RESUMEN
An expansion of poly-alanine up to +13 residues in the C-terminus of the transcription factor PHOX2B underlies the onset of congenital central hypoventilation syndrome (CCHS). Recent studies demonstrated that the alanine tract expansion influences PHOX2B folding and activity. Therefore, structural information on PHOX2B is an important target for obtaining clues to elucidate the insurgence of the alanine expansion-related syndrome and also for defining a viable therapy. Here we report by NMR spectroscopy the structural characterization of the homeodomain (HD) of PHOX2B and HD + C-terminus PHOX2B protein, free and in the presence of the target DNA. The obtained structural data are then exploited to obtain a structural model of the PHOX2B-DNA interaction. In addition, the variant +7Ala, responsible for one of the most frequent forms of the syndrome, was analysed, showing different conformational proprieties in solution and a strong propensity to aggregation. Our data suggest that the elongated poly-alanine tract would be related to disease onset through a loss-of-function mechanism. Overall, this study paves the way for the future rational design of therapeutic drugs, suggesting as a possible therapeutic route the use of specific anti-aggregating molecules capable of preventing variant aggregation and possibly restoring the DNA-binding activity of PHOX2B.
RESUMEN
The α7 nicotinic receptor (α7 nAChR) is an important entry point for Ca2+ into the cell, which has broad and important effects on gene expression and function. The gene (CHRNA7), mapping to chromosome (15q14), has been genetically linked to a large number of diseases, many of which involve defects in cognition. While numerous mutations in CHRNA7 are associated with mental illness and inflammation, an important control point may be the function of a recently discovered partial duplication CHRNA7, CHRFAM7A, that negatively regulates the function of the α7 receptor, through the formation of heteropentamers; other functions cannot be excluded. The deregulation of this human specific gene (CHRFAM7A) has been linked to neurodevelopmental, neurodegenerative, and inflammatory disorders and has important copy number variations. Much effort is being made to understand its function and regulation both in healthy and pathological conditions. However, many questions remain to be answered regarding its functional role, its regulation, and its role in the etiogenesis of neurological and inflammatory disorders. Missing knowledge on the pharmacology of the heteroreceptor has limited the discovery of new molecules capable of modulating its activity. Here we review the state of the art on the role of CHRFAM7A, highlighting unanswered questions to be addressed. A possible therapeutic approach based on genome editing protocols is also discussed.
Asunto(s)
Trastornos Mentales , Receptores Nicotínicos , Humanos , Variaciones en el Número de Copia de ADN , Receptor Nicotínico de Acetilcolina alfa 7/genética , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Cognición , Mutación , Receptores Nicotínicos/genéticaRESUMEN
Inflammation caused by infection, tissue trauma, and disease states such as arthritis and inflammatory bowel disease is perceived by the Central nervous System (CNS) through different routes that, by means of neural reflex circuits, regulate the immune system response [...].
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Inflamación , Enfermedades Inflamatorias del Intestino , Sistema Nervioso Central , Colinérgicos , Humanos , Sistema InmunológicoRESUMEN
Heterozygous mutations of the transcription factor PHOX2B are responsible for Congenital Central Hypoventilation Syndrome, a neurological disorder characterized by inadequate respiratory response to hypercapnia and life-threatening hypoventilation during sleep. Although no cure is currently available, it was suggested that a potent progestin drug provides partial recovery of chemoreflex response. Previous in vitro data show a direct molecular link between progestins and PHOX2B expression. However, the mechanism through which these drugs ameliorate breathing in vivo remains unknown. Here, we investigated the effects of chronic administration of the potent progestin drug Etonogestrel (ETO) on respiratory function and transcriptional activity in adult female rats. We assessed respiratory function with whole-body plethysmography and measured genomic changes in brain regions important for respiratory control. Our results show that ETO reduced metabolic activity, leading to an enhanced chemoreflex response and concurrent increased breathing cycle variability at rest. Furthermore, ETO-treated brains showed reduced mRNA and protein expression of PHOX2B and its target genes selectively in the dorsal vagal complex, while other areas were unaffected. Histological analysis suggests that changes occurred in the solitary tract nucleus (NTS). Thus, we propose that the NTS, rich in both progesterone receptors and PHOX2B, is a good candidate for ETO-induced respiratory modulation.
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Apnea Central del Sueño , Núcleo Solitario , Animales , Desogestrel , Femenino , Proteínas de Homeodominio/metabolismo , Hipoventilación/congénito , Hipoventilación/genética , Mutación , Progestinas/farmacología , Ratas , Apnea Central del Sueño/genética , Núcleo Solitario/metabolismoRESUMEN
CHRFAM7A is a relatively recent and exclusively human gene arising from the partial duplication of exons 5 to 10 of the α7 neuronal nicotinic acetylcholine receptor subunit (α7 nAChR) encoding gene, CHRNA7. CHRNA7 is related to several disorders that involve cognitive deficits, including neuropsychiatric, neurodegenerative, and inflammatory disorders. In extra-neuronal tissues, α7nAChR plays an important role in proliferation, differentiation, migration, adhesion, cell contact, apoptosis, angiogenesis, and tumor progression, as well as in the modulation of the inflammatory response through the "cholinergic anti-inflammatory pathway". CHRFAM7A translates the dupα7 protein in a multitude of cell lines and heterologous systems, while maintaining processing and trafficking that are very similar to the full-length form. It does not form functional ion channel receptors alone. In the presence of CHRNA7 gene products, dupα7 can assemble and form heteromeric receptors that, in order to be functional, should include at least two α7 subunits to form the agonist binding site. When incorporated into the receptor, in vitro and in vivo data showed that dupα7 negatively modulated α7 activity, probably due to a reduction in the number of ACh binding sites. Very recent data in the literature report that the presence of the duplicated gene may be responsible for the translational gap in several human diseases. Here, we will review the studies that have been conducted on CHRFAM7A in different pathologies, with the intent of providing evidence regarding when and how the expression of this duplicated gene may be beneficial or detrimental in the pathogenesis, and eventually in the therapeutic response, to CHRNA7-related neurological and non-neurological diseases.
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Genes Duplicados , Inflamación , Enfermedades Neurodegenerativas , Receptor Nicotínico de Acetilcolina alfa 7 , Sitios de Unión , Humanos , Inflamación/genética , Inflamación/metabolismo , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Neuronas/metabolismo , Isoformas de Proteínas/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7/genética , Receptor Nicotínico de Acetilcolina alfa 7/metabolismoRESUMEN
Congenital Central Hypoventilation Syndrome (CCHS) is a rare disorder of the autonomic nervous system (ANS), characterized by inadequate control of autonomic ventilation and global autonomic dysfunction. Heterozygous polyalanine repeat expansion mutations in exon 3 of the transcription factor Paired-like homeobox 2B (PHOX2B) gene occur in 90% of CCHS cases. In this study, we describe the generation and characterization of two human induced pluripotent stem cell (hiPSC) lines from female CCHS patients carrying a heterozygous + 5 alanine expansion mutation. The generated iPSC lines show a normal karyotype, express pluripotency markers and are able to differentiate into the three germ layers.
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Células Madre Pluripotentes Inducidas , Femenino , Proteínas de Homeodominio/genética , Humanos , Hipoventilación/congénito , Mutación/genética , Péptidos , Apnea Central del Sueño , Factores de Transcripción/genéticaRESUMEN
Glioblastomas (GBMs), the most frequent brain tumours, are highly invasive and their prognosis is still poor despite the use of combination treatment. MG624 is a 4-oxystilbene derivative that is active on α7- and α9-containing neuronal nicotinic acetylcholine receptor (nAChR) subtypes. Hybridisation of MG624 with a non-nicotinic resveratrol-derived pro-oxidant mitocan has led to two novel compounds (StN-4 and StN-8) that are more potent than MG624 in reducing the viability of GBM cells, but less potent in reducing the viability of mouse astrocytes. Functional analysis of their activity on α7 receptors showed that StN-4 is a silent agonist, whereas StN-8 is a full antagonist, and neither alters intracellular [Ca2+] levels when acutely applied to U87MG cells. After 72 h of exposure, both compounds decreased U87MG cell proliferation, and pAKT and oxphos ATP levels, but only StN-4 led to a significant accumulation of cells in phase G1/G0 and increased apoptosis. One hour of exposure to either compound also decreased the mitochondrial and cytoplasmic ATP production of U87MG cells, and this was not paralleled by any increase in the production of reactive oxygen species. Knocking down the α9 subunit (which is expressed at relatively high levels in U87MG cells) decreased the potency of the effects of both compounds on cell viability, but cell proliferation, ATP production, pAKT levels were unaffected by the presence of the noncell-permeable α7/α9-selective antagonist αBungarotoxin. These last findings suggest that the anti-tumoral effects of StN-4 and StN-8 on GBM cells are not only due to their action on nAChRs, but also to other non-nicotinic mechanisms.
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Compuestos de Amonio/farmacología , Antineoplásicos/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Estilbenos/farmacología , Adenosina Trifosfato/metabolismo , Animales , Astrocitos/efectos de los fármacos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Fenómenos Fisiológicos Celulares/efectos de los fármacos , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Ligandos , Ratones , Proteínas Proto-Oncogénicas c-akt/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores Nicotínicos/genética , Receptor Nicotínico de Acetilcolina alfa 7/genéticaRESUMEN
Neuroinflammation and cholinergic dysfunction, leading to cognitive impairment, are hallmarks of aging and neurodegenerative disorders, including Alzheimer's disease (AD). Acetylcholinesterase inhibitors (AChEI), the symptomatic therapy in AD, attenuate and delay the cognitive deficit by enhancing cholinergic synapses. The α7 nicotinic acetylcholine (ACh) receptor has shown a double-edged sword feature, as it binds with high affinity Aß1-42, promoting intracellular accumulation and Aß-induced tau phosphorylation, but also exerts neuroprotection by stimulating anti-apoptotic pathways. Moreover, it mediates peripheral and central anti-inflammatory response, being the effector player of the activation of the cholinergic anti-inflammatory pathway (CAIP), that, by decreasing the release of TNF-α, IL-1ß, and IL-6, it may have a role in improving cognition. The finding in preclinical models that, in addition to their major function (choline esterase inhibition) AChEIs have neuroprotective properties mediated via α7nAChR and modulate innate immunity, possibly as a result of the increased availability of acetylcholine activating the CAIP, pave the way for new pharmacological intervention in AD and other neurological disorders that are characterized by neuroinflammation. CHRFAM7A is a human-specific gene acting as a dominant negative inhibitor of α7nAChR function, also suggesting a role in affecting human cognition and memory by altering α7nAChR activities in the central nervous system (CNS). This review will summarize the current knowledge on the cholinergic anti-inflammatory pathway in aging-related disorders, and will argue that the presence of the human-restricted CHRFAM7A gene might play a fundamental role in the regulation of CAIP and in the response to AChEI.
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Enfermedad de Alzheimer , Inhibidores de la Colinesterasa , Envejecimiento , Enfermedad de Alzheimer/tratamiento farmacológico , Inhibidores de la Colinesterasa/farmacología , Inhibidores de la Colinesterasa/uso terapéutico , Humanos , Neuroinmunomodulación , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
Glioblastomas (GBMs), the most frequent and aggressive human primary brain tumours, have altered cell metabolism, and one of the strongest indicators of malignancy is an increase in choline compounds. Choline is also a selective agonist of some neuronal nicotinic acetylcholine receptor (nAChR) subtypes. As little is known concerning the expression of nAChR in glioblastoma cells, we analysed in U87MG human grade-IV astrocytoma cell line and GBM5 temozolomide-resistant glioblastoma cells selected from a cancer stem cell-enriched culture, molecularly, pharmacologically and functionally which nAChR subtypes are expressed and,whether choline and nicotine can affect GBM cell proliferation. We found that U87MG and GBM5 cells express similar nAChR subtypes, and choline and nicotine increase their proliferation rate and activate the anti-apoptotic AKT and pro-proliferative ERK pathways. These effects are blocked by the presence of non-cell-permeable peptide antagonists selective for α7- and α9-containing nicotinic receptors. siRNA-mediated silencing of α7 or α9 subunit expression also selectively prevents the effects of nicotine and choline on GBM cell proliferation. Our findings indicate that nicotine and choline activate the signalling pathways involved in the proliferation of GBM cells, and that these effects are mediated by α7 and α9-containing nAChRs. This suggests that these nicotinic receptors may contribute to the aggressive behaviour of this tumor and may indicate new therapeutic strategies against high-grade human brain tumours.
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Neoplasias Encefálicas/metabolismo , Colina/farmacología , Glioblastoma/metabolismo , Nicotina/farmacología , Receptores Nicotínicos/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Interferente Pequeño/genética , Receptores Nicotínicos/genética , Receptor Nicotínico de Acetilcolina alfa 7/genéticaRESUMEN
Congenital central hypoventilation syndrome (CCHS) is a genetic disorder of neurodevelopment, with an autosomal dominant transmission, caused by heterozygous mutations in the PHOX2B gene. CCHS is a rare disorder characterized by hypoventilation due to the failure of autonomic control of breathing. Until now no curative treatment has been found. PHOX2B is a transcription factor that plays a crucial role in the development (and maintenance) of the autonomic nervous system, and in particular the neuronal structures involved in respiratory reflexes. The underlying pathogenetic mechanism is still unclear, although studies in vivo and in CCHS patients indicate that some neuronal structures may be damaged. Moreover, in vitro experimental data suggest that transcriptional dysregulation and protein misfolding may be key pathogenic mechanisms. This review summarizes latest researches that improved the comprehension of the molecular pathogenetic mechanisms responsible for CCHS and discusses the search for therapeutic intervention in light of the current knowledge about PHOX2B function.
RESUMEN
The α7 nicotinic acetylcholine receptor (CHRNA7) modulates the inflammatory response by activating the cholinergic anti-inflammatory pathway. CHRFAM7A, the human-restricted duplicated form of CHRNA7, has a negative effect on the functioning of α7 receptors, suggesting that CHRFAM7A expression regulation may be a key step in the modulation of inflammation in the human setting. The analysis of the CHRFAM7A gene's regulatory region reveals some of the mechanisms driving its expression and responsiveness to LPS in human immune cell models. Moreover, given the immunomodulatory potential of donepezil we show that it differently modulates CHRFAM7A and CHRNA7 responsiveness to LPS, thus contributing to its therapeutic potential.
Asunto(s)
Antiinflamatorios/farmacología , Agonistas Colinérgicos/farmacología , Inhibidores de la Colinesterasa/farmacología , Donepezilo/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Factores Inmunológicos/farmacología , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Neuroinmunomodulación/efectos de los fármacos , Receptor Nicotínico de Acetilcolina alfa 7/biosíntesis , Secuencia de Bases , Regulación hacia Abajo/efectos de los fármacos , Sinergismo Farmacológico , Humanos , Macrófagos/metabolismo , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Isoformas de Proteínas/genética , Secuencias Reguladoras de Ácidos Nucleicos , Células THP-1 , Transcripción Genética/efectos de los fármacos , Receptor Nicotínico de Acetilcolina alfa 7/genéticaRESUMEN
About 90% of congenital central hypoventilation syndrome (CCHS) patients show polyalanine triplet expansions in the coding region of transcription factor PHOX2B, which renders this protein an intriguing target to understand the insurgence of this syndrome and for the design of a novel therapeutical approach. Consistently with the role of PHOX2B as a transcriptional regulator, it is reasonable that a general transcriptional dysregulation caused by the polyalanine expansion might represent an important mechanism underlying CCHS pathogenesis. Therefore, this study focused on the biochemical characterization of different PHOX2B variants, such as a variant containing the correct C-terminal (20 alanines) stretch, one of the most frequent polyalanine expansions (+7 alanines), and a variant lacking the complete alanine stretch (0 alanines). Comparison of the different variants by a multidisciplinary approach based on different methodologies (including circular dichroism, spectrofluorimetry, light scattering, and Atomic Force Microscopy studies) highlighted the propensity to aggregate for the PHOX2B variant containing the polyalanine expansion (+7-alanines), especially in the presence of DNA, while the 0-alanines variant resembled the protein with the correct polyalanine length. Moreover, and unexpectedly, the formation of fibrils was revealed only for the pathological variant, suggesting a plausible role of such fibrils in the insurgence of CCHS.
Asunto(s)
Proteínas de Homeodominio/química , Multimerización de Proteína , Factores de Transcripción/química , Secuencias de Aminoácidos , Células HeLa , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Mutación , Péptidos/química , Péptidos/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The paired-like homeobox 2B gene (PHOX2B) encodes a key transcription factor that plays a role in the development of the autonomic nervous system and the neural structures involved in controlling breathing. In humans, PHOX2B over-expression plays a role in the pathogenesis of tumours arising from the sympathetic nervous system such as neuroblastomas, and heterozygous PHOX2B mutations cause Congenital Central Hypoventilation Syndrome (CCHS), a life-threatening neurocristopathy characterised by the defective autonomic control of breathing and involving altered CO2/H+ chemosensitivity. The recovery of CO2/H+ chemosensitivity and increased ventilation have been observed in two CCHS patients using the potent contraceptive progestin desogestrel. Given the central role of PHOX2B in the pathogenesis of CCHS, and the progesterone-mediated effects observed in the disease, we generated progesterone-responsive neuroblastoma cells, and evaluated the effects of 3-Ketodesogestrel (3-KDG), the biologically active metabolite of desogestrel, on the expression of PHOX2B and its target genes. Our findings demonstrate that, through progesterone nuclear receptor PR-B, 3-KDG down-regulates PHOX2B gene expression, by a post-transcriptional mechanism, and its target genes and open up the possibility that this mechanism may contribute to the positive effects observed in some CCHS patients.
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Desogestrel/farmacología , Proteínas de Homeodominio/efectos de los fármacos , Células-Madre Neurales/efectos de los fármacos , Progesterona/genética , Factores de Transcripción/efectos de los fármacos , Núcleo Celular/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Hipoventilación/congénito , Hipoventilación/genética , Células-Madre Neurales/metabolismo , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/genética , Apnea Central del Sueño/genética , Factores de Transcripción/metabolismoRESUMEN
Heterozygous mutations in the PHOX2B gene are causative of congenital central hypoventilation syndrome (CCHS), a neurocristopathy characterized by defective autonomic control of breathing due to the impaired differentiation of neural crest cells. Among PHOX2B mutations, polyalanine (polyAla) expansions are almost exclusively associated with isolated CCHS, whereas frameshift variants, although less frequent, are often more severe than polyAla expansions and identified in syndromic CCHS. This article provides a complete review of all the frameshift mutations identified in cases of isolated and syndromic CCHS reported in the literature as well as those identified by us and not yet published. These were considered in terms of both their structure, whether the underlying indels induced frameshifts of either 1 or 2 steps ("frame 2" and "frame 3" mutations respectively), and clinical associations. Furthermore, we evaluated the structural and functional effects of one "frame 3" mutation identified in a patient with isolated CCHS, and one "frame 2" mutation identified in a patient with syndromic CCHS, also affected with Hirschsprung's disease and neuroblastoma. The data thus obtained confirm that the type of translational frame affects the severity of the transcriptional dysfunction and the predisposition to isolated or syndromic CCHS.
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Mutación del Sistema de Lectura/genética , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Hipoventilación/congénito , Apnea Central del Sueño/genética , Factores de Transcripción/química , Factores de Transcripción/genética , Ensayo de Cambio de Movilidad Electroforética , Células HeLa , Humanos , Hipoventilación/genética , Hipoventilación/metabolismo , Microscopía Fluorescente , Mutación , Apnea Central del Sueño/metabolismoAsunto(s)
Discinesia Inducida por Medicamentos/genética , Levodopa/efectos adversos , Enfermedad de Parkinson/tratamiento farmacológico , Receptores Opioides kappa/genética , Adulto , Anciano , Discinesia Inducida por Medicamentos/etiología , Femenino , Humanos , Modelos Lineales , Masculino , Persona de Mediana Edad , Análisis Multivariante , Variantes Farmacogenómicas , Factores de TiempoRESUMEN
BACKGROUND AND PURPOSE: Tobacco smoke contains many classes of carcinogens and although nicotine is unable to initiate tumourigenesis in humans and rodents, it promotes tumour growth and metastasis in lung tumours by acting on neuronal nicotinic ACh receptors (nAChRs). The aim of this study was to identify molecularly, biochemically and pharmacologically which nAChR subtypes are expressed and functionally activated by nicotine in lung cancer cell lines. EXPERIMENTAL APPROACH: We used A549 and H1975 adenocarcinoma cell lines derived from lung tumours to test the in vitro effects of nicotine, and nAChR subtype-specific peptides and compounds. KEY RESULTS: The two adenocarcinoma cell lines express distinctive nAChR subtypes, and this affects their nicotine-induced proliferation. In A549 cells, nAChRs containing the α7 or α9 subunits not only regulate nicotine-induced cell proliferation but also the activation of the Akt and ERK pathways. Blocking these nAChRs by means of subtype-specific peptides, or silencing their expression by means of subunit-specific siRNAs, abolishes nicotine-induced proliferation and signalling. Moreover, we found that the α7 antagonist MG624 also acts on α9-α10 nAChRs, blocks the effects of nicotine on A549 cells and has dose-dependent cytotoxic activity. CONCLUSIONS AND IMPLICATIONS: These results highlight the pathophysiological role of α7- and α9-containing receptors in promoting non-small cell lung carcinoma cell growth and intracellular signalling and provide a framework for the development of new drugs that specifically target the receptors expressed in lung tumours. LINKED ARTICLES: This article is part of a themed section on Nicotinic Acetylcholine Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.11/issuetoc.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Nicotina/farmacología , Receptores Nicotínicos/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7/antagonistas & inhibidores , Células A549 , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Compuestos de Amonio Cuaternario/farmacología , Estilbenos/farmacología , Relación Estructura-Actividad , Receptor Nicotínico de Acetilcolina alfa 7/metabolismoRESUMEN
Alterations in the balance of inhibitory and excitatory synaptic transmission have been implicated in the pathogenesis of neurological disorders such as epilepsy. Eukaryotic elongation factor 2 kinase (eEF2K) is a highly regulated, ubiquitous kinase involved in the control of protein translation. Here, we show that eEF2K activity negatively regulates GABAergic synaptic transmission. Indeed, loss of eEF2K increases GABAergic synaptic transmission by upregulating the presynaptic protein Synapsin 2b and α5-containing GABAA receptors and thus interferes with the excitation/inhibition balance. This cellular phenotype is accompanied by an increased resistance to epilepsy and an impairment of only a specific hippocampal-dependent fear conditioning. From a clinical perspective, our results identify eEF2K as a potential novel target for antiepileptic drugs, since pharmacological and genetic inhibition of eEF2K can revert the epileptic phenotype in a mouse model of human epilepsy.
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Quinasa del Factor 2 de Elongación/metabolismo , Epilepsia/enzimología , Neuronas/enzimología , Transmisión Sináptica/fisiología , Animales , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/enzimología , Corteza Cerebral/patología , Condicionamiento Psicológico/fisiología , Modelos Animales de Enfermedad , Quinasa del Factor 2 de Elongación/antagonistas & inhibidores , Quinasa del Factor 2 de Elongación/genética , Epilepsia/patología , Miedo/fisiología , Hipocampo/efectos de los fármacos , Hipocampo/enzimología , Hipocampo/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Inhibición Neural/efectos de los fármacos , Inhibición Neural/fisiología , Neuronas/efectos de los fármacos , Neuronas/patología , Ratas Sprague-Dawley , Receptores de GABA-A/metabolismo , Sinapsinas/genética , Sinapsinas/metabolismo , Transmisión Sináptica/efectos de los fármacos , Ácido gamma-Aminobutírico/metabolismoRESUMEN
The GET (guided entry of tail-anchored proteins)/TRC (transmembrane recognition complex) pathway for tail-anchored protein targeting to the endoplasmic reticulum (ER) has been characterized in detail in yeast and is thought to function similarly in mammals, where the orthologue of the central ATPase, Get3, is known as TRC40 or Asna1. Get3/TRC40 function requires an ER receptor, which in yeast consists of the Get1/Get2 heterotetramer and in mammals of the WRB protein (tryptophan-rich basic protein), homologous to yeast Get1, in combination with CAML (calcium-modulating cyclophilin ligand), which is not homologous to Get2. To better characterize the mammalian receptor, we investigated the role of endogenous WRB and CAML in tail-anchored protein insertion as well as their association, concentration, and stoichiometry in rat liver microsomes and cultured cells. Functional proteoliposomes, reconstituted from a microsomal detergent extract, lost their activity when made with an extract depleted of TRC40-associated proteins or of CAML itself, whereas in vitro synthesized CAML and WRB together were sufficient to confer insertion competence to liposomes. CAML was found to be in â¼5-fold excess over WRB, and alteration of this ratio did not inhibit insertion. Depletion of each subunit affected the levels of the other one; in the case of CAML silencing, this effect was attributable to destabilization of the WRB transcript and not of WRB protein itself. These results reveal unanticipated complexity in the mutual regulation of the TRC40 receptor subunits and raise the question as to the role of the excess CAML in the mammalian ER.
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ATPasas Transportadoras de Arsenitos/química , ATPasas Transportadoras de Arsenitos/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Animales , ATPasas Transportadoras de Arsenitos/genética , Línea Celular , Células Cultivadas , Síndrome de Down/genética , Síndrome de Down/metabolismo , Retículo Endoplásmico/metabolismo , Regulación de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células HeLa , Humanos , Microsomas Hepáticos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Subunidades de Proteína , Transporte de Proteínas , Proteolípidos/metabolismo , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
BACKGROUND: Impulse control disorders and compulsive medication intake may occur in a minority of patients with Parkinson's disease (PD). We hypothesize that genetic polymorphisms associated with addiction in the general population may increase the risk for addictive behaviors also in PD. METHODS: Sixteen polymorphisms in candidate genes belonging to five neurotransmitter systems (dopaminergic, catecholaminergic, serotonergic, glutamatergic, opioidergic) and the BDNF were screened in 154 PD patients with addictive behaviors and 288 PD control subjects. Multivariate analysis investigated clinical and genetic predictors of outcome (remission vs. persistence/relapse) after 1 year and at the last follow-up (5.1 ± 2.5 years). RESULTS: Addictive behaviors were associated with tryptophan hydroxylase type 2 (TPH2) and dopamine transporter gene variants. A subsequent analysis within the group of cases showed a robust association between TPH2 genotype and the severity of addictive behaviors, which survived Bonferroni correction for multiple testing. At multivariate analysis, TPH2 genotype resulted the strongest predictor of no remission at the last follow-up (OR[95%CI], 7.4[3.27-16.78] and 13.2[3.89-44.98] in heterozygous and homozygous carriers, respectively, p < 0.001). The extent of medication dose reduction was not a predictor. TPH2 haplotype analysis confirmed the association with more severe symptoms and lower remission rates in the short- and the long-term (p < 0.005 for all analyses). CONCLUSION: The serotonergic system is likely to be involved in the pathophysiology of addictive behaviors in PD, modulating the severity of symptoms and the rate of remission at follow-up. If confirmed in larger independent cohorts, TPH2 genotype may become a useful biomarker for the identification of at-risk individuals.