ABSTRACT
High doses of ionising radiation damage the heart by an as yet unknown mechanism. A concern for radiological protection is the recent epidemiological data indicating that doses as low as 100-500 mGy may induce cardiac damage. The aim of this study was to identify potential molecular targets and/or mechanisms involved in the pathogenesis of low-dose radiation-induced cardiovascular disease. The vascular endothelium plays a pivotal role in the regulation of cardiac function and is therefore a potential target tissue. We report here that low-dose radiation induced rapid and time-dependent changes in the cytoplasmic proteome of the human endothelial cell line EA.hy926. The proteomes were investigated at 4 and 24 h after irradiation at two different dose rates (Co-60 gamma ray total dose 200 mGy; 20 mGy/min and 190 mGy/min) using 2D-DIGE technology. Differentially expressed proteins were identified, after in-gel trypsin digestion, by MALDI-TOF/TOF tandem mass spectrometry, and peptide mass fingerprint analyses. We identified 15 significantly differentially expressed proteins, of which 10 were up-regulated and 5 down-regulated, with more than ±1.5-fold difference compared with unexposed cells. Pathways influenced by the low-dose exposures included the Ran and RhoA pathways, fatty acid metabolism and stress response.
Subject(s)
Endothelial Cells/diagnostic imaging , Endothelial Cells/metabolism , Proteome/metabolism , Apoptosis/radiation effects , Cell Proliferation/radiation effects , Cytosol/metabolism , Cytosol/radiation effects , Dose-Response Relationship, Drug , Endothelial Cells/cytology , Gene Expression Profiling , Humans , Proteomics , Radiography , Time FactorsABSTRACT
Calcitonin gene-related peptide is a 37 amino acid neuropeptide. Although calcitonin gene-related peptide activates a G-protein-coupled receptor, recent evidence suggests that calcitonin gene-related peptide induces more complex signaling cascades including the activation of MAP kinases [Eur J Pharmacol; 389:125-130 (2000), Neuropeptides; 34:229-233 (2000)]. However, the molecular mechanisms of this activation still remain to be elucidated. For the first time we applied a proteomics approach in order to identify molecular targets of calcitonin gene-related peptide downstream signaling in the neuroblastoma cell line SK-N-MC and identified proteins that changed either their expression, location, or their post-translational modifications in a time-dependent manner after calcitonin gene-related peptide stimulation.
Subject(s)
Calcitonin Gene-Related Peptide/pharmacology , Cell Proliferation/drug effects , Gene Expression Profiling , Protein Processing, Post-Translational/drug effects , Proteomics , Signal Transduction/drug effects , Amino Acid Sequence , Cell Line, Tumor/cytology , Cell Line, Tumor/drug effects , Humans , Mitogen-Activated Protein Kinases/genetics , Mitogen-Activated Protein Kinases/metabolism , Molecular Sequence Data , Neuroblastoma/metabolism , Neuroblastoma/pathology , Neuropeptides/genetics , Neuropeptides/metabolism , Protein Processing, Post-Translational/physiology , Receptors, Calcitonin Gene-Related Peptide/genetics , Receptors, Calcitonin Gene-Related Peptide/metabolism , Signal Transduction/physiology , Time FactorsABSTRACT
G protein-coupled receptors (GPCR) play an important role in drug development. Although many classical signal transduction pathways have been elucidated, more and more cross-talk to other cascades, e.g. MAP-kinase have been reported. In order to identify the overall function of receptor stimulation in a specific cell type or under certain conditions proteome analysis has been shown to be a very successful and powerful approach. Here, we will summarize the current state of the art of proteome analysis applied to GPCR.
Subject(s)
Receptors, G-Protein-Coupled/genetics , Animals , Humans , Protein Array Analysis , Proteomics , Receptors, G-Protein-Coupled/metabolism , Signal TransductionABSTRACT
SK-N-MC neuroepithelioma cells are routinely cultured and widely used as a model system in biochemical and pharmacological experiments. To clarify the gene expression patterns of SK-N-MC cells with respect to G protein-coupled receptors and signaling network components, we describe in this report the transcription profile of the cell line. Following the traditional pathway from genome to proteome, selected examples are further examined at the level of protein expression and by functional assays. cRNA targets derived from total RNA extracts were hybridized to Affymetrix Human Genome U133A GeneChip arrays, and the data were analyzed and grouped according to functional aspects. Results obtained for neuropeptide Y (NPY) Y1, Y5, and orexin Ox1 receptors were confirmed by RT-PCR. It is surprising that we found the presence of both NPY receptor subtypes and the absence of the orexin receptor at the mRNA level. Receptor-binding experiments confirmed NPY binding of the Y1 receptor in the nanomolar range but gave no evidence for high expression levels of Y5 receptor subtypes on the cell surface. Protein expression was assayed with immunoblots by using antibodies directed against selected Galpha protein subunits. The presence of at least Galphas, Galphai3, and Galphai2 subunits was indicated.
