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1.
RNA ; 2021 May 05.
Artículo en Inglés | MEDLINE | ID: mdl-33952671

RESUMEN

The function of an RNA is intimately linked to its three-dimensional structure. X-ray crystallography or NMR allow the fine structural characterization of small RNA (e.g., aptamers) with a precision down to atomic resolution. Yet, these technics are time consuming, laborious and do not inform on mutational robustness and the extent to which a sequence can be modified without altering RNA function, an important set of information to assist RNA engineering. On another hand, thought powerful, in silico predictions still lack the required accuracy. These limitations can be overcome by using high-throughput microfluidic-assisted functional screening technologies, as they allow exploring large mutant libraries in a rapid and cost-effective manner. Among them, we recently introduced the microfluidic-assisted In Vitro Compartmentalization (µIVC), an efficient screening strategy in which reactions are performed in picoliter droplets at rates of several thousand per second. We later improved µIVC efficiency by using in tandem with high throughput sequencing, thought a laborious bioinformatic step was still required at the end of the process. In the present work, we strongly increased the automation level of the pipeline by implementing an artificial neural network enabling unsupervised bioinformatic analysis. We demonstrate the efficiency of this "µIVC-Useq" technology by rapidly identifying a set of sequences readily accepted by a key domain of the light-up RNA aptamer SRB-2. This work not only shed some new light on the way this aptamer can be engineered, but it also allowed us to easily identify new variants with an up-to 10-fold improved performance.

2.
Adv Exp Med Biol ; 1379: 445-460, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35761003

RESUMEN

DNA is widely used as a biomarker of contamination, infection, or disease, which has stimulated the development of a wide palette of detection and quantification methods. Even though several analytical approaches based on isothermal amplification have been proposed, DNA is still mainly detected and quantified by quantitative PCR (qPCR). However, for some analyses (e.g., in cancer research) qPCR may suffer from limitations arising from competitions between highly similar template DNAs, the presence of inhibitors, or suboptimal primer design. Nevertheless, digitalizing the analysis (i.e., individualizing DNA molecules into compartments prior to amplifying them in situ) allows to address most of these issues. By its capacity to generate and manipulate millions of highly similar picoliter volume water-in-oil droplets, microfluidics offers both the required miniaturization and parallelization capacity, and led to the introduction of digital droplet PCR (ddPCR). This chapter aims at introducing the reader to the basic principles behind ddPCR while also providing the key guidelines to fabricate, set up, and use his/her own ddPCR platform. We further provide procedures to detect and quantify DNA either purified in solution or directly from individualized cells. This approach not only gives access to DNA absolute concentration with unrivaled sensitivity, but it may also be the starting point of more complex in vitro analytical pipelines discussed at the end of the chapter.


Asunto(s)
ADN , Microfluídica , ADN/genética , Femenino , Humanos , Masculino , Reacción en Cadena en Tiempo Real de la Polimerasa
3.
Methods ; 161: 46-53, 2019 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-30902664

RESUMEN

Biosensors are biological molecules able to detect and report the presence of a target molecule by the emission of a signal. Nucleic acids are particularly appealing for the design of such molecule since their great structural plasticity makes them able to specifically interact with a wide range of ligands and their structure can rearrange upon recognition to trigger a reporting event. A biosensor is typically made of three main domains: a sensing domain that is connected to a reporting domain via a communication module in charge of transmitting the sensing event through the molecule. The communication module is therefore an instrumental element of the sensor. This module is usually empirically developed through a trial-and-error strategy with the testing of only a few combinations judged relevant by the experimenter. In this work, we introduce a novel method combining the use of droplet-based microfluidics and next generation sequencing. This method allows to functionally characterize up to a million of different sequences in a single set of experiments and, by doing so, to exhaustively test every possible sequence permutations of the communication module. Here, we demonstrate the efficiency of the approach by isolating a set of optimized RNA biosensors able to sense theophylline and to convert this recognition into fluorescence emission.


Asunto(s)
Técnicas Biosensibles/métodos , Colorantes Fluorescentes/análisis , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Microfluídica/métodos , ARN/análisis , ARN/genética , Técnicas Biosensibles/normas , Secuenciación de Nucleótidos de Alto Rendimiento/normas , Microfluídica/normas
4.
Cell Microbiol ; 19(8)2017 08.
Artículo en Inglés | MEDLINE | ID: mdl-28256794

RESUMEN

Dormant liver stage forms (hypnozoites) of the malaria parasite Plasmodium vivax present major hurdles to control and eradicate infection. Despite major research efforts, the molecular composition of hypnozoites remains ill defined. Here, we applied a combination of state-of-the-art technologies to generate the first transcriptome of hypnozoites. We developed a robust laser dissection microscopy protocol to isolate individual Plasmodium cynomolgi hypnozoites and schizonts from infected monkey hepatocytes and optimized RNA-seq analysis to obtain the first transcriptomes of these stages. Comparative transcriptomic analysis identified 120 transcripts as being differentially expressed in the hypnozoite stage relative to the dividing liver schizont, with 69 and 51 mRNAs being up- or down-regulated, respectively, in the hypnozoites. This lead to the identification of potential markers of commitment to and maintenance of the dormant state of the hypnozoite including three transcriptional regulators of the ApiAP2 family, one of which is unique to P. cynomolgi and P. vivax, and the global translational repressor, eIF2a kinase eIK2, all of which are upregulated in the hypnozoite. Together, this work not only provides a primary experimentally-derived list of molecular markers of hypnozoites but also identifies transcriptional and posttranscriptional regulation of gene expression as potentially being key to establishing and maintaining quiescence.


Asunto(s)
Perfilación de la Expresión Génica , Interacciones Huésped-Patógeno , Hígado/parasitología , Plasmodium cynomolgi/fisiología , Animales , Haplorrinos , Hepatocitos/parasitología , Captura por Microdisección con Láser
5.
J Neurochem ; 137(6): 913-30, 2016 06.
Artículo en Inglés | MEDLINE | ID: mdl-26991551

RESUMEN

Astroglial cells possess an array of cellular defense mechanisms, including superoxide dismutase (SOD) and catalase antioxidant enzymes, to prevent damages caused by oxidative stress. Nevertheless, astroglial cell viability and functionality can be affected by significant oxidative stress. We have previously shown that pituitary adenylate cyclase-activating polypeptide (PACAP) is a potent glioprotective agent that prevents hydrogen peroxide (H2 O2 )-induced apoptosis in cultured astrocytes. The purpose of this study was to investigate the potential protective effect of PACAP against oxidative-generated alteration of astrocytic antioxidant systems. Incubation of cells with subnanomolar concentrations of PACAP inhibited H2 O2 -evoked reactive oxygen species accumulation, mitochondrial respiratory burst, and caspase-3 mRNA level increase. PACAP also stimulated SOD and catalase activities in a concentration-dependent manner, and counteracted the inhibitory effect of H2 O2 on the activity of these two antioxidant enzymes. The protective action of PACAP against H2 O2 -evoked inhibition of antioxidant systems in astrocytes was protein kinase A, PKC, and MAP-kinase dependent. In the presence of H2 O2 , the SOD blocker NaCN and the catalase inhibitor 3-aminotriazole, both suppressed the protective effects of PACAP on SOD and catalase activities, mitochondrial function, and cell survival. Taken together, these results indicate that the anti-apoptotic effect of PACAP on astroglial cells can account for the activation of endogenous antioxidant enzymes and reduction in respiration rate, thus preserving mitochondrial integrity and preventing caspase-3 expression provoked by oxidative stress. Considering its powerful anti-apoptotic and anti-oxidative properties, the PACAPergic signaling system should thus be considered for the development of new therapeutical approaches to cure various pathologies involving oxidative neurodegeneration. We propose the following cascade for the glioprotective action of Pituitary adenylate cyclase-activating polypeptide (PACAP) against H2 O2 -induced astrocyte damages and cell apoptosis in cultured rat astrocytes. PACAP, through activation of its receptor, PAC1-R, and the protein kinase A (PKA), protein kinase C (PKC), and MAP-kinases signaling pathways, prevents accumulation of ROS and inhibition of SOD and catalase activities. This allows the preservation of mitochondrial membrane integrity and the reduction in caspase-3 activation induced by H2 O2 . These data may lead to the development of new strategies for cerebral injury treatment. Cat, catalase; Cyt. C, cytochrome C; SOD, superoxide dismutase.


Asunto(s)
Antioxidantes/farmacología , Astrocitos/efectos de los fármacos , Peróxido de Hidrógeno/toxicidad , Oxidantes/toxicidad , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/farmacología , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Antioxidantes/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Catalasa/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/citología , Femenino , Proteína Ácida Fibrilar de la Glía/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Proteínas del Tejido Nervioso/metabolismo , Factor de Transcripción 2 de los Oligodendrocitos , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Superóxido Dismutasa/metabolismo , Superóxidos/metabolismo , Factores de Tiempo
6.
Biochim Biophys Acta ; 1828(11): 2385-93, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-23817010

RESUMEN

Detergent-resistant membranes (DRM), an experimental model used to study lipid rafts, are typically extracted from cells by means of detergent treatment and subsequent ultracentrifugation in density gradients, Triton X-100 being the detergent of choice in most of the works. Since lipid rafts are membrane microdomains rich in cholesterol, depletion of this component causes solubilization of DRM with detergent. In previous works from our group, the lack of effect of cholesterol depletion on DRM solubilization with Triton X-100 was detected in isolated rat brain synaptosomes. In consequence, the aim of the present work is to explore reasons for this observation, analyzing the possible role of the actin cytoskeleton, as well as the use of an alternative detergent, Brij 98, to overcome the insensitivity to Triton X-100 of cholesterol-depleted DRM. Brij 98 yields Brij-DRM that are highly dependent on cholesterol, since marker proteins (Flotillin-1 and Thy-1), as well as actin, appear solubilized after MCD treatment. Pretreatment with Latrunculin A results in a significant increase in Flotillin-1, Thy-1 and actin solubilization by Triton X-100 after cholesterol depletion. Studies with transmission electron microscopy show that combined treatment with MCD and Latrunculin A leads to a significant increase in solubilization of DRM with Triton X-100. Thus, Triton-DRM resistance to cholesterol depletion can be explained, at least partially, thanks to the scaffolding action of the actin cytoskeleton, without discarding differential effects of Brij 98 and Triton X-100 on specific membrane components. In conclusion, the detergent of choice is important when events that depend on the actin cytoskeleton are going to be studied.


Asunto(s)
Citoesqueleto de Actina/efectos de los fármacos , Detergentes/farmacología , Animales , Ratas , Ratas Sprague-Dawley , Solubilidad , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismo , Temperatura
7.
Biochim Biophys Acta ; 1811(12): 1124-35, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-22020259

RESUMEN

Brain specific kinases 1 and 2 (BRSK1/2, also named SAD kinases) are serine-threonine kinases specifically expressed in the brain, and activated by LKB1-mediated phosphorylation of a threonine residue at their T-loop (Thr189/174 in human BRSK1/2). BRSKs are crucial for establishing neuronal polarity, and BRSK1 has also been shown to regulate neurotransmitter release presynaptically. How BRSK1 exerts this latter function is unknown, since its substrates at the synaptic terminal and the mechanisms modulating its activity remain to be described. Key regulators of neurotransmitter release, such as SNARE complex proteins, are located at membrane rafts. Therefore we initially undertook this work to check whether BRSK1 also locates at these membrane microdomains. Here we show that brain BRSK1, but not BRSK2, is palmitoylated, and provide biochemical and pharmacological evidences demonstrating that a pool of BRSK1, but not BRSK2 or LKB1, localizes at membrane lipid rafts. We also show that raft-associated BRSK1 has higher activity than BRSK1 from non-raft environment, based on a higher T-loop phosphorylation at Thr-189. Further, recombinant BRSK1 activity increased 3-fold when assayed with small multilamellar vesicles (SMV) generated with lipids extracted from synaptosomal raft fractions. A similar BRSK1-activating effect was obtained with synthetic SMV made with phosphatidylcholine, cholesterol and sphingomyelin, mixed in the same molar ratio at which these three major lipids are present in rafts. Importantly, SMV also enhanced the activity of a constitutively active BRSK1 (T189E), underpinning that interaction with lipid rafts represents a new mechanism of BRSK1 activity modulation, additional to T-loop phosphorylation.


Asunto(s)
Encéfalo/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Microdominios de Membrana/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes/metabolismo , Transducción de Señal/fisiología , Transmisión Sináptica/fisiología , Sinaptosomas/metabolismo , Quinasas de la Proteína-Quinasa Activada por el AMP , Animales , Baculoviridae , Escherichia coli , Feto , Células HEK293 , Humanos , Lipoilación , Membranas Artificiales , Ratones , Fosforilación , Estructura Secundaria de Proteína , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/genética , Treonina/metabolismo
8.
Methods Mol Biol ; 2300: 203-237, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33792882

RESUMEN

For a long time, artificial RNAs have been developed by in vitro selection methodologies like Systematic Evolution of Ligands by EXponential enrichment (SELEX). Yet, even though this technology is extremely powerful to isolate specific and high-affinity binders, it is less suited for the isolation of RNAs optimized for more complex functions such as fluorescence emission or multiple-turnover catalysis. Whereas such RNAs should ideally be developed by screening approaches, conventional microtiter plate assays become rapidly cost-prohibitive. However, the advent of droplet-based microfluidics recently enabled us to devise microfluidic-assisted In Vitro Compartmentalization (µIVC), a strongly miniaturized and highly parallelized screening technology allowing to functionally screen millions of mutants in a single day while using a very low amount of reagent. Used in combination with high-throughput sequencing, the resulting µIVC-seq pipeline described in this chapter now allows rapid and semiautomated screening to be performed at low cost and in an ultrahigh-throughput regime.


Asunto(s)
Microfluídica/métodos , ARN Mensajero/análisis , Análisis de Secuencia de ARN/métodos , Biología Computacional/métodos , Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Miniaturización , Mutación
9.
FEBS Lett ; 581(9): 1851-8, 2007 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-17433308

RESUMEN

Protein ectodomain shedding is the proteolytic release of the extracellular domain of membrane-bound proteins. Neurotrophin receptor p75(NTR) is known to be affected by shedding. The present work provides evidence, in rat brain synaptosomes, that p75(NTR) is present in detergent-resistant membranes (DRM), also known as lipid rafts, only in its full-length form. Disrupting the integrity of lipid rafts causes solubilization of p75(NTR) after detergent treatment and enhancement of the shedding. Analyses of the enzymes described as being responsible for p75(NTR) shedding, i.e. tumor necrosis factor alpha convertase (TACE) and presenilin-1 (PS1), revealed that TACE is absent in DRM, while variable proportions of the C-terminal and N-terminal fragments of PS1 are found. In summary, our results point to a role of lipid rafts in the modulation of the shedding of the p75(NTR) receptor.


Asunto(s)
Microdominios de Membrana/fisiología , Receptor de Factor de Crecimiento Nervioso/metabolismo , Proteínas ADAM/metabolismo , Proteína ADAM17 , Animales , Colesterol/fisiología , Detergentes/farmacología , Humanos , Terminaciones Nerviosas/efectos de los fármacos , Terminaciones Nerviosas/metabolismo , Presenilina-1/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Ratas , Ratas Sprague-Dawley
10.
J Med Chem ; 48(6): 1781-95, 2005 Mar 24.
Artículo en Inglés | MEDLINE | ID: mdl-15771424

RESUMEN

Based on a medicinal chemistry guided hypothetical pharmacophore model, novel series of indolyl sulfonamides have been designed and prepared as selective and high-affinity serotonin 5-HT(6) receptor ligands. Furthermore, based on a screening approach of a discovery library, a series of benzoxazinepiperidinyl sulfonamides were identified as selective 5-HT(6) ligands. Many of the compounds described in this paper possess excellent affinities, displaying pK(i) values greater than 8 (some even >9) and high selectivities against a wide range (>50) of other CNS relevant receptors. First, structure-affinity relationships of these ligands are discussed. In terms of functionality, high-affinity antagonists, as well as agonists and even partial agonists, were prepared. Compounds 19c and 19g represent the highest-affinity 5-HT(6) agonists ever reported in the literature. These valuable tool compounds should allow for the detailed study of the role of the 5-HT(6) receptor in relevant animal models of disorders such as cognition deficits, depression, anxiety, or obesity.


Asunto(s)
Indoles/síntesis química , Receptores de Serotonina/efectos de los fármacos , Antagonistas de la Serotonina/síntesis química , Agonistas de Receptores de Serotonina/síntesis química , Sulfonamidas/síntesis química , Adenilil Ciclasas/biosíntesis , Benzoxazinas/síntesis química , Benzoxazinas/química , Línea Celular , Humanos , Indoles/química , Indoles/farmacología , Ligandos , Piperidinas/síntesis química , Piperidinas/química , Ensayo de Unión Radioligante , Antagonistas de la Serotonina/química , Antagonistas de la Serotonina/farmacología , Agonistas de Receptores de Serotonina/química , Agonistas de Receptores de Serotonina/farmacología , Relación Estructura-Actividad , Sulfonamidas/química , Sulfonamidas/farmacología
11.
FEBS Lett ; 588(1): 167-74, 2014 Jan 03.
Artículo en Inglés | MEDLINE | ID: mdl-24316227

RESUMEN

Neurotrophins are a group of secreted polypeptides, which comprises Nerve Growth Factor (NGF) and Brain-Derived Neurotrophic Factor (BDNF). Each neurotrophin can bind specifically to a tyrosine kinase Trk receptor (TrkA, TrkB or TrkC), while all of the neurotrophins can bind, with similar affinity, to the p75 neurotrophin receptor (p75(NTR)). Experiments on cell viability promotion by BDNF in granule neurons or by NGF in PC12 cells show that neurotrophin-exerted cell viability is neutral sphingomyelinase (nSMase)-dependent, since GW4869 or siRNA knockdown abrogates the protective effects, as well as neurotrophin-induced Akt phosphorylation. Finally, the assessment of nSMase activity promotion drives to the conclusion that neurotrophins can promote cell viability through Trk receptors in a manner depending on basal nSMase but not through SMase activity enhancement.


Asunto(s)
Neuronas/metabolismo , Receptor trkA/metabolismo , Receptor trkB/metabolismo , Receptor trkC/metabolismo , Esfingomielina Fosfodiesterasa/metabolismo , Compuestos de Anilina/farmacología , Animales , Apoptosis/efectos de los fármacos , Compuestos de Bencilideno/farmacología , Western Blotting , Factor Neurotrófico Derivado del Encéfalo/farmacología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cerebelo/citología , Microscopía Fluorescente , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Factor de Crecimiento Nervioso/farmacología , Neuronas/citología , Neuronas/efectos de los fármacos , Células PC12 , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Interferencia de ARN , Ratas , Esfingomielina Fosfodiesterasa/antagonistas & inhibidores , Esfingomielina Fosfodiesterasa/genética
12.
PLoS One ; 8(6): e68055, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23826362

RESUMEN

Tetanus toxin (TeTx) is the protein, synthesized by the anaerobic bacteria Clostridium tetani, which causes tetanus disease. TeTx gains entry into target cells by means of its interaction with lipid rafts, which are membrane domains enriched in sphingomyelin and cholesterol. However, the exact mechanism of host membrane binding remains to be fully established. In the present study we used the recombinant carboxyl terminal fragment from TeTx (Hc-TeTx), the domain responsible for target neuron binding, showing that Hc-TeTx induces a moderate but rapid and sustained increase in the ceramide/sphingomyelin ratio in primary cultures of cerebellar granule neurons and in NGF-differentiated PC12 cells, as well as induces the formation of ceramide platforms in the plasma membrane. The mentioned increase is due to the promotion of neutral sphingomyelinase activity and not to the de novo synthesis, since GW4869, a specific neutral sphingomyelinase inhibitor, prevents neutral sphingomyelinase activity increase and formation of ceramide platforms. Moreover, neutral sphingomyelinase inhibition with GW4869 prevents Hc-TeTx-triggered signaling (Akt phosphorylation), as well as the protective effect of Hc-TeTx on PC12 cells subjected to oxidative stress, while siRNA directed against nSM2 prevents protection by Hc-TeTx of NSC-34 cells against oxidative insult. Finally, neutral sphingomyelinase activity seems not to be related with the internalization of Hc-TeTx into PC12 cells. Thus, the presented data shed light on the mechanisms triggered by TeTx after membrane binding, which could be related with the events leading to the neuroprotective action exerted by the Hc-TeTx fragment.


Asunto(s)
Ceramidas/metabolismo , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Estrés Oxidativo/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Toxina Tetánica/farmacología , Compuestos de Anilina/farmacología , Animales , Compuestos de Bencilideno/farmacología , Supervivencia Celular/efectos de los fármacos , Fármacos del Sistema Nervioso Central/farmacología , Cerebelo/efectos de los fármacos , Cerebelo/metabolismo , Cerebelo/patología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Neuronas/metabolismo , Neuronas/patología , Estrés Oxidativo/fisiología , Células PC12 , Fragmentos de Péptidos/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Esfingomielina Fosfodiesterasa/antagonistas & inhibidores , Esfingomielina Fosfodiesterasa/metabolismo , Toxina Tetánica/metabolismo
13.
FEBS Lett ; 585(2): 414-20, 2011 Jan 21.
Artículo en Inglés | MEDLINE | ID: mdl-21187092

RESUMEN

In the present work we report the presence of protein kinase CK2 in lipid raft preparations from rat brain synaptosomes, obtained after detergent extraction and subsequent isolation of detergent-resistant membranes using sucrose gradient ultracentrifugation. Moreover, the phosphorylation of syntaxin-1 at Ser14, a specific CK2 target, has been detected in lipid rafts, as assessed by a phospho-specific antibody. Treatment with DMAT, a specific CK2 inhibitor, results in a decrease of syntaxin-1 Ser14 phosphorylation in lipid rafts, while the glutamate release from synaptosomes is enhanced. In conclusion, CK2 might control neurotransmitter release by acting on SNARE proteins attached to cholesterol-enriched microdomains.


Asunto(s)
Química Encefálica/fisiología , Quinasa de la Caseína II/antagonistas & inhibidores , Quinasa de la Caseína II/metabolismo , Microdominios de Membrana/metabolismo , Neurotransmisores/metabolismo , Sinaptosomas/química , Animales , Quinasa de la Caseína II/análisis , Microdominios de Membrana/química , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Proteínas SNARE/metabolismo , Sintaxina 1/metabolismo
14.
Biochem Biophys Res Commun ; 348(4): 1334-42, 2006 Oct 06.
Artículo en Inglés | MEDLINE | ID: mdl-16920068

RESUMEN

Although the high presence of cholesterol in nerve terminals is well documented, specific roles of this lipid in transmitter release have remained elusive. Since cholesterol is a highly enriched component in the membrane microdomains known as lipid rafts, it is probable that these domains are very important in synaptic function. The extraction of lipid rafts using Brij 98 at 37 degrees C avoids the formation of nonspecific membrane aggregates at low temperature, allowing the isolation of more physiologically relevant lipid rafts. In the present work, we examine, by means of buoyancy analysis in sucrose gradients after solubilization of the membranes with Brij 98 or with Lubrol WX, the presence of proteins involved in exocytosis in detergent-resistant membranes (DRM) using rat brain synaptosomes as a neurological model. Significant proportions of the proteins tested in the present work, which are involved in neurotransmitter release, are found in Brij 98 raft fractions, demonstrating that significant pools of synaptic proteins are segregated in specific parts of the membrane at physiological temperature. On the other hand, Lubrol WX is unable to solubilize the major fraction of the proteins tested. Treatment of synaptosomes with methyl-beta-cyclodextrin (mbetaCD) causes alteration in the buoyancy properties of proteins initially present in Brij- as well as in Lubrol-resistant membranes, indicating the cholesterol-dependency of both kinds of microdomains. Finally, we detect the depolarization-induced enhancement of the cholesterol-dependent association of syntaxin 1 with Brij 98-rafts, under the same conditions in which prolonged neurotransmitter release is stimulated.


Asunto(s)
Microdominios de Membrana/química , Terminales Presinápticos/química , Proteínas SNARE/análisis , Animales , Calcio/farmacología , Detergentes , Proteínas de la Membrana/química , Aceites de Plantas/química , Polietilenglicoles/química , Potasio/farmacología , Ratas , Ratas Sprague-Dawley , Solubilidad , Membranas Sinápticas/química , Sinaptosomas/efectos de los fármacos , Sintaxina 1/análisis , Temperatura , beta-Ciclodextrinas/farmacología
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