RESUMEN
INTRODUCTION: The diagnosis of bilateral papilledema implies emergency medical care to look for intracranial hypertension and arteritic ischemic neuropathy. However, other causes must also be mentioned, including drugs. Too often underrated because of their usual benignity, drug side ophthalmological effects can be severe and are typically bilateral. CASE REPORT: An 80-year-old woman was hospitalized for bilateral papilledema, predominantly in the left eye, with lowered visual acuity. After ruling out intracranial hypertension, arteritic ischemic optic neuropathy, non-arteritic, and inflammatory bilateral papilledema, the diagnosis was toxic optic neuropathy. CONCLUSION: Bilateral edematous optic neuropathy is a known side effect of amiodarone, uncommon but to be known because of the large number of patients benefiting from this treatment.
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Amiodarona/efectos adversos , Antiarrítmicos/efectos adversos , Enfermedades del Nervio Óptico/inducido químicamente , Anciano de 80 o más Años , Femenino , Humanos , Enfermedad Iatrogénica , Enfermedades del Nervio Óptico/diagnóstico , Papiledema/inducido químicamente , Papiledema/diagnóstico , Trastornos de la Visión/inducido químicamente , Trastornos de la Visión/diagnóstico , Agudeza Visual/efectos de los fármacosRESUMEN
Food allergy is a growing health problem worldwide; thus, there is an urgent need for robust, specific, and sensitive analytical methods for detecting allergens. Mass spectrometry is an alternative to the existing methods, and it can overcome their limitations. One of the first steps in the development of any analytical method is the identification of the analytes to be further studied. In the case of allergen detection by mass spectrometry, the analytes are peptides. In this study, a strategy was developed for identifying potential peptide biomarkers in processed food products. This strategy was applied to processed egg matrices, and 16 potential peptide biomarkers were identified for the further detection and quantification of egg by means of mass spectrometry. With an empirical approach based on dedicated sample preparation, including tandem Lys-C/trypsin enzymatic digestion and high-resolution mass spectrometry analysis, hundreds of peptides from egg proteins were identified. This list of peptides was further refined with a series of criteria, obtained from empirical evidence, to identify the ideal biomarkers for the development of a quantitative method. These criteria include the resistance to food processing and the specificity of the peptides for eggs but also the effects of amino acid modifications and enzymatic digestion efficiency.
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Alérgenos/análisis , Biomarcadores/análisis , Proteínas del Huevo/análisis , Huevos/análisis , Contaminación de Alimentos/análisis , Fragmentos de Péptidos/análisis , Espectrometría de Masas en Tándem/métodos , Alérgenos/química , Animales , Biomarcadores/química , Pollos , Hipersensibilidad al Huevo/inmunología , Hipersensibilidad al Huevo/prevención & control , Proteínas del Huevo/inmunología , Manipulación de Alimentos , Humanos , Fragmentos de Péptidos/inmunologíaRESUMEN
Worldwide, mass spectrometry is widely used to detect and quantify food allergens, especially in complex and processed food products. Yet, the absence of a regulatory framework for the developed methods has led to a lack of harmonization between laboratories. In this study, ten allergens were analyzed in eight food products by UHPLC-MS/MS, in order to establish criteria for the retention time, variation tolerance, the ion ratio deviation, and the signal-to-noise ratio for allergen detection. The set of criteria should help laboratories to compare results and avoid false positives and negatives. Furthermore, a strategy combining standard addition and labeled peptide correction was used to quantify milk, soy, peanut, and egg allergens in eight food products. This strategy is particularly interesting for routine laboratories, which receive hundreds of samples and cannot use an external calibration curve for each sample.
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Alérgenos/análisis , Análisis de los Alimentos/métodos , Péptidos/análisis , Espectrometría de Masas en Tándem/métodos , Animales , Arachis/química , Calibración , Cromatografía Líquida de Alta Presión/métodos , Hipersensibilidad al Huevo , Huevos/análisis , Análisis de los Alimentos/normas , Hipersensibilidad a los Alimentos , Humanos , Laboratorios , Leche/química , Reproducibilidad de los Resultados , Relación Señal-Ruido , Espectrometría de Masas en Tándem/normasRESUMEN
Food allergy is a considerable heath problem, as undesirable contaminations by allergens during food production are still widespread and may be dangerous for human health. To protect the population, laboratories need to develop reliable analytical methods in order to detect allergens in various food products. Currently, a large majority of allergen-related food recalls concern bakery products. It is therefore essential to detect allergens in unprocessed and processed foodstuffs. In this study, we developed a method for detecting ten allergens in complex (chocolate, ice cream) and processed (cookie, sauce) foodstuffs, based on ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). Using a single protocol and considering a signal-to-noise ratio higher than 10 for the most abundant multiple reaction monitoring (MRM) transition, we were able to detect target allergens at 0.5mg/kg for milk proteins, 2.5mg/kg for peanut, hazelnut, pistachio, and cashew proteins, 3mg/kg for egg proteins, and 5mg/kg for soy, almond, walnut, and pecan proteins. The ability of the method to detect 10 allergens with a single protocol in complex and incurred food products makes it an attractive alternative to the ELISA method for routine laboratories.
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Alérgenos/análisis , Cromatografía Líquida de Alta Presión/métodos , Análisis de los Alimentos/métodos , Espectrometría de Masas en Tándem , Chocolate/análisis , Proteínas del Huevo/análisis , Ensayo de Inmunoadsorción Enzimática , Hipersensibilidad a los Alimentos , Helados/análisis , Proteínas de la Leche/análisis , Nueces/química , Relación Señal-RuidoRESUMEN
Sensitive detection of food allergens is affected by food processing and foodstuff complexity. It is therefore a challenge to detect cross-contamination in food production that could endanger an allergic customer's life. Here we used ultra-high performance liquid chromatography coupled to tandem mass spectrometry for simultaneous detection of traces of milk (casein, whey protein), egg (yolk, white), soybean, and peanut allergens in different complex and/or heat-processed foodstuffs. The method is based on a single protocol (extraction, trypsin digestion, and purification) applicable to the different tested foodstuffs: chocolate, ice cream, tomato sauce, and processed cookies. The determined limits of quantitation, expressed in total milk, egg, peanut, or soy proteins (and not soluble proteins) per kilogram of food, are: 0.5mg/kg for milk (detection of caseins), 5mg/kg for milk (detection of whey), 2.5mg/kg for peanut, 5mg/kg for soy, 3.4mg/kg for egg (detection of egg white), and 30.8mg/kg for egg (detection of egg yolk). The main advantage is the ability of the method to detect four major food allergens simultaneously in processed and complex matrices with very high sensitivity and specificity.
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Alérgenos/química , Cromatografía Líquida de Alta Presión/métodos , Análisis de los Alimentos/métodos , Contaminación de Alimentos/análisis , Espectrometría de Masas en Tándem/métodos , Animales , Arachis/química , Arachis/inmunología , Pollos , Huevos , Manipulación de Alimentos , Leche/química , Leche/inmunología , Proteínas de Soja/química , Proteínas de Soja/inmunologíaRESUMEN
The identification of the regulatory proteins that control DNA transcription as well as RNA stability and translation represents a key step in the comprehension of gene expression regulation. Those proteins can be purified by DNA- or RNA-affinity chromatography, followed by identification by mass spectrometry. Although very simple in the concept, this represents a real technological challenge due to the low abundance of regulatory proteins compared to the highly abundant proteins binding to nucleic acids in a nonsequence-specific manner. Here we review the different strategies that have been set up to reach this purpose, discussing the key parameters that should be considered to increase the chances of success. Typically, two categories of biological questions can be distinguished: the identification of proteins that specifically interact with a precisely defined binding site, mostly addressed by quantitative mass spectrometry, and the identification in a non-comparative manner of the protein complexes recruited by a poorly characterized long regulatory region of nucleic acids. Finally, beside the numerous studies devoted to in vitro-assembled nucleic acid-protein complexes, the scarce data reported on proteomic analyses of in vivo-assembled complexes are described, with a special emphasis on the associated challenges.
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Cromatografía de Afinidad/métodos , Proteínas de Unión al ADN , ADN/química , Espectrometría de Masas/métodos , Proteínas de Unión al ARN , ARN/química , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/aislamiento & purificaciónRESUMEN
Cancer cell resistance against chemotherapy is still a heavy burden to improve anticancer treatments. Autophagy activation and the development of hypoxic regions within the tumors are known to promote cancer cell resistance. Therefore, we sought to evaluate the role of autophagy and hypoxia on the taxol-induced apoptosis in MDA-MB-231 breast cancer cells. The results showed that taxol induced apoptosis after 16 h of incubation, and that hypoxia protected MDA-MB-231 cells from taxol-induced apoptosis. In parallel, taxol induced autophagy activation already after 2 h of incubation both under normoxia and hypoxia. Autophagy activation after taxol exposure was shown to be a protective mechanism against taxol-induced cell death both under normoxia and hypoxia. However, at longer incubation time, the autophagic process reached a saturation point under normoxia leading to cell death, whereas under hypoxia, autophagy flow still correctly took place allowing the cells to survive. Autophagy induction is induced after taxol exposure via mechanistic target of rapamycin (mTOR) inhibition, which is more important in cells exposed to hypoxia. Taxol also induced c-Jun N-terminal kinase (JNK) activation and phosphorylation of its substrates B-cell CLL/lymphoma 2 (Bcl2) and BCL2-like 1 (BclXL) under normoxia and hypoxia very early after taxol exposure. Bcl2 and BclXL phosphorylation was decreased more importantly under hypoxia after long incubation time. The role of JNK in autophagy and apoptosis induction was studied using siRNAs. The results showed that JNK activation promotes resistance against taxol-induced apoptosis under normoxia and hypoxia without being involved in induction of autophagy. In conclusion, the resistance against taxol-induced cell death observed under hypoxia can be explained by a more effective autophagic flow activated via the classical mTOR pathway and by a mechanism involving JNK, which could be dependent on Bcl2 and BclXL phosphorylation but independent of JNK-induced autophagy activation.
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Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Hipoxia de la Célula , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Paclitaxel/farmacología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Proteínas de la Membrana/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Proteína bcl-X/metabolismoRESUMEN
To depict the largest picture of a core promoter interactome, we developed a one-step DNA-affinity capture method coupled with an improved mass spectrometry analysis process focused on the identification of low abundance proteins. As a proof of concept, this method was developed through the analysis of 230 bp contained in the 5'long terminal repeat (LTR) of the human immunodeficiency virus 1 (HIV-1). Beside many expected interactions, many new transcriptional regulators were identified, either transcription factors (TFs) or co-regulators, which interact directly or indirectly with the HIV-1 5'LTR. Among them, the homeodomain-containing TF myeloid ectopic viral integration site was confirmed to functionally interact with a specific binding site in the HIV-1 5'LTR and to act as a transcriptional repressor, probably through recruitment of the repressive Sin3A complex. This powerful and validated DNA-affinity approach could also be used as an efficient screening tool to identify a large set of proteins that physically interact, directly or indirectly, with a DNA sequence of interest. Combined with an in silico analysis of the DNA sequence of interest, this approach provides a powerful approach to select the interacting candidates to validate functionally by classical approaches.
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Duplicado del Terminal Largo de VIH , VIH-1/genética , Proteínas de Homeodominio/metabolismo , Proteínas de Neoplasias/metabolismo , Proteómica/métodos , Factores de Transcripción/metabolismo , Sitios de Unión , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/aislamiento & purificación , Células HeLa , Proteínas de Homeodominio/fisiología , Humanos , Espectrometría de Masas , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide , FN-kappa B/análisis , Proteínas de Neoplasias/fisiología , Proteínas Represoras/análisis , Transcripción GenéticaRESUMEN
To explain why mitochondrial DNA (mtDNA)-depleted or rho0 cells still keep a mitochondrial membrane potential (Delta(psi)m) in the absence of respiration, several hypotheses have been proposed. The principal and well accepted one involves a reverse of action for ANT combined to F1-ATPase activity. However, the existence of other putative electrogenic channels has been speculated. Here, using mRNA differential display reverse transcriptase-polymerase chain reaction on L929 mtDNA-depleted cells, we identified mtCLIC as a differentially expressed gene in cells deprived from mitochondrial ATP production. Mitochondrial chloride intracellular channel (mtCLIC), a member of a recently discovered and expanding family of chloride intracellular channels, is up-regulated in mtDNA-depleted and rho0 cells. We showed that its expression is dependent on CREB and p53 and is sensitive to calcium and tumor necrosis factor alpha. Interestingly, up- or down-regulation of mtCLIC protein expression changes Delta(psi)m whereas the chloride channel inhibitor NPPB reduces the Delta(psi)m in mtDNA-depleted L929 cells, measured with the fluorescent probe rhodamine 123. Finally, we demonstrated that purified mitochondria from mtDNA-depleted cells incorporate, in a NPPB-sensitive manner, more 36chloride than parental mitochondria. These findings suggest that mtCLIC could be involved in mitochondrial membrane potential generation in mtDNA-depleted cells, a feature required to prevent apoptosis and to drive continuous protein import into mitochondria.
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Canales de Cloruro/biosíntesis , Canales de Cloruro/fisiología , Mitocondrias/fisiología , Proteínas Mitocondriales/biosíntesis , Proteínas Mitocondriales/fisiología , Animales , Calcio/fisiología , Línea Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , ADN Mitocondrial/genética , Regulación de la Expresión Génica , Potenciales de la Membrana , Ratones , Mitocondrias/efectos de los fármacos , Modelos Biológicos , ARN Mensajero/biosíntesis , Regulación hacia ArribaRESUMEN
We characterized a new signaling pathway leading to the activation of cAMP-responsive element-binding protein (CREB) in several cell lines affected by mitochondrial dysfunction. In vitro kinase assays, inhibitors of several kinase pathways and overexpression of a dominant-negative mutant for calcium/calmodulin kinase IV (CaMKIV), which blocks the activation of CREB, showed that CaMKIV is activated by a mitochondrial activity impairment. A high calcium concentration leading to the disruption of the protein interaction with protein phosphatase 2A explains CaMKIV activation in these conditions. Transcrip tionally active phosphorylated CREB was also found in a rho0 143B human osteosarcoma cell line and in a MERRF cybrid cell line mutated for tRNA(Lys) (A8344G). We also showed that phosphorylated CREB is involved in the proliferation defect induced by a mitochondrial dysfunction. Indeed, cell proliferation inhibition can be prevented by CaMKIV inhibition and CREB dominant-negative mutants. Finally, our data suggest that phosphorylated CREB recruits p53 tumor suppressor protein, modifies its transcriptional activity and increases the expression of p21(Waf1/Cip1), a p53-regulated cyclin-dependent kinase inhibitor.
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División Celular/fisiología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Mitocondrias/metabolismo , Transducción de Señal/fisiología , Animales , Secuencia de Bases , Proteína Quinasa Tipo 4 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Línea Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/genética , Ciclinas/metabolismo , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Activación Enzimática , Humanos , Ratones , Mutación , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Proteína Fosfatasa 2 , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Hypoxia is a common denominator of many vascular disorders, especially those associated with ischemia. To study the effect of oxygen depletion on endothelium, we developed an in vitro model of hypoxia on human umbilical vein endothelial cells (HUVEC). Hypoxia strongly activates HUVEC, which then synthesize large amounts of prostaglandins and platelet-activating factor. The first step of this activation is a decrease in ATP content of the cells, followed by an increase in the cytosolic calcium concentration ([Ca(2+)](i)) which then activates the phospholipase A(2) (PLA(2)). The link between the decrease in ATP and the increase in [Ca(2+)](i) was not known and is investigated in this work. We first showed that the presence of extracellular Na(+) was necessary to observe the hypoxia-induced increase in [Ca(2+)](i) and the activation of PLA(2). This increase was not due to the release of Ca(2+) from intracellular stores, since thapsigargin did not inhibit this process. The Na(+)/Ca(2+) exchanger was involved since dichlorobenzamil inhibited the [Ca(2+)](i) and the PLA(2) activation. The glycolysis was activated, but the intracellular pH (pH(i)) in hypoxic cells did not differ from control cells. Finally, the hypoxia-induced increase in [Ca(2+)](i) and PLA(2) activation were inhibited by phlorizin, an inhibitor of the Na(+)-glucose cotransport. The proposed biochemical mechanism occurring under hypoxia is the following: glycolysis is first activated due to a requirement for ATP, leading to an influx of Na(+) through the activated Na(+)-glucose cotransport followed by the activation of the Na(+)/Ca(2+) exchanger, resulting in a net influx of Ca(2+).
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Calcio/metabolismo , Endotelio Vascular/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Adenosina Trifosfato/metabolismo , Hipoxia de la Célula/fisiología , Endotelio Vascular/citología , Glucólisis/fisiología , Humanos , Concentración de Iones de Hidrógeno , Modelos Biológicos , Fosfolipasas A/metabolismo , Sodio/metabolismo , Venas Umbilicales/citologíaRESUMEN
Despite increasing evidence supporting the involvement of neutrophils in ischemic and postischemic damages, the mechanisms underlying the early recruitment of these cells are not completely understood. In this report, the effects of conditioned media from hypoxic endothelial cells on neutrophil chemotaxis were investigated by biochemical and morphological studies. We showed that conditioned media collected from several endothelial cell origins submitted to hypoxia as well as ischemic rat liver perfusion liquids have a chemotactic activity for neutrophils. The role of various chemoattractant molecules like HETEs, platelet-activating factor, and cytokines such as interleukin-8 and interleukin-1 was examined in the same model. Chemotactic peptide contribution was ruled out as boiled conditioned media still trigger chemotaxis. However, cell treatment with cyclooxygenase inhibitors, neutralization of PGF(2alpha) biological activity with polyclonal antibodies, and the neutrophil preincubation with a specific PGF(2alpha) antagonist, all dramatically inhibited neutrophil chemotaxis. A strong chemoattractant effect of pure exogenous PGF(2alpha) or of a synthetic analog was also observed. The major effect of PGF(2alpha) on neutrophil chemotaxis was confirmed ex vivo in a rat liver perfusion ischemic model. These results suggest that PGF(2alpha), a prostanoid abundantly released by the endothelium of hypoxic or ischemic tissues, is a chemoattractant molecule that might be involved in the early recruitment of neutrophils in ischemic organs.
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Factores Quimiotácticos/fisiología , Infiltración Neutrófila/fisiología , Prostaglandinas F/fisiología , Animales , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , Dinoprost/análogos & derivados , Dinoprost/farmacología , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Femenino , Humanos , Ácidos Hidroxieicosatetraenoicos/biosíntesis , Hipoxia/metabolismo , Hipoxia/patología , Interleucina-1/metabolismo , Interleucina-8/metabolismo , Isquemia/metabolismo , Circulación Hepática , Infiltración Neutrófila/efectos de los fármacos , Antagonistas de Prostaglandina/farmacología , Prostaglandinas F/antagonistas & inhibidores , Ratas , Ratas WistarRESUMEN
The origin of several vascular pathologies involves sudden or recurrent oxygen deficiency. In this review, we examine what the biochemical and molecular responses of the endothelial cells to the lack of oxygen are and how these responses may account for the features observed in pathological situations, mainly by modifications of cell-cell interactions. Two major responses of the endothelial cells have been observed depending on the degree and duration of the oxygen deficiency. Firstly, acute hypoxia rapidly activates the endothelial cells to release inflammatory mediators and growth factors. These inflammatory mediators are able to recruit and promote the adherence of neutrophils to the endothelium where they become activated. The synthesis of platelet-activating factor plays a key role in this adherence process. Secondly, longer periods of hypoxia increase the expression of specific genes such as those encoding some cytokines as well as for the growth factors platelet-derived growth factor and vascular endothelial growth factor. The transcriptional induction of these genes is mediated through the activation of several transcription factors, the most important one being hypoxia inducible factor-1. The link between our knowledge of the signalling cascade of the cellular and molecular events initiated by hypoxia and their involvement in several vascular pathological situations, varicose veins, tumor angiogenesis and pulmonary hypertension is discussed briefly.
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Endotelio Vascular/metabolismo , Hipoxia/fisiopatología , Comunicación Celular , Citocinas/genética , Citocinas/metabolismo , Endotelio Vascular/citología , Regulación de la Expresión Génica , Humanos , Neutrófilos/citologíaRESUMEN
Regulator of G protein signaling (RGS) proteins function as GTPase-activating proteins (GAPs) that stimulate the inactivation of heterotrimeric G proteins. We have recently shown that RGS proteins may be regulated on a post-translational level (Benzing, T., Brandes, R., Sellin, L., Schermer, B., Lecker, S., Walz, G., and Kim, E. (1999) Nat. Med. 5, 913-918). However, mechanisms controlling the GAP activity of RGS proteins are poorly understood. Here we show that 14-3-3 proteins associate with RGS7 and RGS3. Binding of 14-3-3 is mediated by a conserved phosphoserine located in the Galpha-interacting portion of the RGS domain; interaction with 14-3-3 inhibits the GAP activity of RGS7, depends upon phosphorylation of a conserved residue within the RGS domain, and results in inhibition of GAP function. Collectively, these data indicate that phosphorylation-dependent binding of 14-3-3 may act as molecular switch that controls the GAP activity keeping a substantial fraction of RGS proteins in a dormant state.
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Proteínas de Unión al GTP , Proteínas Activadoras de GTPasa/metabolismo , Proteínas/metabolismo , Proteínas RGS/metabolismo , Proteínas Represoras , Tirosina 3-Monooxigenasa , Proteínas 14-3-3 , Secuencia de Aminoácidos , Sitios de Unión , Línea Celular , Humanos , Cinética , Datos de Secuencia Molecular , Mutagénesis Insercional , Mutagénesis Sitio-Dirigida , Fosforilación , Proteínas RGS/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Estaurosporina/farmacología , TransfecciónRESUMEN
Hypoxia-inducible factor-1 (HIF-1) is a transcription factor activated by hypoxia. The HIF-1 activation transduction pathway is poorly understood. In this report, we investigated the activation of extracellular regulated kinases (ERK) in hypoxia and their involvement in HIF-1 activation. We demonstrated that in human microvascular endothelial cells-1 (HMEC-1), ERK kinases are activated during hypoxia. Using dominant negative mutants, we showed that ERK1 is needed for hypoxia-induced HIF-1 transactivation activity. Moreover, using a kinase assay and Western blot experiments, we showed that HIF-1alpha is phosphorylated in hypoxia by an ERK-dependent pathway. These results evidence the role of mitogen-activated protein kinase in the transcriptional response to hypoxia.
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Hipoxia de la Célula/fisiología , Proteínas de Unión al ADN/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Nucleares/metabolismo , Western Blotting , Línea Celular , Núcleo Celular/enzimología , Endotelio Vascular/citología , Endotelio Vascular/enzimología , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Humanos , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Microcirculación/citología , Microcirculación/enzimología , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Fosforilación , Pruebas de Precipitina , Estructura Terciaria de Proteína , Factores de Transcripción/metabolismo , Tirosina/metabolismoRESUMEN
Regulators of G protein signaling (RGS) proteins accelerate the intrinsic GTPase activity of certain Galpha subunits and thereby modulate a number of G protein-dependent signaling cascades. Currently, little is known about the regulation of RGS proteins themselves. We identified a short-lived RGS protein, RGS7, that is rapidly degraded through the proteasome pathway. The degradation of RGS7 is inhibited by interaction with a C-terminal domain of polycystin, the protein encoded by PKD1, a gene involved in autosomal-dominant polycystic kidney disease. Furthermore, membranous expression of C-terminal polycystin relocalized RGS7. Our results indicate that rapid degradation and interaction with integral membrane proteins are potential means of regulating RGS proteins.
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Proteínas/genética , Proteínas/metabolismo , Proteínas RGS , Secuencia de Aminoácidos , Linfocitos B/metabolismo , Sitios de Unión , Cisteína Endopeptidasas/metabolismo , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/metabolismo , Biblioteca de Genes , Humanos , Datos de Secuencia Molecular , Complejos Multienzimáticos/metabolismo , Riñón Poliquístico Autosómico Dominante/genética , Riñón Poliquístico Autosómico Dominante/metabolismo , Complejo de la Endopetidasa Proteasomal , Biosíntesis de Proteínas , Proteínas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Canales Catiónicos TRPP , Transcripción Genética , Ubiquitinas/metabolismoRESUMEN
Autosomal dominant polycystic kidney disease (ADPKD) is caused by germ line mutations in at least three ADPKD genes. Two recently isolated ADPKD genes, PKD1 and PKD2, encode integral membrane proteins of unknown function. We found that PKD2 upregulated AP-1-dependent transcription in human embryonic kidney 293T cells. The PKD2-mediated AP-1 activity was dependent upon activation of the mitogen-activated protein kinases p38 and JNK1 and protein kinase C (PKC) epsilon, a calcium-independent PKC isozyme. Staurosporine, but not the calcium chelator BAPTA [1,2-bis(o-aminophenoxy)ethane-N,N,N', N'-tetraacetate], inhibited PKD2-mediated signaling, consistent with the involvement of a calcium-independent PKC isozyme. Coexpression of PKD2 with the interacting C terminus of PKD1 dramatically augmented PKD2-mediated AP-1 activation. The synergistic signaling between PKD1 and PKD2 involved the activation of two distinct PKC isozymes, PKC alpha and PKC epsilon, respectively. Our findings are consistent with others that support a functional connection between PKD1 and PKD2 involving multiple signaling pathways that converge to induce AP-1 activity, a transcription factor that regulates different cellular programs such as proliferation, differentiation, and apoptosis. Activation of these signaling cascades may promote the full maturation of developing tubular epithelial cells, while inactivation of these signaling cascades may impair terminal differentiation and facilitate the development of renal tubular cysts.
Asunto(s)
Proteínas de la Membrana/genética , Proteínas Quinasas Activadas por Mitógenos , Riñón Poliquístico Autosómico Dominante/genética , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Línea Celular , Proteínas de Unión al ADN/genética , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Proteínas de Unión al GTP/genética , Regulación de la Expresión Génica/genética , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Mutación/genética , Fosforilación , Proteína Quinasa C/metabolismo , Proteínas/genética , Proteínas Proto-Oncogénicas c-jun/genética , Transducción de Señal , Estaurosporina/farmacología , Canales Catiónicos TRPP , Factor de Transcripción AP-1/genéticaRESUMEN
The recently discovered family of regulators of G protein signaling (RGS) accelerates the intrinsic GTPase activity of certain Galpha subunits, thereby terminating G protein signaling. Particularly high mRNA levels of one family member, RGS3, are found in the adult kidney. To establish the temporal and spatial renal expression pattern of RGS3, a polyclonal antiserum was raised against the COOH terminus of RGS3. Staining of mouse renal tissue at different gestational stages revealed high levels of RGS3 within the developing and mature tubular epithelial cells. We tested whether RGS3 can modulate tubular migration, an important aspect of tubular development, in response to G protein-mediated signaling. Several mouse intermedullary collecting duct (mIMCD-3) cell lines were generated that expressed RGS3 under the control of an inducible promoter. Lysophosphatidic acid (LPA) is a potent chemoattractant that mediates its effects through heterotrimeric G proteins. We found that induction of RGS3 significantly reduced LPA-mediated cell migration in RGS3-expressing mIMCD-3 clones, whereas chemotaxis induced by hepatocyte growth factor remained unaffected by RGS3. Our findings suggest that RGS3 modulates tubular functions during renal development and in the adult kidney.