Changes In S100B Research Articles

A New Pathophysiological Aspect of S100B in Schizophrenia: Potential Regulation of S100B by Its Scavenger Soluble RAGE

Several studies have reported elevated S100B serum levels in schizophrenia. Our study focused on its scavenger, soluble receptor for advanced glycation end products (sRAGE). Given the benefits of sRAGE in metabolic and inflammatory diseases, we hypothesized a similar effect in schizophrenia. S100B and sRAGE concentrations were explored during acute paranoid schizophrenia and during reconvalescence. Serum samples from 26 inpatients were investigated on hospital admission (T0) and 6 weeks posttreatment (T6) by S100B-immunoluminometry and sRAGE-ELISA. Thirty-two matched healthy individuals served as controls. Psychopathology was monitored using the Positive and Negative Syndrome Scale (PANSS). S100B (p = .021) and sRAGE (p = .020) were elevated in schizophrenic patients at T0. S100B levels normalized under antipsychotic treatment (p = .003), whereas sRAGE increased further by T6 (p = .005). Changes of S100B during treatment correlated inversely with DeltasRAGE (r = -.422, p = .032). PANSS was negatively associated with sRAGE at T0 (positive score: r = -.415, p = .035; total score: r = -.395, p = .046). Our results provide support for a reduction of S100B levels during reconvalescence from acute paranoid schizophrenia that is regulated by its scavenger sRAGE. This mechanism could provide novel treatment strategies.

Divalent Metal Ion Complexes of S100B in the Absence and Presence of Pentamidine

As part of an effort to inhibit S100B, structures of pentamidine (Pnt) bound to Ca2+-loaded and Zn2+,Ca2+-loaded S100B were determined by X-ray crystallography at 2.15 Å (Rfree=0.266) and 1.85 Å (Rfree=0.243) resolution, respectively. These data were compared to X-ray structures solved in the absence of Pnt, including Ca2+-loaded S100B and Zn2+,Ca2+-loaded S100B determined here (1.88 Å; Rfree=0.267). In the presence and absence of Zn2+, electron density corresponding to two Pnt molecules per S100B subunit was mapped for both drug-bound structures. One Pnt binding site (site 1) was adjacent to a p53 peptide binding site on S100B (±Zn2+), and the second Pnt molecule was mapped to the dimer interface (site 2; ±Zn2+) and in a pocket near residues that define the Zn2+ binding site on S100B. In addition, a conformational change in S100B was observed upon the addition of Zn2+ to Ca2+–S100B, which changed the conformation and orientation of Pnt bound to sites 1 and 2 of Pnt–Zn2+,Ca2+–S100B when compared to Pnt–Ca2+–S100B. That Pnt can adapt to this Zn2+-dependent conformational change was unexpected and provides a new mode for S100B inhibition by this drug. These data will be useful for developing novel inhibitors of both Ca2+- and Ca2+,Zn2+-bound S100B.

Amide Exchange Shows Calcium-Induced Conformational Changes Are Transmitted to the Dimer Interface of S100B

S100B is a 21 kDa member of the S100 calcium-binding protein family. This protein comprises a symmetric homodimer with each subunit having two EF-hands arranged from four alpha-helices (I-IV). S100B binds calcium and undergoes a conformation change leading to the exposure of hydrophobic surface residues that enable the protein to interact with biological target molecules. The most significant structural change that occurs during calcium binding results in a change in the orientation of helix III with respect to helices II and IV. In this work, the calcium-sensitive conformational change has been studied by utilizing fast 1H-15N HSQC experiments and water-transfer methods to follow the amide exchange in apo-S100B and Ca-S100B at 35 degrees C. In apo-S100B, the protection factors are 2-3 orders of magnitude lower for helix III than for helix I, II, or IV. In addition, the exchange stability measured here for the dimer interface helices (I, I', IV, and IV'), in the absence of calcium, is similar to the stability obtained from chemical denaturation experiments. When calcium binds, significant decreases in the protection factors for helices I and IV indicate a modification in the stability of the dimer interface has occurred. In contrast, helix II protection factors increase slightly, which is consistent with a decreased level of surface exposure of this helix. These data have been compared with those of the monomeric S100 protein, calbindin D9k, to illustrate that upon calcium binding there is a balance maintained between the amide exchange rates in helices II and III, although largely the rates are dissimilar for each of these proteins. This distinguishing feature may be important for the calcium-induced conformational change in S100B, where calcium binding is transmitted to the dimer-forming helices.

RAGE Ligand Upregulation of VEGF Secretion in ARPE-19 Cells

The importance of VEGF in stimulating neovascular age-related macular degeneration (AMD) is well-recognized, but the initiating factors that induce local upregulation of VEGF remain unclear. The current study was conducted to test the hypothesis that activation of RAGE (receptor for advanced glycation end products [AGEs]) by its ligands, including AGEs, amyloid-beta peptide (Abeta), and S100B/calgranulins, some of which are known components of drusen and Bruch's membrane deposits, modulate secretion of VEGF by retinal pigment epithelial (RPE) cells. ARPE-19 cells were used for all experiments. The cells were transfected with constructs encoding a signal transduction mutant of human RAGE to assess the RAGE-dependence of intracellular signaling. VEGF secretion and gene expression were assessed by ELISA and quantitative real-time PCR. SDS-PAGE and size exclusion chromatography were performed to analyze the structural changes of S100B after oxidation of its thiol groups under denaturing and nondenaturing conditions, respectively. NF-kappaB activation was assessed via electrophoretic mobility shift assay (EMSA). The impact of the NF-kappaB inhibition was assessed by using parthenolide. ARPE-19 cells basally secreted VEGF under normal cell culture conditions. Immobilized ligands of RAGE increased VEGF secretion in a RAGE-dependent manner. In contrast, soluble AGE-BSA, fresh Abeta, and S100B were less effective in increasing VEGF secretion. Studies with Abeta demonstrated that oligomeric and surface-immobilized forms of Abeta, but not soluble monomeric forms of Abeta, were effective upregulators of VEGF secretion via RAGE. Oxidation of S100B's thiol groups resulted in the formation of oligomers that displayed distinct RAGE biological activity compared with the simple dimeric form. RAGE-mediated upregulation of VEGF secretion by ARPE-19 cells was largely dependent on NF-kappaB, as indicated by studies with parthenolide. Immobilized or oligomerized ligands for RAGE induce RPE cells to increase VEGF secretion. NF-kappaB plays a central role in RAGE-dependent RPE secretion of VEGF. In AMD, activation of the RAGE axis in RPE cells may contribute to upregulation of VEGF, potentially inciting or propagating neovascular macular disease.

Conformation Changes in S-100 Proteins Caused by K+-Binding

Abstract The conformation changes in S-100a.a′ and S-100b caused by K+-binding were determined by several methods in the apo-state and in the Ca2+-bound state. The reactivities of the cysteine (Cys) residues in S-100a.a′ and S-100b to a thiol-specific reagent, 2,2′-dinitro-5,5′-dithiodibenzoic acid (DTNB), were not affected by the K+-binding. On the contrary, the reactivities of those in Ca2+/S-100a.a′ and Ca2+/S-100b toward DTNB were enhanced or decreased, respectively, by the K+-binding. The conformation changes in S-100b, Ca2+/S-100a.a′, and Ca2+/S-100b caused by the K+-binding were also detected using a fluorescence environmental probe, 2-p-toluidinonaphthalene-6-sulfonate (TNS). A clear difference in the decrease of the TNS fluorescence intensity obtained by the K+-binding between S-100a.a′ and S-100b was demonstrated in the Ca2+-bound state. The measurements of the difference absorption spectrum of S-100a.a′ obtained by the K+-binding showed a positive spectrum, while those of S-100b showed a negative spectrum, regardless of the Ca2+-binding.

Ketogenic diet fed rats have low levels of S100B in cerebrospinal fluid

Ketogenic diets have been used to treat seizure disorders of children resistant to conventional anti-epileptic drug treatment. The mechanism of action of this diet, however, is unknown. Gliosis is a very common characteristic in tissues associated with epileptogenesis and glial cytokines may be involved in the pathology of seizure disorders. We investigate herein, whether ketogenic diet fed rats demonstrate changes in the immunocontent of S100B, an astrocyte-derived cytokine elevated in the temporal lobe of refractory epilepsy. Lower levels of S100B were observed in cerebrospinal fluid with no significant changes in S100B and GFAP content in brain tissue. Ketogenic fed rats presented a lower seizure severity induced by pentylenetetrazole and no change in cerebrospinal fluid S100B after pentylenetetrazole administration. These results support the concept that the ketogenic diet is neuroprotective in seizure disorders. Since S100B has an extracellular activity in neuronal excitability and synaptic plasticity, it would be reasonable to conceive that a decrease in the S100B could be involved in the mechanism of action of the ketogenic diet. However, it is not possible to establish a direct link between reduced CSF S100B and decreased severity of PTZ-induced attacks at present moment. Regardless of this, CSF S100B could be proposed as an index of efficacy of ketogenic diet for seizure disorders.

Heterochronous involvement of neurotrophic factors in the neurochemical organization of learning and memory processes in adult organisms.

Studies were performed on the involvement of neurotrophic factors in the neurochemical mechanisms of the integrative functions of the brain. The effects of various intrahippocampal doses of antibodies to neurotrophic factors--protein S100 and lectin CSL--were studied on the formation, retention, and reproduction of a habituated acoustic startle response and conditioned fear in adult rats. S100b contents in the hippocampus, hypothalamus, frontal cortex, and cerebellar hemispheres and vermis, and in the basal nuclei were measured in rat brains 0.5, 1, 2, 4, 6, 8, 24, and 48 h after long-term habituation to the startle response. Antibodies to neurotrophic factors had selective and dose-dependent effects on the different memory and learning processes underlying these types of behavior. Changes in S100b in brain structures were seen, which were specific in terms of quantitative levels and dynamics, after acquisition of the behavioral habit. The results obtained here, along with previously reported data on the effects of antibodies to S100b and CSL given into the cerebellum, are discussed as experimental support for the hypothesis of the heterochronous neurochemical organization of integrative brain activity.

Solution NMR Structure of S100B Bound to the High-affinity Target Peptide TRTK-12

The solution NMR structure is reported for Ca 2+-loaded S100B bound to a 12-residue peptide, TRTK-12, from the actin capping protein CapZ (α1 or α2 subunit, residues 265–276: TRTKIDWNKILS). This peptide was discovered by Dimlich and co-workers by screening a bacteriophage random peptide display library, and it matches exactly the consensus S100B binding sequence ((K/R)(L/I)XWXXIL). As with other S100B target proteins, a calcium-dependent conformational change in S100B is required for TRTK-12 binding. The TRTK-12 peptide is an amphipathic helix (residues W7 to S12) in the S100B–TRTK complex, and helix 4 of S100B is extended by three or four residues upon peptide binding. However, helical TRTK-12 in the S100B–peptide complex is uniquely oriented when compared to the three-dimensional structures of other S100–peptide complexes. The three-dimensional structure of the S100B–TRTK peptide complex illustrates that residues in the S100B binding consensus sequence (K4, I5, W7, I10, L11) are all involved in the S100B–peptide interface, which can explain its orientation in the S100B binding pocket and its relatively high binding affinity. A comparison of the S100B–TRTK peptide structure to the structures of apo- and Ca 2+-bound S100B illustrates that the binding site of TRTK-12 is buried in apo-S100B, but is exposed in Ca 2+-bound S100B as necessary to bind the TRTK-12 peptide.

S100A6 and S100A11 Are Specific Targets of the Calcium- and Zinc-binding S100B Protein in Vivo

In solution, S100B protein is a noncovalent homodimer composed of two subunits associated in an antiparallel manner. Upon calcium binding, the conformation of S100B changes dramatically, leading to the exposure of hydrophobic residues at the surface of S100B. The residues in the C-terminal domain of S100B encompassing Phe(87) and Phe(88) have been implicated in interaction with target proteins. In this study, we used two-hybrid technology to identify specific S100B target proteins. Using S100B as bait, we identify S100A6 and S100A11 as specific targets for S100B. S100A1, the closest homologue of S100B, is capable of interaction with S100B but does not interact with S100A6 or S100A11. S100B, S100A6, and S100A11 isoforms are co-regulated and co-localized in astrocytoma U373 cells. Furthermore, co-immunoprecipitation experiments demonstrated that Ca(2+)/Zn(2+) stabilizes S100B-S100A6 and S100B-S100A11 heterocomplexes. Deletion of the C-terminal domain or mutation of Phe(87) and Phe(88) residues has no effect on S100B homodimerization and heterodimerization with S100A1 but drastically decreases interaction between S100B and S100A6 or S100A11. Our data suggest that the interaction between S100B and S100A6 or S100A11 should not be viewed as a typical S100 heterodimerization but rather as a model of interaction between S100B and target proteins.

Effects of in utero ethanol exposure and maternal treatment with a 5-HT 1A agonist on S100B-containing glial cells

This laboratory previously showed that in utero ethanol exposure severely impairs the development of the cell bodies and projections of serotonin (5-HT) neurons, and that maternal treatment with a 5-HT 1A agonist prevents many of these abnormalities. Others demonstrated that stimulation of fetal astroglial 5-HT 1A receptors increases production and release of S100B, a glial trophic factor that is essential for the development of 5-HT neurons. The present study investigated a potential mechanism by which ethanol hinders development of 5-HT neurons, and by which maternal 5-HT 1A agonist treatment prevents this damage. This study tested the hypothesis that in utero ethanol exposure reduces the number of S100B immunopositive glia and that maternal 5-HT 1A agonist treatment prevents ethanol-associated changes in S100B. To test our hypothesis, we determined the effects of in utero ethanol exposure and maternal treatments with the 5-HT 1A agonists ipsapirone and buspirone on S100B immunopositive glial cells. On gestation day 20 (G20), S100B immunopositive cells were quantified in the midline raphe glial structure (MRGS), a large transient structure that contains substantial numbers of S100B-positive glial cells and that spans the dorsal raphe, median raphe, and B9 complex of 5-HT neurons. S100B immunopositive glial cells were also determined in an area proximal to the dorsal raphe in postnatal day 2 (PN2) rats. In utero ethanol exposure significantly reduced S100B immunopositive glial cells in the MRGS at G20 and in the dorsal raphe at PN2. In addition, treatment of pregnant rats with a 5-HT 1A agonist between G13 and G20 prevented the ethanol-associated reduction in S100B immunopositive glial cells. These studies demonstrated that part of ethanol’s damaging effects on developing 5-HT neurons is mediated by a reduction of S100B and that some of the protective effects of maternal 5-HT 1A agonist treatment are related to the actions of these drugs on glial cells.

Serum S100b changes during intracranial vascular surgery

Department of Neuroanesthesia, The National Hospital for Neurology & Neurosurgery, Queen Square, London, UK.