Estelle Leclerc

S100B and S100A6 Differentially Modulate Cell Survival by Interacting with Distinct RAGE (Receptor for Advanced Glycation End Products) Immunoglobulin Domains

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-κB, 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 C1 domains and S100A6 specifically interacted with the C1 and C2 RAGE domains. Altogether these results highlight the complexity of S100/RAGE cellular signaling.

Structural and functional insights into RAGE activation by multimeric S100B

Nervous system development and plasticity require regulation of cell proliferation, survival, neurite outgrowth and synapse formation by specific extracellular factors. The EF‐hand protein S100B is highly expressed in human brain. In the extracellular space, it promotes neurite extension and neuron survival via the receptor RAGE (receptor for advanced glycation end products). The X‐ray structure of human Ca2+‐loaded S100B was determined at 1.9 Å resolution. The structure revealed an octameric architecture of four homodimeric units arranged as two tetramers in a tight array. The presence of multimeric forms in human brain extracts was confirmed by size‐exclusion experiments. Recombinant tetrameric, hexameric and octameric S100B were purified from Escherichia coli and characterised. Binding studies show that tetrameric S100B binds RAGE with higher affinity than dimeric S100B. Analytical ultracentrifugation studies imply that S100B tetramer binds two RAGE molecules via the V‐domain. In line with these experiments, S100B tetramer caused stronger activation of cell growth than S100B dimer and promoted cell survival. The structural and the binding data suggest that tetrameric S100B triggers RAGE activation by receptor dimerisation.

Site-Specific Blockade of RAGE-Vd Prevents Amyloid-β Oligomer Neurotoxicity

In the genesis of Alzheimers disease (AD), converging lines of evidence suggest that amyloid-β peptide (Aβ) triggers a pathogenic cascade leading to neuronal loss. It was long assumed that Aβ had to be assembled into extracellular amyloid fibrils or aggregates to exert its cytotoxic effects. Over the past decade, characterization of soluble oligomeric Aβ species in the brains of AD patients and in transgenic models has raised the possibility that different conformations of Aβ 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 Aβ. Here, we investigate the role of RAGE in apoptosis induced by distinct well characterized Aβ conformations: Aβ oligomers (AβOs), Aβ fibrils (AβFs), and Aβ aggregates (AβAs). In our in vitro system, treatment with polyclonal anti-RAGE antibodies significantly improves SHSY-5Y cell and neuronal survival exposed to either AβOs or AβAs but does not affect AβF toxicity. Interestingly, using site-specific antibodies, we demonstrate that targeting of the Vd domain of RAGE attenuates AβO-induced toxicity in both SHSY-5Y cells and rat cortical neurons, whereas inhibition of AβA-induced apoptosis requires the neutralization of the C1d domain of the receptor. Thus, our data indicate that distinct regions of RAGE are involved in Aβ-induced cellular and neuronal toxicity with respect to the Aβ aggregation state, and they suggest the blockage of particular sites of the receptor as a potential therapeutic strategy to attenuate neuronal death.

The S100B/RAGE Axis in Alzheimer's Disease

Increasing evidence suggests that the small EF-hand calcium-binding protein S100B plays an important role in Alzheimers disease. Among other evidences are the increased levels of both S100B and its receptor, the Receptor for Advanced Glycation Endproducts (RAGEs) in the AD diseased brain. The regulation of RAGE signaling by S100B is complex and probably involves other ligands including the amyloid beta peptide (Aβ), the Advanced Glycation Endproducts (AGEs), or transtheyretin. In this paper we discuss the current literature regarding the role of S100B/RAGE activation in Alzheimers disease.

The importance of Ca2+/Zn2+ signaling S100 proteins and RAGE in translational medicine.

The Receptor for Advanced Glycation Endproducts (RAGE) is a multiligand receptor involved in a large number of human disorders. Identified first as the receptor for the Advanced Glycation Endproducts (AGEs), RAGE has emerged in recent years as a major receptor for many members of the S100 calcium and zinc binding protein family. The interaction with and the signaling triggered by several S100 proteins such as S100B and S100A12 have been studied in details and have shown concentration and cell type dependent signaling cascades. The S100 protein family consists of more than 20 members which present high amino-acid sequence and structural similarities. These small EF-hand calcium binding proteins interact with a large number of protein targets and are almost all been shown to be involved in cancer. In this review we discuss the recent knowledge about the role of S100 proteins and RAGE in human disorders.

Polymeric Nanoparticles with Sequential and Multiple FRET Cascade Mechanisms for Multicolor and Multiplexed Imaging

The ability to map multiple biomarkers at the same time has far-reaching biomedical and diagnostic applications. Here, a series of biocompatible poly(D,L-lactic-co-glycolic acid) and polyethylene glycol particles for multicolor and multiplexed imaging are reported. More than 30 particle formulations that exhibit distinct emission signatures (ranging from the visible to NIR wavelength region) are designed and synthesized. These particles are encapsulated with combinations of carbocyanine-based fluorophores DiO, Dil, DiD, and DiR, and are characterized as <100 nm in size and brighter than commercial quantum dots. A particle formulation is identified that simultaneously emits fluorescence at three different wavelengths upon a single excitation at 485 nm via sequential and multiple FRET cascade events for multicolor imaging. Three other particles that display maximum fluorescence intensities at 570, 672, or 777 nm for multiplexed imaging are also identified. These particles are individually conjugated with specific (Herceptin or IgG2A11 antibody) or nonspecific (heptaarginine) ligands for targeting and, thus, could be applied to differentiate different cancer cells from a cell mixture according to the expressions of cell-surface human epidermal growth factor receptor 2 and the receptor for advanced glycation endproducts. Using an animal model subcutaneously implanted with the particles, it is further demonstrated that the developed platform could be useful for in vivo multiplexed imaging.

Arabinoxylans, gut microbiota and immunity.

Arabinoxylan (AX) is a non-starch polysaccharide found in many cereal grains and is considered as a dietary fiber. Despite their general structure, there is structural heterogeneity among AX originating from different botanical sources. Furthermore, the extraction procedure and hydrolysis by xylolytic enzymes can further render differences to theses AX. The aim of this review was to address the effects of AX on the gut bacteria and their immunomodulatory properties. Given the complex structure of AX, we also aimed to discuss how the structural heterogeneity of AX affects its role in bacterial growth and immunomodulation. The existing literature indicates the role of fine structural details of AX on its potential as polysaccharides that can impact the gut associated microbial growth and immune system.

Crosstalk between calcium, amyloid beta and the receptor for advanced glycation endproducts in Alzheimer's disease.

Hallmarks of Alzheimers disease (AD) include the accumulation of amyloid beta peptide (Abeta), hyperphosphorylation of tau protein, and increased inflammatory activity in the hippocampus and cerebral cortex. The receptor for advanced glycation endproducts (RAGE) has been shown to interact with Abeta and to modulate Abeta transport across the blood-brain barrier. Furthermore, RAGE is upregulated at sites of inflammation and its activation results in distinct intracellular signaling cascades in respect to Abeta conformers. Besides Abeta, RAGE interacts with several members of the calcium binding S100 protein family, amphoterin and advanced glycation endproducts. Mounting evidence suggests that RAGE is a key player in the signaling pathways triggered by Abeta and S100 proteins in AD. In this review, we discuss recent discoveries about the crosstalk between RAGE, Abeta and S100 proteins in the pathophysiology of AD.

RAGE overexpression confers a metastatic phenotype to the WM115 human primary melanoma cell line.

The formation of melanoma metastases from primary tumor cells is a complex phenomenon that involves the regulation of multiple genes. We have previously shown that the receptor for advanced glycation end products (RAGE) was up-regulated in late metastatic stages of melanoma patient samples and we hypothesized that up-regulation of RAGE in cells forming a primary melanoma tumor could contribute to the metastatic switch of these cells. To test our hypothesis, we overexpressed RAGE in the WM115 human melanoma cell line that was established from a primary melanoma tumor of a patient. We show here that overexpression of RAGE in these cells is associated with mesenchymal-like morphologies of the cells. These cells demonstrate higher migration abilities and reduced proliferation properties, suggesting that the cells have switched to a metastatic phenotype. At the molecular level, we show that RAGE overexpression is associated with the up-regulation of the RAGE ligand S100B and the down-regulation of p53, ERK1/2, cyclin E and NF-kB. Our study supports a role of RAGE in the metastatic switch of melanoma cells.

Interaction between glycated serum albumin and AGE-receptors depends on structural changes and the glycation reagent

Physiologically relevant reactive carbonyl compounds vary greatly in their glycation reactivity and the resulting advanced glycation endproducts (AGE) are likely to have distinct structural and biological properties. We characterized a panel of twenty AGE-BSA preparations in terms of (i) their biophysical properties, (ii) their binding to the receptor for advanced glycation endproducts (RAGE) and galectin-3, and (iii) their effects on cellular proliferation. We could establish correlations between lysine glycation and changes in secondary structure. Circular dichroism and differential scanning calorimetry experiments showed that glycation causes albumin to adopt folding properties of a molten globule. Binding studies between AGE-albumin and RAGE or galectin-3 indicate that binding to the isolated receptor domains was weak. Only AGE compounds derived by glycation with ribose were able to bind tightly (K(d) < 10 μM) to both AGE receptors. Cell based assays using an engineered melanoma cell line demonstrated correlations between the extent of (i) lysine side chain modification, (ii) β-sheet content and (iii) albumin multimerization with stimulation of cell proliferation. However, in addition to structural properties of the protein, the chemical structures of the AGE-modifications were important for receptor binding and biological activity as well.