Stefan W. Vetter

Binding of S100 proteins to RAGE: an update.

The Receptor for Advanced Glycation Endproducts (RAGE) is a multi-ligand receptor of the immunoglobulin family. RAGE interacts with structurally different ligands probably through the oligomerization of the receptor on the cell surface. However, the exact mechanism is unknown. Among RAGE ligands are members of the S100 protein family. S100 proteins are small calcium binding proteins with high structural homology. Several members of the family have been shown to interact with RAGE in vitro or in cell-based assays. Interestingly, many RAGE ligands appear to interact with distinct domains of the extracellular portion of RAGE and to trigger various cellular effects. In this review, we summarize the modes of S100 protein-RAGE interaction with regard to their cellular functions.

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.

Moderate glycation of serum albumin affects folding, stability, and ligand binding.

BACKGROUND Serum protein glycation and formation of advanced glycation endproducts (AGE) correlates with diabetic complications. Highly AGE-modified albumin is frequently used to study the biochemical and cellular activities of AGE-proteins. However, moderately modified albumin samples are expected to be (patho)physiologically more relevant for diabetes research. We produced a panel of nine moderately modified albumin samples and characterized these in terms of side chain modifications, secondary structure, folding stability, and spectroscopic properties. METHODS A panel of nine albumin samples modified with glucose, methylglyoxal, glyoxylic acid and carboxymethyl lysine was characterized in terms of side chain modifications, thermal folding stability, secondary structure, aggregation, surface charge, and ligand binding. The analytical tools employed included chemical analysis, biochemical and immunochemical assays for side chain modifications, near UV circular dichroism, differential scanning calorimetry, analytical size exclusion and ion exchange HPLC, and a ligand binding assay. CONCLUSION Moderate glycation and AGE modification of serum albumin causes structural changes that depend on the chemical reactivity of the modifying reagent and the concentration used for in-vitro glycation. In general, the α-helical content is decreased and thermal unfolding behavior is altered. However, moderate glycation does not cause aggregation or formation of amyloid structures as previous reported for highly modified albumin. A structural characterization of in vitro produced AGE-proteins will be useful to correctly interpret the pathophysiological significance of AGE products in diabetes.

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.

The role of S100 proteins and their receptor RAGE in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with low survival rates. Current therapeutic treatments have very poor response rates due to the high inherent chemoresistance of the pancreatic-cancer cells. Recent studies have suggested that the receptor for advanced glycation end products (RAGE) and its S100 protein ligands play important roles in the progression of PDAC. We will discuss the potential role of S100 proteins and their receptor, RAGE, in the development and progression of pancreatic cancer.

Replacement of the axial histidine heme ligand with cysteine in nitrophorin 1: spectroscopic and crystallographic characterization

To evaluate the potential of using heme-containing lipocalin nitrophorin 1 (NP1) as a template for protein engineering, we have replaced the native axial heme-coordinating histidine residue with glycine, alanine, and cysteine. We report here the characterization of the cysteine mutant H60C_NP1 by spectroscopic and crystallographic methods. The UV/vis, resonance Raman, and magnetic circular dichroism spectra suggest weak thiolate coordination of the ferric heme in the H60C_NP1 mutant. Reduction to the ferrous state resulted in loss of cysteine coordination, while addition of exogenous imidazole ligands gave coordination changes that varied with the ligand. Depending on the substitution of the imidazole, we could distinguish three heme coordination states: five-coordinate monoimidazole, six-coordinate bisimidazole, and six-coordinate imidazole/thiolate. Ligand binding affinities were measured and found to be generally 2–3 orders of magnitude lower for the H60C mutant relative to NP1. Two crystal structures of the H60C_NP1 in complex with imidazole and histamine were solved to 1.7- and 1.96-Å resolution, respectively. Both structures show that the H60C mutation is well tolerated by the protein scaffold and suggest that heme–thiolate coordination in H60C_NP1 requires some movement of the heme within its binding cavity. This adjustment may be responsible for the ease with which the engineered heme–thiolate coordination can be displaced by exogenous ligands.

Calorimetric investigation of diclofenac drug binding to a panel of moderately glycated serum albumins.

Glycation alters the drug binding properties of serum proteins and could affect free drug concentrations in diabetic patients with elevated glycation levels. We investigated the effect of bovine serum albumin glycation by eight physiologically relevant glycation reagents (glucose, ribose, carboxymethyllysine, acetoin, methylglyoxal, glyceraldehyde, diacetyl and glycolaldehyde) on diclofenac drug binding. We used this non-steroidal anti-inflammatory drug diclofenac as a paradigm for acidic drugs with high serum binding and because of its potential cardiovascular risks in diabetic patients. Isothermal titration calorimetry showed that glycation reduced the binding affinity Ka of serum albumin and diclofenac 2 to 6-fold by reducing structural rigidity of albumin. Glycation affected the number of drug binding sites in a glycation reagent dependent manner and lead to a 25% decrease for most reagent, expect for ribose, with decreased by 60% and for the CML-modification, increased the number of binding sites by 60%. Using isothermal titration calorimetry and differential scanning calorimetry we derived the complete thermodynamic characterization of diclofenac binding to all glycated BSA samples. Our results suggest that glycation in diabetic patients could significantly alter the pharmacokinetics of the widely used over-the-counter NSDAI drug diclofenac and with possibly negative implications for patients.

Glycated Serum Albumin and AGE Receptors

In vivo modification of proteins by molecules with reactive carbonyl groups leads to intermediate and advanced glycation end products (AGE). Glucose is a significant glycation reagent due to its high physiological concentration and poorly controlled diabetics show increased albumin glycation. Increased levels of glycated and AGE-modified albumin have been linked to diabetic complications, neurodegeneration, and vascular disease. This review discusses glycated albumin formation, structural consequences of albumin glycation on drug binding, removal of circulating AGE by several scavenger receptors, as well as AGE-induced proinflammatory signaling through activation of the receptor for AGE. Analytical methods for quantitative detection of protein glycation and AGE formation are compared. Finally, the use of glycated albumin as a novel clinical marker to monitor glycemic control is discussed and compared to glycated hemoglobin (HbA1c) as long-term indicator of glycemic status.