RESUMEN
In the genesis of Alzheimer's disease (AD), converging lines of evidence suggest that amyloid-beta peptide (Abeta) triggers a pathogenic cascade leading to neuronal loss. It was long assumed that Abeta had to be assembled into extracellular amyloid fibrils or aggregates to exert its cytotoxic effects. Over the past decade, characterization of soluble oligomeric Abeta species in the brains of AD patients and in transgenic models has raised the possibility that different conformations of Abeta may contribute to AD pathology via different mechanisms. The receptor for advanced glycation end products (RAGE), a member of the Ig superfamily, is a cellular binding site for Abeta. Here, we investigate the role of RAGE in apoptosis induced by distinct well characterized Abeta conformations: Abeta oligomers (AbetaOs), Abeta fibrils (AbetaFs), and Abeta aggregates (AbetaAs). In our in vitro system, treatment with polyclonal anti-RAGE antibodies significantly improves SHSY-5Y cell and neuronal survival exposed to either AbetaOs or AbetaAs but does not affect AbetaF toxicity. Interestingly, using site-specific antibodies, we demonstrate that targeting of the V(d) domain of RAGE attenuates AbetaO-induced toxicity in both SHSY-5Y cells and rat cortical neurons, whereas inhibition of AbetaA-induced apoptosis requires the neutralization of the C(1d) domain of the receptor. Thus, our data indicate that distinct regions of RAGE are involved in Abeta-induced cellular and neuronal toxicity with respect to the Abeta aggregation state, and they suggest the blockage of particular sites of the receptor as a potential therapeutic strategy to attenuate neuronal death.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Neuronas/metabolismo , Receptores Inmunológicos/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/fisiopatología , Péptidos beta-Amiloides/antagonistas & inhibidores , Animales , Anticuerpos/farmacología , Especificidad de Anticuerpos/inmunología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Sitios de Unión/efectos de los fármacos , Sitios de Unión/fisiología , Encéfalo/efectos de los fármacos , Encéfalo/fisiopatología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Humanos , Neuronas/efectos de los fármacos , Neurotoxinas/antagonistas & inhibidores , Neurotoxinas/metabolismo , Placa Amiloide/efectos de los fármacos , Placa Amiloide/metabolismo , Conformación Proteica/efectos de los fármacos , Estructura Terciaria de Proteína/efectos de los fármacos , Estructura Terciaria de Proteína/fisiología , Ratas , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/química , Receptores Inmunológicos/efectos de los fármacosRESUMEN
The receptor for advanced glycation endproducts (RAGE) interacts with several ligands and is involved in various human diseases. RAGE_v1 or sRAGE, a RAGE splice variant, is secreted and contributes to the removal of RAGE ligands. Because RAGE blockade by specific antibodies directed against RAGE extracellular domains and the use of sRAGE have been proven to be beneficial in the context of pathological settings, both RAGE and sRAGE are considered as therapeutic target. Here, we show that sRAGE is also produced through regulated intramembrane proteolysis of the RAGE receptor, which is catalyzed by ADAM10 and the gamma-secretase and that calcium is an essential regulator of RAGE processing. Furthermore, RAGE intracellular domain localizes both in the cytoplasm and the nucleus and induces apoptosis when expressed in cells. These findings reveal new aspects of RAGE regulation and signaling and also provide a new interaction between RAGE and human pathologies.
Asunto(s)
Proteínas ADAM/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Calcio/metabolismo , Membrana Celular/metabolismo , Proteínas de la Membrana/metabolismo , Receptores Inmunológicos/metabolismo , Proteína ADAM10 , Anticuerpos/farmacología , Línea Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Dimetilsulfóxido/farmacología , Humanos , Ionomicina/farmacología , Estructura Terciaria de Proteína , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/antagonistas & inhibidores , Receptores Inmunológicos/genética , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
S100 proteins are EF-hand calcium-binding proteins with various intracellular functions including cell proliferation, differentiation, migration, and apoptosis. Some S100 proteins are also secreted and exert extracellular paracrine and autocrine functions. Experimental results suggest that the receptor for advanced glycation end products (RAGE) plays important roles in mediating S100 protein-induced cellular signaling. Here we compared the interaction of two S100 proteins, S100B and S100A6, with RAGE by in vitro assay and in culture of human SH-SY5Y neuroblastoma cells. Our in vitro binding data showed that S100B and S100A6, although structurally very similar, interact with different RAGE extracellular domains. Our cell assay data demonstrated that S100B and S100A6 differentially modulate cell survival. At micromolar concentration, S100B increased cellular proliferation, whereas at the same concentration, S100A6 triggered apoptosis. Although both S100 proteins induced the formation of reactive oxygen species, S100B recruited phosphatidylinositol 3-kinase/AKT and NF-kappaB, whereas S100A6 activated JNK. More importantly, we showed that S100B and S100A6 modulate cell survival in a RAGE-dependent manner; S100B specifically interacted with the RAGE V and C(1) domains and S100A6 specifically interacted with the C(1) and C(2) RAGE domains. Altogether these results highlight the complexity of S100/RAGE cellular signaling.
Asunto(s)
Supervivencia Celular/fisiología , Receptores Inmunológicos/inmunología , Proteínas S100/fisiología , Apoptosis , Western Blotting , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Línea Celular Tumoral , Medio de Cultivo Libre de Suero , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Técnica del Anticuerpo Fluorescente Directa , Glioblastoma/patología , Humanos , Etiquetado Corte-Fin in Situ , Mediciones Luminiscentes , Modelos Biológicos , FN-kappa B/metabolismo , Neuroblastoma/patología , Estructura Terciaria de Proteína , Especies Reactivas de Oxígeno/metabolismo , Receptor para Productos Finales de Glicación Avanzada , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas S100/genética , Proteínas S100/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Factores de Tiempo , TransfecciónRESUMEN
S100A16 protein is a new and unique member of the EF-hand Ca(2+)-binding proteins. S100 proteins are cell- and tissue-specific and are involved in many intra- and extracellular processes through interacting with specific target proteins. In the central nervous system S100 proteins are implicated in cell proliferation, differentiation, migration, and apoptosis as well as in cognition. S100 proteins became of major interest because of their close association with brain pathologies, for example depression or Alzheimer's disease. Here we report for the first time the purification and biochemical characterization of human and mouse recombinant S100A16 proteins. Flow dialysis revealed that both homodimeric S100A16 proteins bind two Ca(2+) ions with the C-terminal EF-hand of each subunit, the human protein exhibiting a 2-fold higher affinity. Trp fluorescence variations indicate conformational changes in the orthologous proteins upon Ca(2+) binding, whereas formation of a hydrophobic patch, implicated in target protein recognition, only occurs in the human S100A16 protein. In situ hybridization analysis and immunohistochemistry revealed a widespread distribution in the mouse brain. Furthermore, S100A16 expression was found to be astrocyte-specific. Finally, we investigated S100A16 intracellular localization in human glioblastoma cells. The protein was found to accumulate within nucleoli and to translocate to the cytoplasm in response to Ca(2+) stimulation.
Asunto(s)
Proteínas S100/metabolismo , Animales , Secuencia de Bases , Western Blotting , Cationes , Células Cultivadas , Cartilla de ADN , ADN Complementario , Humanos , Inmunohistoquímica , Ratones , Reacción en Cadena de la Polimerasa , Unión Proteica , ARN Mensajero/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas S100/genética , Espectrometría de FluorescenciaRESUMEN
RAGE is a multi-ligand receptor involved in various human diseases including diabetes, cancer or Alzheimer's disease. Engagement of RAGE by its ligands triggers activation of key cellular signalling pathways such as the MAP kinase and NF-kappaB pathways. Whereas the main isoform of RAGE is a transmembrane receptor with both extra- and intracellular domains, a secreted soluble isoform (sRAGE), corresponding to the extracellular part only, has the ability to block RAGE signalling and suppress cellular activation. Administration of sRAGE to animal models of cancer or multiple sclerosis blocked successfully tumour growth and the course of the autoimmune disease. These findings demonstrate that sRAGE may have a potential as therapeutic. We present here a fast and simple purification protocol of sRAGE from the yeast Pichia pastoris. The identity of the protein was confirmed by mass spectrometry and Western blot. The protein was N-glycosylated and 95-98% pure as judged by SDS-PAGE.
Asunto(s)
Pichia/metabolismo , Ingeniería de Proteínas/métodos , Receptores Inmunológicos/aislamiento & purificación , Receptores Inmunológicos/metabolismo , Secuencia de Aminoácidos , Humanos , Datos de Secuencia Molecular , Peso Molecular , Pichia/genética , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/química , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , SolubilidadRESUMEN
Head and neck squamous cell carcinoma express high levels of the EF-hand calcium-binding protein S100A2 in contrast to other tumorigenic tissues and cell lines where the expression of this protein is reduced. Subtractive hybridization of tumorigenic versus normal tumor-derived mammary epithelial cells has previously identified the S100A2 protein as potential tumor suppressor. The biological function of S100A2 in carcinogenesis, however, has not been elucidated to date. Here, we report for the first time that during recovery from hydroxyurea treatment, the S100A2 protein translocated from the cytoplasm to the nucleus and co-localized with the tumor suppressor p53 in two different oral carcinoma cells (FADU and SCC-25). Co-immunoprecipitation experiments and electrophoretic mobility shift assay showed that the interaction between S100A2 and p53 is Ca(2+)-dependent. Preliminary characterization of this interaction indicated that the region in p53 involved with binding to S100A2 is located at the C terminus of p53. Finally, luciferase-coupled transactivation assays, where a p53-reporter construct was used, indicated that interaction with S100A2 increased p53 transcriptional activity. Our data suggest that in oral cancer cells the Ca(2+)- and cell cycle-dependent p53-S100A2 interaction might modulate proliferation.