Günter Fritz

S100 proteins: structure, functions and pathology.

S100 proteins regulate intracellular processes such as cell growth and motility, cell cycle regulation, transcription and differentiation. Twenty members have been identified so far, and altogether, S100 proteins represent the largest subgroup in the EF-hand Ca2+ -binding protein family. A unique feature of these proteins is that individual members are localized in specific cellular compartments from which some are able to relocate upon Ca2+ activation, transducing the Ca2+ signal in a temporal and spacial manner by interacting with different targets specific for each S100 protein. Some members are even secreted from cells exerting extracellular, cytokine-like activities partially via the surface receptor RAGE (receptor for advanced glycation endproducts) with paracrine effects e.g. on neurons, promoting their survival during development or after injury. Another important aspect is that 14 bona fide S100 genes are found in a gene cluster on human chromosome 1q21 where a number of chromosomal abnormalities occur. This results in a deregulated expression of some S100 genes associated with neoplasias. Recently, S100 proteins have received increasing attention due to their close association with several human diseases including cardiomyopathy, neurodegenerative disorders and cancer. They have also been proven to be valuable in the diagnostic of these diseases, as predictive markers of improving clinical management, outcome and survival of patients and are considered having a potential as drug targets to improve therapies.

Microglia emerge from erythromyeloid precursors via Pu.1- and Irf8-dependent pathways

Microglia are crucial for immune responses in the brain. Although their origin from the yolk sac has been recognized for some time, their precise precursors and the transcription program that is used are not known. We found that mouse microglia were derived from primitive c-kit+ erythromyeloid precursors that were detected in the yolk sac as early as 8 d post conception. These precursors developed into CD45+ c-kitlo CX3CR1− immature (A1) cells and matured into CD45+ c-kit− CX3CR1+ (A2) cells, as evidenced by the downregulation of CD31 and concomitant upregulation of F4/80 and macrophage colony stimulating factor receptor (MCSF-R). Proliferating A2 cells became microglia and invaded the developing brain using specific matrix metalloproteinases. Notably, microgliogenesis was not only dependent on the transcription factor Pu.1 (also known as Sfpi), but also required Irf8, which was vital for the development of the A2 population, whereas Myb, Id2, Batf3 and Klf4 were not required. Our data provide cellular and molecular insights into the origin and development of microglia.

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.

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.

RAGE: a single receptor fits multiple ligands

The receptor for advanced glycation end products (RAGE) is a central signaling molecule in the innate immune system and is involved in the onset and sustainment of the inflammatory response. RAGE belongs to a class of pattern recognition receptors that recognize common features rather than a specific ligand. Recent structural information on the extracellular portion (ectodomain) of RAGE shed new light on this unusual ability. X-ray crystallographic, NMR and biochemical data suggest that ligand binding is driven largely by electrostatic interactions between the positively charged surface of the ectodomain and negatively charged ligands. In this article, I propose a putative mechanism of RAGE ligand recognition of receptor activation.

Molecular basis for manganese sequestration by calprotectin and roles in the innate immune response to invading bacterial pathogens

The S100A8/S100A9 heterodimer calprotectin (CP) functions in the host response to pathogens through a mechanism termed “nutritional immunity.” CP binds Mn2+ and Zn2+ with high affinity and starves bacteria of these essential nutrients. Combining biophysical, structural, and microbiological analysis, we identified the molecular basis of Mn2+ sequestration. The asymmetry of the CP heterodimer creates a single Mn2+-binding site from six histidine residues, which distinguishes CP from all other Mn2+-binding proteins. Analysis of CP mutants with altered metal-binding properties revealed that, despite both Mn2+ and Zn2+ being essential metals, maximal growth inhibition of multiple bacterial pathogens requires Mn2+ sequestration. These data establish the importance of Mn2+ sequestration in defense against infection, explain the broad-spectrum antimicrobial activity of CP relative to other S100 proteins, and clarify the impact of metal depletion on the innate immune response to infection.

RAGE regulation and signaling in inflammation and beyond

RAGE is a key molecule in the onset and sustainment of the inflammatory response. New studies indicate that RAGE might represent a new link between the innate and adaptive immune system. RAGE belongs to the superfamily of Ig cell‐surface receptors and is expressed on all types of leukocytes promoting activation, migration, or maturation of the different cells. RAGE expression is prominent on the activated endothelium, where it mediates leukocyte adhesion and transmigration. Moreover, proinflammatory molecules released from the inflamed or injured vascular system induce migration and proliferation of SMCs. RAGE binds a large number of different ligands and is therefore considered as a PRR, recognizing a structural motif rather than a specific ligand. In this review, we summarize the current knowledge about the signaling pathways activated in the different cell types and discuss a potential activation mechanism of RAGE, as well as putative options for therapeutic intervention.

Natural and amyloid self‐assembly of S100 proteins: structural basis of functional diversity

The S100 proteins are 10–12 kDa EF‐hand proteins that act as central regulators in a multitude of cellular processes including cell survival, proliferation, differentiation and motility. Consequently, many S100 proteins are implicated and display marked changes in their expression levels in many types of cancer, neurodegenerative disorders, inflammatory and autoimmune diseases. The structure and function of S100 proteins are modulated by metal ions via Ca2+ binding through EF‐hand motifs and binding of Zn2+ and Cu2+ at additional sites, usually at the homodimer interfaces. Ca2+ binding modulates S100 conformational opening and thus promotes and affects the interaction with p53, the receptor for advanced glycation endproducts and Toll‐like receptor 4, among many others. Structural plasticity also occurs at the quaternary level, where several S100 proteins self‐assemble into multiple oligomeric states, many being functionally relevant. Recently, we have found that the S100A8/A9 proteins are involved in amyloidogenic processes in corpora amylacea of prostate cancer patients, and undergo metal‐mediated amyloid oligomerization and fibrillation in vitro. Here we review the unique chemical and structural properties of S100 proteins that underlie the conformational changes resulting in their oligomerization upon metal ion binding and ultimately in functional control. The possibility that S100 proteins have intrinsic amyloid‐forming capacity is also addressed, as well as the hypothesis that amyloid self‐assemblies may, under particular physiological conditions, affect the S100 functions within the cellular milieu.

Mutations in POGLUT1, Encoding Protein O-Glucosyltransferase 1, Cause Autosomal-Dominant Dowling-Degos Disease

Dowling-Degos disease (DDD) is an autosomal-dominant genodermatosis characterized by progressive and disfiguring reticulate hyperpigmentation. We previously identified loss-of-function mutations in KRT5 but were only able to detect pathogenic mutations in fewer than half of our subjects. To identify additional causes of DDD, we performed exome sequencing in five unrelated affected individuals without mutations in KRT5. Data analysis identified three heterozygous mutations from these individuals, all within the same gene. These mutations, namely c.11G>A (p.Trp4*), c.652C>T (p.Arg218*), and c.798-2A>C, are within POGLUT1, which encodes protein O-glucosyltransferase 1. Further screening of unexplained cases for POGLUT1 identified six additional mutations, as well as two of the above described mutations. Immunohistochemistry of skin biopsies of affected individuals with POGLUT1 mutations showed significantly weaker POGLUT1 staining in comparison to healthy controls with strong localization of POGLUT1 in the upper parts of the epidermis. Immunoblot analysis revealed that translation of either wild-type (WT) POGLUT1 or of the protein carrying the p.Arg279Trp substitution led to the expected size of about 50 kDa, whereas the c.652C>T (p.Arg218*) mutation led to translation of a truncated protein of about 30 kDa. Immunofluorescence analysis identified a colocalization of the WT protein with the endoplasmic reticulum and a notable aggregating pattern for the truncated protein. Recently, mutations in POFUT1, which encodes protein O-fucosyltransferase 1, were also reported to be responsible for DDD. Interestingly, both POGLUT1 and POFUT1 are essential regulators of Notch activity. Our results furthermore emphasize the important role of the Notch pathway in pigmentation and keratinocyte morphology.