966 resultados para 14-3-3 Proteins
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G-protein-coupled receptors are desensitized by a two-step process. In a first step, G-protein-coupled receptor kinases (GRKs) phosphorylate agonist-activated receptors that subsequently bind to a second class of proteins, the arrestins. GRKs can be classified into three subfamilies, which have been implicated in various diseases. The physiological role(s) of GRKs have been difficult to study as selective inhibitors are not available. We have used SELEX (systematic evolution of ligands by exponential enrichment) to develop RNA aptamers that potently and selectively inhibit GRK2. This process has yielded an aptamer, C13, which bound to GRK2 with a high affinity and inhibited GRK2-catalyzed rhodopsin phosphorylation with an IC50 of 4.1 nM. Phosphorylation of rhodopsin catalyzed by GRK5 was also inhibited, albeit with 20-fold lower potency (IC50 of 79 nM). Furthermore, C13 reveals significant specificity, since almost no inhibitory activity was detectable testing it against a panel of 14 other kinases. The aptamer is two orders of magnitude more potent than the best GRK2 inhibitors described previously and shows high selectivity for the GRK family of protein kinases.
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Retrograde transport of NF-κB from the synapse to the nucleus in neurons is mediated by the dynein/dynactin motor complex and can be triggered by synaptic activation. The calibre of axons is highly variable ranging down to 100 nm, aggravating the investigation of transport processes in neurites of living neurons using conventional light microscopy. In this study we quantified for the first time the transport of the NF-κB subunit p65 using high-density single-particle tracking in combination with photoactivatable fluorescent proteins in living mouse hippocampal neurons. We detected an increase of the mean diffusion coefficient (Dmean) in neurites from 0.12 ± 0.05 µm2/s to 0.61 ± 0.03 µm2/s after stimulation with glutamate. We further observed that the relative amount of retrogradely transported p65 molecules is increased after stimulation. Glutamate treatment resulted in an increase of the mean retrograde velocity from 10.9 ± 1.9 to 15 ± 4.9 µm/s, whereas a velocity increase from 9 ± 1.3 to 14 ± 3 µm/s was observed for anterogradely transported p65. This study demonstrates for the first time that glutamate stimulation leads to an increased mobility of single NF-κB p65 molecules in neurites of living hippocampal neurons.
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Aims: In our previous work, we reported that the insulin potentiating effect on melatonin synthesis is regulated by a post-transcriptional mechanism. However, the major proteins of the insulin signaling pathway (ISP) and the possible pathway component recruited on the potentiating effect of insulin had not been characterized. A second question raised was whether windows of sensitivity to insulin exist in the pineal gland due to insulin rhythmic secretion pattern. Main methods: Melatonin content from norepinephrine(NE)-synchronized pineal gland cultures was quantified by high performance liquid chromatography with electrochemical detection and arylalkylamine-N-acetyltransferase (AANAT) activity was assayed by radiometry. Immunoblotting and immunoprecipitation techniques were performed to establish the ISP proteins expression and the formation of 14-3-3: AANAT complex, respectively. Key findings: The temporal insulin susceptibility protocol revealed two periods of insulin potentiating effect, one at the beginning and another one at the end of the in vitro induced ""night"". In some Timed-insulin Stimulation (TSs), insulin also promoted a reduction on melatonin synthesis, showing its dual action in cultured pineal glands. The major ISP components, such as IR beta, IGF-1R, IRS-1, IRS-2 and PI3K(p85), as well tyrosine phosphorylation of pp85 were characterized within pineal glands. Insulin is not involved in the 14-3-3:AANAT complex formation. The blockage of PI3K by LY 294002 reduced melatonin synthesis and AANAT activity. Significance: The present study demonstrated windows of differential insulin sensitivity, a functional ISP and the PI3K-dependent insulin potentiating effect on NE-mediated melatonin synthesis, supporting the hypothesis of a crosstalk between noradrenergic and insulin pathways in the rat pineal gland. (C) 2010 Elsevier Inc. All rights reserved.
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Sugarcane is one of the most important products of the world and Brazil is responsible for 25 % of the world production. One problem of this culture at northeast of Brazil is the early flowering. In our laboratory, it has been made before four subtractive libraries using early and late flowering genotypes in order to identify messages related to the flowering process. In this work, two cDNAs were chosen to make in silico analysis and overexpression constructs. Another approach to understand the flowering process in sugarcane was to use proteomic tools. First, the protocol for protein extraction using apical meristem was set up. After that, these proteins were separated on two bidimensional gels. It was possible to observe some difference for some regions of these gels as well as some proteins that can be found in all conditions. The next step, spots will be isolated and sequence on MS spectrometry in order to understand this physiological process in sugarcane
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The capture web of N. clavipes presents viscous droplets, which play important roles in web mechanics and prey capture. By using scanning and transmission electron microscopy, it was demonstrated that the web droplets are constituted of different chemical environments, provided by the existence both of an aqueous and a lipid layer, which, in turn, present a suspension of tenths of vesicles containing polypeptides and/or tipids. GC/EI-MS Analysis of the contents of these vesicles led to the identification of some saturated fatty acids, such as decanoic acid, undecanoic acid, dodecanoic acid, tetradecanoic acid, octadecanoic acid, and icosanoic acid, while other components were unsaturated fatty acids, such as (Z)-tetradec-9-enoic acid, (Z)-octadec-9-enoic acid, and (Z)-icosa-11-enoic acid; and polyunsaturated fatty acids like (9Z,12Z)-octadeca-9,12-dienoic acid, (9Z,12Z,15Z)-octadeca-9,12,15-trienoic acid, and (11Z,14Z)-icosa-11,14-dienoic acid. Toxic proteins such as calcium-activated proteinase and metalloproteinase jararhagin-like precursor were also identified by using a proteomic approach, indicating the possible involvement of these enzymes in the pre-digestion of spiders' preys web-captured. Apparently, the mixture of fatty acids are relatively toxic to insects by topical application (LD50 64.3 +/- 7.6 ng mg(-1) honeybee), while the proteins alone present no topical effect; however, when injected into the prey-insects, these proteins presented a moderate toxicity (LD50 40.3 +/- 4.8 ng mg(-1) honeybee); the mixture of fatty acids and proteins is very toxic to the preys captured by the web droplets of the viscid spiral of Nephila clavipes when topically applied on them (LD50 14.3 +/- 1.8ng mg(-1) honeybee).
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In the yeast Saccharomyces cerevisiae a novel control exerted by TPS1 (=GGS1=FDP1=BYP1=CIF1=GLC6=TSS1)-encoded trehalose-6-phosphate synthase, is essential for restriction of glucose influx into glycolysis apparently by inhibiting hexokinase activity in vivo. We show that up to 50-fold overexpression of hexokinase does not noticeably affect growth on glucose or fructose in wild-type cells. However, it causes higher levels of glucose-6-phosphate, fructose-6-phosphate and also faster accumulation of fructose-1,6-bisphosphate during the initiation of fermentation. The levels of ATP and Pi correlated inversely with the higher sugar phosphate levels. In the first minutes after glucose addition, the metabolite pattern observed was intermediate between those of the tps1Δ mutant and tile wild-type strain. Apparently, during the start-up of fermentation hexokinase is more rate-limiting in the first section of glycolysis than phosphofructokinase. We have developed a method to measure the free intracellular glucose level which is based on the simultaneous addition of D-glucose and an equal concentration of radiolabelled L-glucose. Since the latter is not transported, the free intracellular glucose level can be calculated as the difference between the total B-glucose measured (intracellular + periplasmic/extracellular) and the total L-glucose measured (periplasmic/extracellular). The intracellular glucose level rose in 5 min after addition of 100 mM-glucose to 0.5-2 mM in the wild-type strain, ± 10 mm in a hxk1Δ hxk2Δ glk1Δ and 2-3 mM in a tps1Δ strain. In the strains overexpressing hexokinase PII the level of free intracellular glucose was not reduced. Overexpression of hexokinase PII never produced a strong effect on the rate of ethanol production and glucose consumption. Our results show that overexpression of hexokinase does not cause the same phenotype as deletion of Tps1. However, it mimics it transiently during the initiation of fermentation. Afterwards, the Tps1-dependent control system is apparently able to restrict Properly up to 50-fold higher hexokinase activity.
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Many plants, even without scientific confirmation, are used in Brazil as hypoglycemic. The objective of the present work was to study the influence of the Allium sativum treatment, plant known popularly as garlic, on characteristic biochemical parameters in streptozotocin-induced diabetic rats. Female Wistar rats were injected with 40 mg kg-1 streptozotocin (STZ). Oral administration of an aqueous extract of A. sativum was given to the diabetic animals in 2 doses: 200 and 400 mg kg-1 (n=6 animals/group). Diabetic rats given distilled water constituted the control group. After 28 days of treatment, the female rats were anesthetized and died by decapitation for collection of the blood for biochemical determinations and retreat of liver samples for hepatic glycogen dosage. The treatment with Allium sativum in the doses of 200 and 400 mg kg-1 alter no concentrations of total proteins, hepatic glycogen, triglycerides and VLDL, but it promoted reduction in the total cholesterol rate (control=280.5 ± 30.9; 200 mg kg-1 A. sativum =169.9 ± 19.5 and 400 mg kg-1 A. sativum =148.4 ± 26.6 mg dL-1) and LDL (control=128.8± 25.3; 200 mg kg-1 A. sativum = 41.4 ± 16.2 and 400 mg kg-1 A. sativum=42.0 ± 26.0 mg dL-1). The extract presented beneficial effect because it decreased 13.0% of glycemia in the highest dose. Therefore, the of garlic extract reduced the HDL concentration in two tested doses (control= 81.4 ± 30.2; 200 mg kg-1 A. sativum=49.6 ± 14.3 and 400 mg kg-1 A. sativum=41.7 ± 16.1 mg dL -1), presenting impaired effect. Thus, the garlic extract showed efficiency in the analyzed experimental conditions, and it could be used as complementary therapy to diabetic patients.
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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
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Akt (also called PKB) is a 63 kDa serine/threonine kinase involved in promotion of cell survival, proliferation a nd metabolic responses downstream the phosphoinositide-3-kinase (PI 3-kinase) signaling pathway. In resting cells, Akt is a predominantly cytosolic enzyme; however generation of PI 3-kinase lipid products recruits Akt to the plasma membrane, resulting in a conformational change which confers full enzymatic activity through the phosphorylation of the membrane-bound protein at two residues, Thr308, and Ser473. Activated Akt redistributes to cytoplasm and nucleus, where phosphorylation of specific substrates occurs. Both the presence and the activity of Akt in the nucleus have been described. An interesting mechanism that mediates nuclear translocation of Akt has been described in human mature T-cell leukemia: the product of TCL1 gene, Tcl1, interacts with the PH domain of phosphorylated Akt, thus driving Akt to the nucleus. In this context, Tcl1 may act as a direct transporter of Akt or may contribute to the formation of a complex that promotes the transport of active Akt to the nucleus, where it can phosphorylate nuclear substrates. A well described nuclear substrate if Foxo. IGF-1 triggers phosphorylation of Foxo by Akt inside the nucleus, where phospho-Foxo associates to 14.3.3 proteins that, in turn, promote its export to the cytoplasm where it is sequestered. Remarkably, Foxo phosphorylation by Akt has been shown to be a crucial event in Akt-dependent myogenesis. However, most Akt nuclear substrates have so far remained elusive, as well as nuclear Akt functions. This lack of information prompted us to undertake a search of substrates of Akt in the nucleus, by the combined use of 2D-separation/mass spectrometry and anti-Akt-phosphosubstrate antibody. This study presents evidence of A-type lamins as novel nuclear substrates of Akt. Lamins are type V intermediate filaments proteins found in the nucleus of higher eukaryotes where, together with lamin-binding proteins, they form the lamina at the nuclear envelope, providing mechanical stability for the nuclear membrane. By coimmunoprecipitation, it is demonstrated here that endogenous lamin A and Akt interact, and that A-type lamins are phosphorylated by Akt both in vitro and in vivo. Moreover, by phosphoaminoacid analysis and mutagenesis, it is further demonstrated that Akt phosphorylates lamin A at Ser404, and, more importantly, that while lamin A/C phosphorylation is stable throughout the cell cycle, phosphorylation of the precursor prelamin A becomes detectable as cells enter the G2 phase, picking at G2/M. This study also shows that lamin phosphorylation by Akt creates a binding site for 14.3.3 adaptors which, in turn, promote prelamin A degradation. While this mechanism is in agreement with a general role of Akt in the regulation of a subset of its substrates, opposite to what has been described, degradation is not mediated through a ubiquitination and proteasomal mechanism but through a lysosomal pathway, as indicated by the reverting action of the lysosomal inhibitor cloroquine. Phosphorylation is a key event in the mitotic breakdown of the nuclear lamina. However, the kinases and the precise sites of phosphorylation are scarcely known. Therefore, these results represent an important breakthrough in this very significant but understudied area. The phosphorylation of the precursor protein prelamin A and its subsequent degradation at G2/M, when both the nuclear envelop and the nuclear lamina disassemble, can be view as part of a mechanism to dispose off the precursor that is not needed in this precise context. The recently reported finding that patients affected by Emery-Dreifuss muscular dystrophy carry a mutation at Arg 401, in the Akt phosphorylation motif, open new perspective that warrant further investigation in this very important field.
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Ziel: Die Radiotherapie hat in der Behandlung von Plattenepithelkarzinomen des Kopf- und Halsbereichs nach wie vor einen hohen Stellenwert. Der Erfolg eines Therapieregimes, das die Behandlung mit ionisierenden Strahlen einschließt, ist jedoch häufig limitiert durch die Entwicklung radioresistenter Tumorzellpopulationen, die nicht selten durch die Bestrahlung selbst induziert wird. Die Mechanismen, die zu einer solchen bestrahlungsinduzierten Radioresistenz führen sind bisher nur unvollständig verstanden und Methoden, durch die die Entwicklung von Radioresistenz verhindert werden könnte, wie beispielsweise der präventive Einsatz von Pharmazeutika, sind bislang nicht systematisch untersucht. Das Ziel der vorliegenden Arbeit war es zu überprüfen, ob der Cyclooxygenase-Inhibitor Flurbiprofen durch Bestrahlung induzierte Veränderungen der Phosphoprotein-Expression verstärken oder abschwächen kann und ob sich aus solchen Modifikationen des Bestrahlungsergebnisses ein radioprotektiver Effekt der Flurbiprofenapplikation ableiten lässt. Methoden: Es wurde ein experimenteller Ansatz gewählt, der mittels 2D PAGE und anschließender MALDI-TOF Massenspektrometrie das Phosphoproteom einer HNSCC-Zelllinie unter verschiedenen Bedingungen untersuchte. Die Zellen wurden entweder mit einer Energiedosis von 8 Gy bestrahlt, mit einer 200 μM Flurbiprofen enthaltenden Lösung inkubiert oder sie wurden mit einer Kombination aus Flurbiprofenapplikation und Bestrahlung behandelt. Vor der 2D PAGE wurden die Phosphoproteine durch IMAC angereichert. Zur Verbesserung der Gel-Analytik wurde die Software Delta 2D angewendet, die zum Ausgleich von Laufweitenunterschieden zwischen den Gelen ein Warping vorsieht. Ergebnisse und Diskussion: Bei der Analyse, der unter den verschiedenen experimentellen Bedingungen differentiell exprimierten Phosphoproteinen mittels bioinformatischer Hilfsprogramme wie z.B. WEBGestalt und STRING, wurden sieben Proteine mit Bedeutung für das Wachstum und die Entdifferenzierung von Tumoren identifiziert und einer ausführlichen Literaturrecherche unterzogen. Auf diese Weise konnten die Ergebnisse der für die vorliegende Arbeit durchgeführten Experimente in den systembiologischen Kontext eingeordnet werden. Besonders hervorzuheben ist die Herabregulierung der möglicherweise Radioresistenz vermittelnden Proteine GRP-75, 14-3-3 sigma und CRT sowie die Herabregulierung des anti-apoptotischen und tumor-begünstigenden Hsp60 durch Flurbiprofen. Die Verminderung der Expression unterstreicht das Potential dieses Pharmakons sowie der Klasse der COX-Inhibitoren als mögliche radiosensitivierende und tumorsuppressive Substanzen.
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The cardiac voltage-gated Na(+) channel Na(v)1.5 generates the cardiac Na(+) current (INa). Mutations in SCN5A, the gene encoding Na(v)1.5, have been linked to many cardiac phenotypes, including the congenital and acquired long QT syndrome, Brugada syndrome, conduction slowing, sick sinus syndrome, atrial fibrillation, and dilated cardiomyopathy. The mutations in SCN5A define a sub-group of Na(v)1.5/SCN5A-related phenotypes among cardiac genetic channelopathies. Several research groups have proposed that Na(v)1.5 may be part of multi-protein complexes composed of Na(v)1.5-interacting proteins which regulate channel expression and function. The genes encoding these regulatory proteins have also been found to be mutated in patients with inherited forms of cardiac arrhythmias. The proteins that associate with Na(v)1.5 may be classified as (1) anchoring/adaptor proteins, (2) enzymes interacting with and modifying the channel, and (3) proteins modulating the biophysical properties of Na(v)1.5 upon binding. The aim of this article is to review these Na(v)1.5 partner proteins and to discuss how they may regulate the channel's biology and function. These recent investigations have revealed that the expression level, cellular localization, and activity of Na(v)1.5 are finely regulated by complex molecular and cellular mechanisms that we are only beginning to understand.
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Major surface protein 5 (Msp5) of Anaplasma marginale is highly conserved in the genus Anaplasma and the antigen used in a commercially available competitive enzyme-linked immunosorbent assay (cELISA) for serologic identification of cattle with anaplasmosis. This study analyzes the degrees of conservation of Msp5 among various isolates of Anaplasma phagocytophilum and the extent of serologic cross-reactivity between recombinant Msp5 (rMsp5) of Anaplasma marginale and A. phagocytophilum. The msp5 genes from various isolates of A. phagocytophilum were sequenced and compared. rMsp5 proteins of A. phagocytophilum and A. marginale were used separately in an indirect ELISA to detect cross-reactivity in serum samples from humans and dogs infected with A. phagocytophilum and cattle infected with A. marginale. Serum samples were also tested with a commercially available competitive ELISA that uses monoclonal antibody ANAF16C1. There were 100% sequence identities in the msp5 genes among all of the A. phagocytophilum isolates from the United States and a horse isolate from Sweden. Sheep isolates from Norway and dog isolates from Sweden were 99% identical to one another but differed in 17 base pairs from the United States isolates and the horse isolate. Serologic cross-reactivity was identified when serum samples from cattle infected with A. marginale were reacted with rMsp5 of A. phagocytophilum and when serum samples from humans and dogs infected with A. phagocytophilum were reacted with rMsp5 of A. marginale in an indirect-ELISA format. Serum samples from dogs or humans infected with A. phagocytophilum did not cross-react with rMsp5 of A. marginale when tested with the commercially available cELISA. These results suggest that rMsp5 of A. phagocytophilum is highly conserved among United States and European isolates and that serologic distinction between A. phagocytophilum and A. marginale infections cannot be accomplished if rMsp5 from either organism is used in an indirect ELISA.