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-B, 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.

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.

No abstract

In the genesis of Alzheimer's disease (AD), converging lines of evidence suggest that amyloid-␤ peptide (A␤) triggers a pathogenic cascade leading to neuronal loss. It was long assumed that A␤ had to be assembled into extracellular amyloid fibrils or aggregates to exert its cytotoxic effects. Over the past decade, characterization of soluble oligomeric A␤ species in the brains of AD patients and in transgenic models has raised the possibility that different conformations of A␤ 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 A␤. Here, we investigate the role of RAGE in apoptosis induced by distinct well characterized A␤ conformations: A␤ oligomers (A␤Os), A␤ fibrils (A␤Fs), and A␤ aggregates (A␤As). In our in vitro system, treatment with polyclonal anti-RAGE antibodies significantly improves SHSY-5Y cell and neuronal survival exposed to either A␤Os or A␤As but does not affect A␤F toxicity. Interestingly, using site-specific antibodies, we demonstrate that targeting of the V d domain of RAGE attenuates A␤O-induced toxicity in both SHSY-5Y cells and rat cortical neurons, whereas inhibition of A␤A-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 A␤-induced cellular and neuronal toxicity with respect to the A␤ aggregation state, and they suggest the blockage of particular sites of the receptor as a potential therapeutic strategy to attenuate neuronal death.

Recombinant adeno-associated viruses (rAAVs) are promising vectors for gene therapy since they efficiently and stably transduce a variety of tissues of immunocompetent animals. The major disadvantage of rAAVs is their limited capacity to package foreign DNA (р5 kb). Often, co-expression of two or more genes from a single viral vector is desirable to achieve maximal therapeutic efficacy or to track transduced cells in vivo by suitable reporter genes. The internal ribosome entry site (IRES) sequence of encephalomyocarditis virus has been widely used to construct bicistronic viral vectors. However, the IRES is rather long and IRES-mediated translation can be relatively inefficient when compared with capdependent translation. As an alternative to the IRES for in vivo gene expression, we studied the 16 amino-acid long 2A peptide of foot and mouth disease virus (FMDV). The 2A peptide mediates the primary cis-'cleavage' of the FMDV polyprotein in a cascade of processing events that ultimately