RESUMEN
Somatic activating mutations in the GNAQ have been recently associated with several congenital genetic disorders and tumors; however, the molecular mechanism/etiology that leads to GNAQ somatic mosaic mutation are unknown. Here, we reported a case of Sturge-Weber Syndrome (SWS) manifesting cutaneous vascular malformations (hemifacial Port-wine stain), cerebral and ocular vascular abnormalities (including epilepsy and glaucoma) and harboring a c.548G>A (p.R183Q) somatic mosaic mutation in GNAQ. Computational modeling studies were performed to assistant with the comprehension of the functional impact of p.R183Q and p.Q209L mutations in GNAQ, which encodes a G protein subunit alpha q (Gαq). The p.R183Q mutation was predicted to abolish hydrogen bonds between R183 residue and GDP molecule, destabilizing the inactive GDP-bound conformation of the Gαq mutants. Furthermore, replacement of R183 by Q183 residue was predicted to promote conformation changes in protein surface features affecting the switch I region, a key region that undergoes conformational changes triggered by receptor binding during signal transduction. In addition, replacement of Q209 by L209 residue was predicted to affect the molecular interaction between Gαq and Gß subunit, impairing formation of the inactive heterotrimeric complex. These findings, in association with PPI network analysis, indicate that p.R183Q and p.Q209L mutations result in the over-activation of different downstream effectors, which in turn will determine the distinct cell responses and phenotype. These findings bring new insights on molecular etiology of vascular malformations associated to SWS and on different mechanisms underlying hyperactivation of downstream pathways to Gαq.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gq-G11/química , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Relación Estructura-Actividad Cuantitativa , Adulto , Alelos , Sitios de Unión , Niño , Análisis Mutacional de ADN , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Humanos , Masculino , Modelos Moleculares , Fenotipo , Unión Proteica , Conformación Proteica , Síndrome de Sturge-Weber/diagnóstico , Síndrome de Sturge-Weber/genéticaRESUMEN
Melanopsin has been implicated in the mammalian photoentrainment by blue light. This photopigment, which maximally absorbs light at wavelengths between 470 and 480 nm depending on the species, is found in the retina of all classes of vertebrates so far studied. In mammals, melanopsin activation triggers a signaling pathway which resets the circadian clock in the suprachiasmatic nucleus (SCN). Unlike mammals, Drosophila melanogaster and Danio rerio do not rely only on their eyes to perceive light, in fact their whole body may be capable of detecting light and entraining their circadian clock. Melanopsin, teleost multiple tissue (tmt) opsin and others such as neuropsin and va-opsin, are found in the peripheral tissues of Danio rerio, however, there are limited data concerning the photopigment/s or the signaling pathway/s directly involved in light detection. Here, we demonstrate that melanopsin is a strong candidate to mediate synchronization of zebrafish cells. The deduced amino acid sequence of melanopsin, although being a vertebrate opsin, is more similar to invertebrate than vertebrate photopigments, and melanopsin photostimulation triggers the phosphoinositide pathway through activation of a G(q/11)-type G protein. We stimulated cultured ZEM-2S cells with blue light at wavelengths consistent with melanopsin maximal absorption, and evaluated the time course expression of per1b, cry1b, per2 and cry1a. Using quantitative PCR, we showed that blue light is capable of slightly modulating per1b and cry1b genes, and drastically increasing per2 and cry1a expression. Pharmacological assays indicated that per2 and cry1a responses to blue light are evoked through the activation of the phosphoinositide pathway, which crosstalks with nitric oxide (NO) and mitogen activated protein MAP kinase (MAPK) to activate the clock genes. Our results suggest that melanopsin may be important in mediating the photoresponse in Danio rerio ZEM-2S cells, and provide new insights about the modulation of clock genes in peripheral clocks.
Asunto(s)
Relojes Circadianos/genética , Fibroblastos/efectos de la radiación , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Retina/efectos de la radiación , Opsinas de Bastones/genética , Proteínas de Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Línea Celular , Criptocromos/genética , Criptocromos/metabolismo , Embrión no Mamífero , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Regulación de la Expresión Génica , Luz , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Datos de Secuencia Molecular , Óxido Nítrico/metabolismo , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Fosfatidilinositoles/metabolismo , Estimulación Luminosa , Retina/citología , Retina/metabolismo , Opsinas de Bastones/metabolismo , Transducción de Señal , Núcleo Supraquiasmático/citología , Núcleo Supraquiasmático/metabolismo , Pez Cebra , Proteínas de Pez Cebra/metabolismoRESUMEN
BACKGROUND: Chronic stress has significant effects on hippocampal structure and function. We have previously identified nerve growth factor (NGF), membrane glycoprotein 6a (M6a), the guanine nucleotide binding protein (G protein) alpha q polypeptide (GNAQ), and CDC-like kinase 1 (CLK-1) as genes regulated by psychosocial stress and clomipramine treatment in the hippocampus of tree shrews. These genes encode proteins involved in neurite outgrowth. METHODS: To analyze whether regulation of the above-mentioned genes is conserved between different species, stressors, and antidepressant drugs, we subjected mice to repeated restraint stress and tianeptine treatment and measured hippocampal messenger RNA (mRNA) levels by real time reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Chronically stressed mice displayed a reduction in transcript levels for NGF, M6a, GNAQ, and CLK-1. In addition, other genes implicated in neuronal plasticity, such as brain-derived neurotrophic factor (BDNF), cyclic adenosine monophosphate (cAMP) response element binding protein (CREB), protein kinase C (PKC), neural cell adhesion molecule (NCAM), and synapsin I were downregulated in stressed mice. Tianeptine treatment reversed the stress effects for the genes analyzed. Alterations in gene expression were dependent on the duration of the stress treatment and, in some cases, were only observed in male mice. CONCLUSIONS: These results suggest that genes involved in neurite remodeling are one of the main targets for regulation by chronic stress. The finding that this regulation is conserved in different stress models and antidepressant treatments highlights the biological relevance of the genes analyzed and suggests that they might be involved in stress-related disorders.