A proteomic study using zebra mussels (D. polymorpha) exposed to benzo(α)pyrene: the role of gender and exposure concentrations.

It has recently been established that the use of proteomics can be a useful tool in the field of ecotoxicology. Despite the fact that the mussel Dreissena polymorpha is a valuable bioindicator for freshwater ecosystems, the application of a proteomic approach with this organism has not been deeply investigated. To this end, several zebra mussel specimens were subjected to a 7-day exposure of two different concentrations (0.

Noonan syndrome associated with both a new Jnk-activating familial SOS1 and a de novo RAF1 mutations.

Noonan syndrome is a genetic condition characterized by congenital heart defects, short stature, and characteristic facial features. Familial or de novo mutations in PTPN11, RAF1, SOS1, KRAS, and NRAS are responsible for 60-75% of the cases, thus, additional genes are expected to be involved in the pathogenesis. In addition, the genotype-phenotype correlation has been hindered by the highly variable expressivity of the disease.

N-terminal interaction domain implicates PAK4 in translational regulation and reveals novel cellular localization signals.

The serine/threonine kinase PAK4 is a Rho GTPases effector protein implicated in many critical biological processes, including regulation of cell morphology and motility, embryonic development, cell survival, response to infection, and oncogenic transformation. Consistently with its pro-oncogenic features, PAK4 was found to be overexpressed in many cancer cell lines and tissues, and to be necessary to promote activation of survival pathways. PAK4, like other Paks, is now considered a promising target for specific therapy.

Proteome profile in Myotonic Dystrophy type 2 myotubes reveals dysfunction in protein processing and mitochondrial pathways.

Myotonic Dystrophy type 2 (DM2) is caused by a DNA microsatellite expansion within the Zinc Finger Protein 9 gene leading to an abnormal splicing pattern largely responsible for the pathological condition. To better define the functional changes occurring in human DM2 myotubes we performed a quantitative proteome comparison between myotubes of DM2 and control patients using two-dimensional gel electrophoresis followed by mass spectrometry. Our results indicate that the proteins, altered in DM2 cultures, belong to two major functional categories: i) mitochondrial components, with a reduction of EFTu, HSP60, GRP75 and Dienoyl-CoA-Isomerase, an enzyme involved in fatty acids degradation; ii) the ubiquitin proteasome system with increase of the 26S proteasome regulatory subunit 13 and a reduction of Proteasome subunit Alfa6 and of Rad23B homolog.

Identification of novel RasGRF1 interacting partners by large-scale proteomic analysis.

The brain-specific Ras guanine nucleotide exchange factor RasGRF1 is a protein harbouring a complex array of structural motifs. It contains a pleckstrin homology (PH1) domain, a coiled coil region (CC) and an ilimaquinone (IQ) one in addition to the catalytic Ras and Rac exchange factor domains. In this study, we used the recombinant N-terminal PH1, CC and IQ region (PHCCIQ) fused to the chitin-binding domain to isolate interacting proteins from mouse brain extracts.

Transcriptomic and proteomic analyses of mouse cerebellum reveals alterations in RasGRF1 expression following in vivo chronic treatment with delta 9-tetrahydrocannabinol.

We have applied transcriptomic and proteomic techniques to identify changes in the RNA and the protein levels in the mouse cerebellum after chronic treatment with Delta(9)-tetrahydrocannabinol (THC). Among approximately 14,000 transcripts in a mouse cDNA microarray library, we found 11 genes with altered expression. RasGRF1, a neuron-specific Ras guanine nucleotide exchange factor, showed a reduction both at the RNA and protein levels with a specific decrease of the protein pool associated to cell membranes.

SCLIP, a microtubule-destabilizing factor, interacts with RasGRF1 and inhibits its ability to promote Rac activation and neurite outgrowth.

RasGRF1 is a neuron-specific guanine nucleotide exchange factor for the small GTPases Ras and Rac. It is implicated in the regulation of memory formation and in the development of tolerance to drug abuse, although the mechanisms have been elucidated only in part. Here we report the isolation, by the yeast two-hybrid screen, of the microtubule-destabilizing factor SCLIP (SCG10-like protein) as a novel RasGRF1-interacting protein.

Changes in the expression of G protein-coupled receptor kinases and beta-arrestins in mouse brain during cannabinoid tolerance: a role for RAS-ERK cascade.

The focus of our study was to determine the role of G protein-coupled receptor kinases (GRKs) and beta-arrestins in agonist-induced CB1 receptor modulation during cannabinoid tolerance and their dependence from the extracellular signal-regulated kinase (ERK) cascade. In wild-type mice, chronic Delta9-tetrahydrocannabinol (THC) exposure significantly activated specific GRK and beta- arrestin subunits in all the considered brain areas (striatum, cerebellum, hippocampus, and prefrontal cortex), suggesting their involvement in the adaptive processes underlying CB1 receptor downregulation and desensitization. These events were ERK-dependent in the striatum and cerebellum, because they were prevented in the genetic (Ras-GRF1 knockout mice) and pharmacological (SL327-pretreated mice) models of ERK activation inhibition, whereas in the hippocampus and prefrontal cortex, they appeared to be mostly ERK-independent.

ERK-dependent modulation of cerebellar synaptic plasticity after chronic Delta9-tetrahydrocannabinol exposure.

Chronic exposure to Delta9-tetrahydrocannabinol (THC) induces tolerance to cannabinoid-induced locomotor effects, which are mediated by cannabinoid receptors (CB1Rs) located in motor control regions, including the cerebellum. There is substantial evidence of cerebellar CB1R molecular adaptation and modifications in receptor signaling after prolonged cannabinoid exposure. However, very little is known about the effects of chronic cannabinoid administration on cerebellar synaptic plasticity, which may contribute to the development of cannabinoid behavioral tolerance.

The guanine nucleotide exchange factor RasGRF1 directly binds microtubules via DHPH2-mediated interaction.

RasGRF is a family of guanine nucleotide exchange factors with dual specificity for both Ras and Rac GTPases. In this study, using mouse brain extracts, we show that both RasGRF1 and RasGRF2 interact with microtubules in an in vitro microtubule assembly system and this binding is very tight. To characterize this association, recombinant purified proteins containing different regions of RasGRF1 were tested for their ability to bind microtubules preassembled from pure tubulin.

Ras/ERK signalling in cannabinoid tolerance: from behaviour to cellular aspects.

We investigated the role of the Ras/extracellular-regulated kinase (ERK) pathway in the development of tolerance to Delta(9)-tetrahydrocannabinol (THC)-induced reduction in spontaneous locomotor activity by a genetic (Ras-specific guanine nucleotide exchange factor (Ras-GRF1) knock-out mice) and pharmacological approach. Pre-treatment of wild-type mice with SL327 (50 mg/kg i.p.

Modulation of extracellular signal-regulated kinases cascade by chronic delta 9-tetrahydrocannabinol treatment.

Acute Delta(9)-tetrahydrocannabinol (THC) injection increased ERK pathway (ERK, pCREB, and c-fos) mostly in the caudate putamen and cerebellum. This effect underwent to homeostatic adaptation after chronic treatment. Moreover, chronic THC exposure induced increases in the ERK cascade (ERK, pCREB, and Fos B) in the prefrontal cortex and hippocampus, suggesting that different neuronal circuits seem to be involved in the early phase and late phase of exposure.

Depolarization-induced signaling to Ras, Rap1 and MAPKs in cortical neurons.

In neurons, membrane depolarization triggers pleiotropic signaling which includes the activation of the small GTPases, Ras and Rap1, and the mitogen-activated protein kinases (MAPKs) Erk1/2. We have studied the intracellular signaling mechanisms which regulate these events in mouse-cultured cortical neurons. We show that depolarization induces activation of both Ras and Rap1, although with different kinetics: Ras activation is strong and fast while Rap1 activation is slower and weaker.

Modulation of the inward rectifier potassium channel IRK1 by the Ras signaling pathway.

In this study, we investigated the role of Ras and the mitogen-activated protein kinase (MAPK) pathway in the modulation of the inward rectifier potassium channel IRK1. We show that although expression of IRK1 in HEK 293 cells leads to the appearance of a potassium current with strong inward rectifying properties, coexpression of the constitutively active form of Ras (Ras-L61) results in a significant reduction of the mean current density without altering the biophysical properties of the channel. The inhibitory effect of Ras-L61 is not due to a decreased expression of IRK1 since Northern analysis indicates that IRK1 mRNA level is not affected by Ras-L61 co-expression.