive features of P2X7R is the gradual increase in current with repetitive stimulation. In the current study, we report an effect of Panx1-CT (amino acid residues 350 to 426) on P2X7R current, which differs from the effect of full-length Panx1. Panx1-CT inhibited P2X7R current, which persisted in all consecutive agonist applications. However, full-length Panx1 reduced P2X7R current at initial stimulations, followed by gradual augmentation. When P2X7R was activated for an extended period, cells expressing Panx1-CT exhibited less mitochondrial depolarization, reactive oxygen species (ROS) generation, Caspase 3 activation and cell death, whereas cells overexpressing full-length Panx1 showed the opposite effect. Taken together, these findings suggest that Panx1 can either attenuate or augment P2X7R-mediated cellular processes depending on the degree of P2X7R activation.
Asunto(s)
Conexinas , Receptores Purinérgicos P2X7 , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/metabolismo , Conexinas/genética , Conexinas/metabolismo , Transducción de Señal , Muerte Celular , Adenosina Trifosfato/metabolismoRESUMEN
Many neurodegenerative diseases, such as Alzheimer's disease (AD) and frontotemporal dementia with Parkinsonism linked to chromosome 17, are characterized by tau pathology. Numerous motor proteins, many of which are involved in synaptic transmission, mediate transport in neurons. Dysfunction in motor protein-mediated neuronal transport mechanisms occurs in several neurodegenerative disorders but remains understudied in AD. Kinesins are the most important molecular motor proteins required for microtubule-dependent transport in neurons, and kinesin-1 is crucial for neuronal transport among all kinesins. Although kinesin-1 is required for normal neuronal functions, the dysfunction of these motor domains leading to neurodegenerative diseases is not fully understood. Here, we reported that the kinesin-I heavy chain (KIF5B), a key molecular motor protein, is involved in tau homeostasis in AD cells and animal models. We found that the levels of KIF5B in P301S tau mice are high. We also found that the knockdown and knockout (KO) of KIFf5B significantly decreased the tau stability, and overexpression of KIF5B in KIF5B-KO cells significantly increased the expression of phosphorylated and total tau levels. This suggested that KIF5B might prevent tau accumulation. By conducting experiments on P301S tau mice, we showed that partially reducing KIF5B levels can reduce hyperphosphorylation of the human tau protein, formation of insoluble aggregates, and memory impairment. Collectively, our results suggested that decreasing KIF5B levels is sufficient to prevent and/or slow down abnormal tau behavior of AD and other tauopathies.
RESUMEN
AIMS: High mobility group box (HMGB) family proteins, HMGB1, HMGB2, HMGB3, and HMGB4 are oncogenic. The oncogenic nature of HMGB1 is characterized by its association with autophagy, ROS, and MMP. Since HMGB3 is its paralog, we hypothesized that it might also modulate autophagy, ROS, and MMP. Hence, we targeted HMGB3 using its shRNA or miR-142-3p and assessed the changes in autophagy, ROS, MMP, and tumorigenic properties of human breast cancer cells. MAIN METHODS: Cell viability was assessed by resazurin staining and annexin-V/PI dual staining was used for confirming apoptosis. Colony formation, transwell migration, invasion and luciferase reporter (for miRNA-target validation) assays were also performed. ROS and MMP were detected using DHE and MitoTracker dyes, respectively. A zebrafish xenograft model was used to assess the role of miR-142-3p on in vivo metastatic potential of breast cancer cells. KEY FINDINGS: Breast cancer tissues from Indian patients and TCGA samples exhibit overexpression of HMGB3. miR-142-3p binds to 3' UTR of HMGB3, leading to its downregulation that subsequently inhibits colony formation and induces apoptosis involving increased ROS accumulation and decreased MMP, phospho-mTOR and STAT3. Our findings show that HMGB3 is directly involved in the miR-142-3p-mediated disruption of autophagy and induction of apoptotic cell death via modulation of LC3, cleaved PARP and Bcl-xL. In addition, miR-142-3p inhibited migration, invasion and metastatic potential of breast cancer cells. SIGNIFICANCE: Our findings highlighted the role of HMGB3, for the first time, in the modulation of autophagy and apoptosis in human breast cancer cells, and these results have therapeutic implications.
Asunto(s)
Neoplasias de la Mama , Proteína HMGB1 , Proteína HMGB3 , MicroARNs , Regiones no Traducidas 3' , Animales , Apoptosis/genética , Autofagia , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular/genética , Femenino , Proteína HMGB1/genética , Proteína HMGB3/genética , Proteína HMGB3/metabolismo , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno , Pez Cebra/genéticaRESUMEN
AIMS: High-mobility group (HMG) proteins are oncogenic in different cancers, including cervical cancer; silencing their individual expression using sh-RNAs, siRNAs, and miRNAs has had anti-tumorigenic effects, but the consequences of their collective downregulation are not known. Since multiple gene targeting is generally very effective in cancer therapy, the present study highlighted the consequences of silencing the expression of HMGA1, A2, B1, and B3 using sh-RNAs or miR-142-3p (that can potentially target HMGA1, A2, B1, and B3) in cervical cancer cell lines. MAIN METHODS: 3' UTR luciferase reporter assays were performed to validate HMGA1, A2, B1, and B3 as targets of miR-142-3p in human cervical cancer cells. Annexin V/PI dual staining and flow cytometry analyses were used to detect apoptotic cells. miR-142-3p-mediated regulation of cell death, colony formation, migration, and invasion was investigated in human cervical cancer cells together with in vivo metastasis in zebrafish. KEY FINDINGS: Concurrent knockdown of HMGA1, A2, B1, and B3 through their corresponding sh-RNAs inhibited cell viability and colony formation but induced apoptosis, and these effects were relatively reduced upon their individual knockdown. miR-142-3p targeted HMGA1, A2, B1, and B3 by binding to their 3'UTRs and induced apoptosis but inhibited proliferation, migration, and invasion of human cervical cancer cells. In addition, miR-142-3p expression decreased phospho-p65 and EMT-related proteins in cervical cancer cells and their in vivo metastatic potential upon implantation in zebrafish. SIGNIFICANCE: These findings suggest that miR-142-3p acts as a tumor-suppressive miRNA by targeting HMGA1, A2, B1, and B3 and may serve as a potential therapeutic agent in human cervical cancer.
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MicroARNs/genética , Neoplasias del Cuello Uterino/metabolismo , Animales , Carcinogénesis/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/efectos de los fármacos , Femenino , Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/genética , Proteína HMGA1a/genética , Proteína HMGA1a/metabolismo , Proteína HMGA2/genética , Proteína HMGA2/metabolismo , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Proteína HMGB3/genética , Proteína HMGB3/metabolismo , Células HeLa , Humanos , MicroARNs/metabolismo , Modelos Animales , Invasividad Neoplásica/genética , Oncogenes , Neoplasias del Cuello Uterino/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Pez CebraRESUMEN
Breast cancer cells evade cell death by overexpressing SLC7A11, which functions by transporting cystine into cells in exchange for intracellular glutamate facilitating glutathione synthesis and reducing reactive oxygen species (ROS)-mediated stress. Using an in silico approach, we predicted an miRNA (miR-5096) that can target and downregulate SLC7A11. We demonstrated SLC7A11 as a target of miR-5096 by 3'UTR luciferase assay and further validated it by identifying reduced mRNA and protein levels of SLC7A11 upon miR-5096 overexpression. miR-5096-induced ferroptotic cell death in human breast cancer cells was confirmed by concurrently increased ROS, OH-, lipid ROS, and iron accumulation levels and decreased GSH and mitochondrial membrane potential (MitoTracker™ Orange) with mitochondrial shrinkage and partial cristae loss (observed by TEM). miR-5096 inhibited colony formation, transwell migration, and breast cancer cell invasion, whereas antimiR-5096 promoted these tumorigenic properties. Ectopic expression of SLC7A11 partly reversed miR-5096-mediated effects on cell survival, ROS, lipid peroxides, iron accumulation, GSH, hydroxyl radicals, mitochondrial membrane potential, and colony formation. miR-5096 modulated the expression of epithelial-mesenchymal transition markers in vitro and inhibited the metastatic potential of MDA-MB-231 cells in a tumor xenograft model of zebrafish larvae. Our results demonstrate that miR-5096 is a tumor-suppressive miRNA in breast cancer cells, and this paper discusses its therapeutic implications.
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Sistema de Transporte de Aminoácidos y+/genética , Neoplasias de la Mama/genética , Carcinogénesis/genética , MicroARNs/genética , Animales , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Transición Epitelial-Mesenquimal/genética , Femenino , Ferroptosis/genética , Regulación Neoplásica de la Expresión Génica/genética , Glutatión/metabolismo , Xenoinjertos , Humanos , Peroxidación de Lípido/genética , Potencial de la Membrana Mitocondrial , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Pez CebraRESUMEN
Extracellular ATP works as an autocrine and/or paracrine signaling molecule by activating plasma membrane-localized purinergic receptors. Stimulation of purinergic P2X7 receptor (P2X7R) increases cytosolic Ca2+ ([Ca2+]c), which in turn activates Pannexin 1 (Panx1) channel. In earlier studies, Panx1 and P2X7R have been shown to interact physically. Also, both the channels have been implicated in similar pathophysiological processes. In this study, we investigated the effect of Panx1 on P2X7R-mediated Ca2+influx. Panx1 attenuated P2X7R-mediated [Ca2+]c rise in CHO-K1 and HEK-293 cells. [Ca2+]c rise was higher in Panx1 knockdown astrocytes. The inhibitory effect was unaffected in the presence of Panx1 blocker, carbenoxolone. The region between 350th and 386th amino acid residues in the carboxyl terminus (CT) of Panx1 was found to be crucial for inhibiting P2X7R. Like full-length Panx1, the CT (350th to 426th amino acids) alone was able to attenuate the [Ca2+]c rise. Further, CT prevented cell death caused by P2X7R overactivation. Based on our results, we propose a novel pro-survival role of Panx1 exerted by modulating P2X7R.
Asunto(s)
Conexinas , Receptores Purinérgicos P2X7 , Adenosina Trifosfato , Muerte Celular , Conexinas/genética , Conexinas/metabolismo , Células HEK293 , Humanos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismoRESUMEN
Pannexin 1 (Panx1) has been implicated in a plethora of physiological and pathophysiological processes. It is one of the major ATP release channels in many cell types. Extracellular ATP, activates purinergic P2X and P2Y receptors, triggering several signaling cascades. A disease-associated mutation, Arg-217-His (R217H) in the 3rd transmembrane domain of Panx1 attenuates channel functions through an unknown mechanism. Since carboxyl terminus (CT) gates the channel, we hypothesized that R217 interacts with the CT, and this interaction is required for optimum channel activities. R217H mutation though reduced the currents in the full-length channel, did not affect CT-truncated Panx1-Δ386. Also, compared to the wild-type, Panx1-R217H expressing cells showed lesser cell death when activated through P2X7 receptor. However, cell death in Panx1-R217H-Δ386 and Panx1-Δ386 expressing cells were similar. The mutation is ineffective unless the channel has an intact CT. Based on our results we propose that R217H mutation perturbs the conformational flexibility of CT, leading to channel dysfunction.
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Conexinas/química , Conexinas/genética , Mutación/genética , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Secuencia de Aminoácidos , Muerte Celular , Conexinas/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Humanos , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/metabolismoRESUMEN
Acid-sensing ion channels (ASICs) have been implicated in many physiological and patho-physiological processes like synaptic plasticity, inflammation, pain perception, stroke-induced brain damage and, drug-seeking behaviour. Although ASICs have been shown to be modulated by gasotransmitters like nitric oxide (NO), their regulation by hydrogen sulfide (H2S) is not known. Here, we present strong evidence that H2S potentiates ASICs-mediated currents. Low pH-induced current in Chinese hamster ovary (CHO) cells, expressing homomeric either ASIC1a, ASIC2a or ASIC3, increased significantly by an H2S donor NaHS. The effect was reversed by washing the cells with NaHS-free external solution of pH 7.4. MTSES, a membrane impermeable cysteine thiol-modifier failed to abrogate the effect of NaHS on ASIC1a, suggesting that the target cysteine residues are not in the extracellular region of the channel. The effect of NaHS is not mediated through NO, as the basal NO level in cells did not change following NaHS application. This previously unknown mechanism of ASICs-modulation by H2S adds a new dimension to the ASICs in health and disease.
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Canales Iónicos Sensibles al Ácido/metabolismo , Sulfuro de Hidrógeno/farmacología , Animales , Células CHO , Cricetulus , Concentración de Iones de Hidrógeno , Óxido Nítrico/metabolismo , Técnicas de Placa-Clamp , Agonistas de los Canales de Sodio/farmacologíaRESUMEN
Alzheimer's disease is a neurodegenerative disease that leads to irreversible neuronal damage. Senile plaques, composed of amyloid beta peptide, is the principal abnormal characteristic of the disease. Among the factors involved, the secretase enzymes, namely, α secretase, beta-site amyloid precursor protein-cleaving enzyme, ß secretase, and γ secretase, hold consequential importance. Beta-site amyloid precursor protein-cleaving enzyme 1 is considered to be the rate-limiting factor in the production of amyloid beta peptide. Research supporting the concept of inhibition of beta-site amyloid precursor protein-cleaving enzyme activity as one of the effective therapeutic targets in the mitigation of Alzheimer's disease is well accepted. The identification of natural compounds, such as ß-amyloid precursor protein-selective beta-site amyloid precursor protein-cleaving enzyme inhibitors, and the idea of compartmentalisation of the beta-site amyloid precursor protein-cleaving enzyme 1 action have caused a dire need to closely examine the natural compounds and their effectiveness in the disease mitigation. Many natural compounds have been reported to effectively modulate beta-site amyloid precursor protein-cleaving enzyme 1. At lower doses, compounds like 2,2',4'-trihydroxychalcone acid, quercetin, and myricetin have been shown to effectively reduce beta-site amyloid precursor protein-cleaving enzyme 1 activity. The currently used five drugs that are marketed and used for the management of Alzheimer's disease have an increased risk of toxicity and restricted therapeutic efficiency, hence, the search for new anti-Alzheimer's disease drugs is of primary concern. A variety of natural compounds having pure pharmacological moieties showing multitargeting activity and others exhibiting specific beta-site amyloid precursor protein-cleaving enzyme 1 inhibition as discussed below have superior biosafety. Many of these compounds, which are isolated from medicinal herbs and marine flora, have been long used for the treatment of various ailments since ancient times in the Chinese and Ayurvedic medical systems. The aim of this article is to review the available data on the selected natural compounds, giving emphasis to the inhibition of beta-site amyloid precursor protein-cleaving enzyme 1 activity as a mode of Alzheimer's disease treatment.
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Enfermedad de Alzheimer/tratamiento farmacológico , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Productos Biológicos/uso terapéutico , Alcaloides/uso terapéutico , Flavonoides/uso terapéutico , Humanos , Fenoles/uso terapéutico , FitoterapiaRESUMEN
Two-dimensional (2D) tungsten disulfide (WS2) quantum dots offer numerous promising applications in materials and optoelectronic sciences. Additionally, the catalytic and photoluminescence properties of ultra-small WS2 nanoparticles are of potential interest in biomedical sciences. Addressing the use of WS2 in the context of infection, the present study describes the conjugation of two potent antimicrobial peptides with WS2 quantum dots, as well as the application of the resulting conjugates in antimicrobial therapy and bioimaging. In doing so, we determined the three-dimensional solution structure of the quantum dot-conjugated antimicrobial peptide by a series of high-resolution nuclear magnetic resonance (NMR) techniques, correlating this to the disruption of both model lipid and bacterial membranes, and to several key biological performances, including antimicrobial and anti-biofilm effects, as well as cell toxicity. The results demonstrate that particle conjugation enhances the antimicrobial and anti-biofilm potency of these peptides, effects inferred to be due to multi-dendate interactions for the conjugated peptides. As such, our study provides information on the mode-of-action of such conjugates, laying the foundation for their potential use in treatment and monitoring of infections.
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Antiinfecciosos/farmacología , Diagnóstico por Imagen , Disulfuros/química , Péptidos/química , Puntos Cuánticos/química , Tungsteno/química , Secuencia de Aminoácidos , Biopelículas/efectos de los fármacos , Candida albicans/efectos de los fármacos , Candida albicans/ultraestructura , Espectroscopía de Resonancia Magnética , Pruebas de Sensibilidad Microbiana , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/ultraestructuraRESUMEN
Leucine-rich repeat-containing 8 (LRRC8) proteins have been proposed to evolutionarily originate from the combination of the channel protein pannexin, and a leucine-rich repeat (LRR) domain. Five paralogs of LRRC8, namely LRRC8A, LRRC8B, LRRC8C, LRRC8D and LRRC8E have been reported. LRRC8A has been shown to be instrumental in cell swelling. Here, we identify LRRC8B as a key player in the cellular Ca2+ signaling network. Overexpression of human LRRC8B in HEK293 cells reduced the Ca2+ level in the endoplasmic reticulum (ER). LRRC8B-overexpressing cells exhibited a lesser release of Ca2+ from the ER in response to ATP, carbachol and intracellular administration of inositol (1,4,5)-trisphosphate (IP3). LRRC8B-knockdown cells showed a slower depletion of the ER Ca2+ stores when sarco-endoplasmic reticulum Ca2+-ATPase was blocked with thapsigargin (TG), while overexpression of LRRC8B had the opposite effect. LRRC8B-overexpressing cells exhibited a higher level of store-operated Ca2+ entry following store-depletion by TG. Collectively, LRRC8B participates in intracellular Ca2+ homeostasis by acting as a leak channel in the ER. This study gives a fundamental understanding of the role of a novel protein in the elemental cellular process of ER Ca2+ leak and expands the known roles for LRRC8 proteins.This article has an associated First Person interview with the first author of the paper.
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Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Adenosina Trifosfato/fisiología , Señalización del Calcio , Células HEK293 , Humanos , Transporte de ProteínasRESUMEN
Aspirin induces apoptotic cell death in various cancer cell lines. Here we showed that silencing of VDAC1 protected HeLa cells from aspirin-induced cell death. Compared to the wild type cells, VDAC1 knocked down cells showed lesser change of mitochondrial membrane potential (Δψm), upon aspirin treatment. Aspirin augmented ATP and ionomycin-induced mitochondrial Ca2+ uptake which was abolished in VDAC1 knocked down cells. Aspirin dissociated bound hexokinase II (HK-II) from mitochondria. Further, aspirin promoted the closure of recombinant human VDAC1, reconstituted in planar lipid bilayer. Taken together, these results imply that VDAC1 serves as a novel target for aspirin. Modulation of VDAC1 is possibly associated with the cell death and anticancer effects of aspirin.
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Antiinflamatorios no Esteroideos/farmacología , Apoptosis/efectos de los fármacos , Aspirina/farmacología , Mitocondrias/efectos de los fármacos , Canal Aniónico 1 Dependiente del Voltaje/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Calcio/metabolismo , Células HeLa , Hexoquinasa/metabolismo , Humanos , Potencial de la Membrana Mitocondrial , Mitocondrias/metabolismo , RatasRESUMEN
Acid-sensing ion channels (ASICs) are associated with many pathophysiological processes, such as neuronal death during ischemic stroke, epileptic seizure and nociception. However, there is a dearth of ASIC-specific therapeutic blockers. Here we report that quercetin, a plant flavonoid, which is known for its neuroprotective effect, reversibly inhibits homomeric rat ASIC1a, ASIC2a and ASIC3 with an IC50 of about 2µM. Also, quercetin prevents low pH-induced intracellular calcium rise and cell death in HEK-293 cells, which have endogenous expression of ASIC1a and 2a. The inhibitory effect of quercetin on ASICs is not due to membrane perturbation, as it did not have any effect on other channels, like NMDA receptor, GABAA receptor and P2X4 receptor. Unlike quercetin, another flavonoid resveratrol had no effect on ASIC1a. Computational analysis revealed that quercetin binds to the channel in a cavity at the central vestibule, lined by several charged residues like Q276, R369, E373 and E416 in ASIC1a. Mutation of Arg369 to Ala or Glu416 to Gln abolished the inhibitory effect of quercetin on rat ASIC1a completely, while Glu373 to Gln showed reduced sensitivity. Our results raise the possibility of using quercetin for targeting ASICs in vivo.
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Bloqueadores del Canal Iónico Sensible al Ácido/farmacología , Canales Iónicos Sensibles al Ácido/metabolismo , Neuronas/efectos de los fármacos , Quercetina/farmacología , Animales , Sitios de Unión , Células CHO , Calcio/metabolismo , Muerte Celular/efectos de los fármacos , Cricetulus , Células HEK293 , Humanos , Modelos Moleculares , Neuronas/metabolismoRESUMEN
Orexin-A and orexin-B (Ox-A, Ox-B) are neuropeptides produced by a small number of neurons that originate in the hypothalamus and project widely in the brain. Only discovered in 1998, the orexins are already known to regulate several behaviours. Most prominently, they help to stabilise the waking state, a role with demonstrated significance in the clinical management of narcolepsy and insomnia. Orexins bind to G-protein-coupled receptors (predominantly postsynaptic) of two subtypes, OX1R and OX2R. The primary effect of Ox-OXR binding is a direct depolarising influence mediated by cell membrane cation channels, but a wide variety of secondary effects, both pre- and postsynaptic, are also emerging. Given that inhibitory GABAergic neurons also influence orexin-regulated behaviours, crosstalk between the two systems is expected, but at the cellular level, little is known and possible mechanisms remain unidentified. Here, we have used an expression system approach to examine the feasibility, and nature, of possible postsynaptic crosstalk between Ox-A and the GABAA receptor (GABAAR), the brain's main inhibitory neuroreceptor. When HEK293 cells transfected with OX1R and the α1, ß1, and γ2S subunits of GABAAR were exposed to Ox-A, GABA-induced currents were inhibited, in a calcium-dependent manner. This inhibition was associated with increased phosphorylation of the ß1 subunit of GABAAR, and the inhibition could itself be attenuated by (1) kinase inhibitors (of protein kinase C and CaM kinase II) and (2) the mutation, to alanine, of serine 409 of the ß1 subunit, a site previously identified in phosphorylation-dependent regulation in other pathways. These results are the first to directly support the feasibility of postsynaptic crosstalk between Ox-A and GABAAR, indicating a process in which Ox-A could promote phosphorylation of the ß1 subunit, reducing the GABA-induced, hyperpolarising current. In this model, Ox-A/GABAAR crosstalk would cause the depolarising influence of Ox-A to be boosted, a type of positive feedback that could, for example, facilitate the ability to abruptly awake.
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Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Calcio/metabolismo , Orexinas/metabolismo , Proteína Quinasa C/metabolismo , Procesamiento Proteico-Postraduccional , Receptores de GABA-A/metabolismo , Potenciales de Acción , Células HEK293 , Humanos , Receptores de Orexina/genética , Receptores de Orexina/metabolismo , Fosforilación , Subunidades de Proteína/metabolismoRESUMEN
Since its discovery, neuroglobin (Ngb), a neuron-specific oxygen binding hemoglobin, distinct from the classical myoglobin and blood hemoglobin, has attracted attention as an endogenous neuroprotectant. Recent reports suggest that Ngb protects neurons from brain stroke, ischemic stress-induced degeneration, and other brain disorders. Proteins with a specific role in neuroprotection are often associated with neurodegeneration, as well, depending on the cellular environment or specific cellular triggers that tilt the balance one way or the other. This investigation explored the potential role of Ngb in amyloid fibril-related neuronal disorder. Ngb was capable of amyloid formation in vitro at neutral pH and ambient temperature, in both apo and holo forms, albeit at a slower rate in the holo form, unlike other hemoglobins that exhibit such behavior exclusively in the apo states. Elevated temperature enhanced the rate of fibril formation significantly. The B-helix, which is known to play a major role in Ngb ligand binding kinetics, was found to be amyloidogenic with the Phe28B10 amino acid side chain as the key inducer of fibrillation. The Ngb amyloid fibril was also significantly cytotoxic to neuroblastoma cell lines, compared to those obtained from reference hemoglobins. The Ngb fibril probably promoted toxicity by inducing channel formation in the cell membrane, as investigated here using synthetic lipid bilayer membranes and the propidium iodide uptake assay. These findings imply that Ngb plays a role in neurodegenerative disorders in vivo, for which there seems to be indirect evidence by association. Ngb thus presents a novel prospect for understanding amyloid-related brain disorders beyond the limited set of proteins currently investigated for such diseases.
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Amiloide/química , Encéfalo/metabolismo , Globinas/química , Hemoglobinas/química , Proteínas del Tejido Nervioso/química , Fenilalanina/química , Línea Celular Tumoral , Dicroismo Circular , Globinas/genética , Humanos , Concentración de Iones de Hidrógeno , Microscopía Electrónica de Transmisión , Mutagénesis Sitio-Dirigida , Proteínas del Tejido Nervioso/genética , Neuroglobina , TemperaturaRESUMEN
Dietary trans fatty acids (TFAs) are known to increase the risk of cardiovascular diseases by altering plasma lipid profile and activating various inflammatory signaling pathways. Here we show that elaidic acid (EA), the most abundant TFA in diet, alters the electrophysiological properties of voltage-dependent anion channel (VDAC) of mitochondria. Purified bovine brain VDAC, when incorporated in the planar lipid bilayer (PLB) composed of 1,2-diphytanoyl-sn-glycero-3 phosphatidyl choline (DPhPC) and EA in a 9 to 1 ratio (wt/wt), exhibited complete closing events at different voltages. The closing events were observed at even -10 mV, a voltage at which VDAC usually remains fully open all the time. Additionally, the voltage sensitivity of VDAC was lost in presence of EA; the channel conductance did not decrease with increasing voltages. In identical experimental conditions, membrane containing oleic acid (OA), the cis isomer of EA did not produce any such effect. We propose that EA possibly exerts its adverse effect by modulating VDAC.
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Mitocondrias/fisiología , Ácido Oléico/farmacología , Canales Aniónicos Dependientes del Voltaje/efectos de los fármacos , Animales , Mitocondrias/efectos de los fármacos , Ácidos Oléicos , Canales Aniónicos Dependientes del Voltaje/fisiologíaRESUMEN
Nitric oxide (NO), a major gaseous signaling molecule, modulates several ion channels and receptors. Here we show that NO attenuates pannexin 1 (Panx1) mediated currents in HEK-293 cells. NO exerts its effect by activating a cGMP-protein kinase G (PKG) dependent pathway. NO donors, sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP) and S-nitrosoglutathione (GSNO), reduced Panx1 currents by 25-41%. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylyl cyclase (sGC), blocked the inhibition completely, whereas sGC activator YC-1 mimicked the effect of NO, suggesting the involvement of a cGMP dependent pathway. Supporting this, NO had no effect in the presence of the PKG inhibitor, KT5823. Further, immuno-precipitated Panx1 was recognized by an anti-phosphoserine antibody in Western blot. Phosphorylation was enhanced significantly when cells were treated with SNP. The target for phosphorylation is possibly Ser 206 of Panx1, as its mutation to Ala completely abolished the NO mediated inhibition.
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Conexinas/antagonistas & inhibidores , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Óxido Nítrico/farmacología , Conexinas/metabolismo , Células HEK293 , Humanos , Proteínas del Tejido Nervioso/metabolismo , Óxido Nítrico/metabolismoRESUMEN
The γ-aminobutyric acid type A receptor (GABAAR) is negatively modulated by two structurally similar neurosteroids, pregnenolone sulfate (PS) and dehydroepiandrosterone sulfate (DHEAS). This study attempted to ascertain the molecular mechanisms of inhibition of the GABA-ergic current by neurosteroids. We demonstrated that the presence of the γ subunit in GABAAR enhances the efficacy of DHEAS without altering its binding affinity. A saturating concentration of DHEAS blocked approximately 75 % of currents mediated by GABAAR, which is composed of human α1, ß1, and γ2S subunits, whereas the inhibition was only 35 % in GABAAR containing only α1 and ß1 subunits. The IC50 values of DHEAS with and without the γ subunit were almost identical. In contrast to DHEAS, neither the affinity nor the efficacy of PS was altered by the γ subunit. When Val256 of α1 subunit was mutated to Ser, the mutant channel became resistant to inhibition by both DHEAS and PS. PS exerted its inhibitory effect by enhancing the desensitization kinetics of GABAAR possibly through promoting the interaction between the M2-M3 linker and extracellular loop 7/loop 2. Mutant α1, containing double Cys in loop 2/loop 7 and the M2-M3 linker, formed disulfide bonds three times as much fast, when treated with saturating GABA+PS, compared with GABA alone or with GABA+DHEAS. We demonstrated that PS, but not DHEAS, mediates GABA-ergic inhibition by promoting collisions between the structural elements involved in receptor desensitization, i.e., loop 2, loop 7, and the M2-M3 linker, thus following different inhibitory mechanisms.
Asunto(s)
Sulfato de Deshidroepiandrosterona/farmacología , Antagonistas de Receptores de GABA-A/farmacología , Pregnenolona/farmacología , Receptores de GABA-A/química , Regulación Alostérica , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión , Células HEK293 , Humanos , Datos de Secuencia Molecular , Unión Proteica , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismoRESUMEN
Voltage dependent anion channel (VDAC) of mitochondria plays a crucial role in apoptosis. Human VDAC-1, reconstituted in planar lipid bilayer showed reduced conductance when treated with curcumin. Curcumin interacts with residues in the α helical N-terminus of VDAC and in the channel wall, as revealed by molecular docking, followed by mutational analysis. N-terminus mimicking peptide showed conformational changes in circular dichroism, upon curcumin treatment. We propose that the interaction of curcumin with amino acids in N-terminus and in channel wall fixes the α helix in closed conformation. This restricts its movement which is required for the opening of the channel.