S100A5 is a novel member of the EF-hand superfamily of calcium-binding proteins that is poorly characterized at the protein level. Immunohistochemical analysis demonstrates that it is expressed in very restricted regions of the adult brain. Here we characterized the human recombinant S100A5, especially its interaction with Ca 2؉ , Zn 2؉ , and Cu 2؉. Flow dialysis revealed that the homodimeric S100A5 binds four Ca 2؉ ions with strong positive cooperativity and an affinity 20 -100-fold higher than the other S100 proteins studied under identical conditions. S100A5 also binds two Zn 2؉ ions and four Cu 2؉ ions per dimer. Cu 2؉ binding strongly impairs the binding of Ca 2؉ ; however, none of these ions change the ␣-helical-rich secondary structure. After covalent labeling of an exposed thiol with 2-(4-(iodoacetamide)anilino)-naphthalene-6-sulfonic acid, binding of Cu 2؉ , but not of Ca 2؉ or Zn 2؉ , strongly decreased its fluorescence. In light of the three-dimensional structure of S100 proteins, our data suggest that in each subunit the single Zn 2؉ site is located at the opposite side of the EF-hands. The two Cu 2؉ -binding sites likely share ligands of the EF-hands. The potential role of S100A5 in copper homeostasis is discussed.Calcium, a versatile second messenger of extracellular signals inside the cell, binds to a multitude of cytosolic Ca 2ϩ -binding proteins each of which can, in turn, regulate several effector proteins. The S100 protein family (18 different members) constitutes the largest group of Ca 2ϩ -binding proteins of the EF-hand type (1, 2). At least 13 S100 genes are clustered on human chromosome 1q21, leading to the designation S100A1 to S100A13 for the protein products of these genes (3). Their expression is cell-and tissue-specific, and different human diseases have been associated with deregulated expression (4, 5). S100 proteins are non-covalent homodimers with the notable exception of the heterodimeric S100A8-S100A9. Each monomer possesses 2 EF-hands as follows: a classical C-terminal EFhand with a canonical Ca 2ϩ -binding loop of 12 amino acids and a N-terminal EF-hand with a loop of 14 amino acids which is specific for S100 proteins. This structural difference likely is the reason for the large difference in the Ca 2ϩ affinities of the N-and C-terminal EF-hands. The affinities of S100 proteins for Ca 2ϩ are in general rather low with [Ca 2ϩ ] 0.5 1 values of 100 -300 mM (for review see Refs. 2 and 6), and in 6 out of 8 studied cases binding of Ca 2ϩ displays positive cooperativity. Zn 2ϩ binds to several S100 proteins; the Zn 2ϩ and Ca 2ϩ sites are distinct and can modify the affinity for Ca 2ϩ . S100B binds four Zn 2ϩ ions with concomitant 10-fold increases of the Ca 2ϩ affinity (7). High affinity binding of two Zn 2ϩ to the S100A12 dimer leads to induction of two high affinity Ca 2ϩ -binding sites (8). S100A3 binds eight Zn 2ϩ , but without effect on the affinity for Ca 2ϩ (9, 10). S100A2 binds four Zn 2ϩ with high affinity, in a manner antagonistic to Ca 2ϩ (11). Recently it was reported...

In recent years, protein translocation has been implicated as the mechanism that controls assembly of signaling complexes and induction of signaling cascades. Several members of the multifunctional Ca 2؉ -(Zn 2؉ -and Cu 2؉ )-binding S100 proteins appear to translocate upon cellular stimulation, and some are even secreted from cells, exerting extracellular functions. We transfected cells with S100B-green fluorescent fusion proteins and followed the relocation in real time. A small number of cells underwent translocation spontaneously. However, the addition of thapsigargin, which increases Ca 2؉ levels, intensified ongoing translocation and secretion or induced these processes in resting cells. On the other hand, EGTA or BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid), the Ca 2؉ -chelating agents, inhibited these processes. In contrast, relocation of S100B seemed to be negatively dependent on Zn 2؉ levels. Treatment of cells with TPEN (N,N,N,N-tetrakis(2-pyridylmethyl)ethylenediamine), a Zn 2؉ -binding drug, resulted in a dramatic redistribution and translocation of S100B. Secretion of S100B, when measured by ELISA, was dependent on cell density. As cells reached confluence the secretion drastically declined. However, an increase in Ca 2؉ levels, and even more so, a decrease in Zn 2؉ concentration, reactivated secretion of S100B. On the other hand, secretion did not decrease by treatment with brefeldin A, supporting the view that this process is independent of the endoplasmic reticulum-Golgi classical secretion pathway. S100B is a small acidic protein containing two distinct EFhands, predominantly expressed in astrocytes, oligodendrocytes, and Schwann cells. Intracellularly, S100B regulates the cytoskeletal dynamics through disassembly of tubulin filaments and binding to fibrillary proteins such as CapZ (1-4). Furthermore, S100B interacts in a Ca 2ϩ -dependent manner with the cytoplasmic domain of myelin-associated glycoprotein and inhibits its phosphorylation by protein kinase A (5). Similarly, interaction of S100B with p53 was shown to inhibit protein kinase C phosphorylation (6, 7). When secreted by astrocytes, in addition to an autocrine effect leading, for example, to the activation of extracellular signal-regulated kinase (8), S100B can have a paracrine effect on neurons, promoting their survival during development and after injury through the NF-B pathway, as well as through neurite outgrowth (1, 9 -11). However, the extracellular concentration of S100B plays a crucial role in the physiological response. Although nanomolar quantities have trophic effects on cells, high levels of this protein have been implicated in glial activation (a prominent feature in Down syndrome and Alzheimer's disease), up-regulation of nitric-oxide synthase, and apoptosis (11-13). Recently, Schmidt and colleagues (14, 15) identified the surface receptor RAGE (receptor for advanced glycation endproducts) for S100B, shedding more light on its extracellular function. Others also demonstrated that the binding of extracel...

The levels of expression of the S100A1, S100A2, S100A3, S100A4, S100A5, S100A6 and S100B proteins were immunohistochemically assayed and quantitatively determined in a series of 95 astrocytic tumors including 26 World Health Organization (WHO) grade I (pilocytic astrocytomas), 23 WHO grade II (astrocytomas), 25 WHO grade III (anaplastic astrocytomas) and 21 WHO grade IV (glioblastomas) cases. The level of the immunohistochemical expression of the S100 proteins was quantitatively determined in the solid tumor tissue (tumor mass). In addition twenty blood vessel walls and their corresponding perivascular tumor astrocytes were also immunohistochemically assayed for 10 cases chosen at random from each of the four histopathological groups. The data showed modifications in the level of S100A3 protein expression; these modifications clearly identified the pilocytic astrocytomas from WHO grade II‐IV astrocytic tumors as a distinct biological group. Modifications in the level of S100A6 protein expression enabled a clear distinction to be made between low (WHO grade I and II) and high (WHO grade III and IV) grade astrocytic tumors. Very significant modifications occurred in the level of S100A1 protein expression (and, to a lesser extent, in their of the S100A4 and S100B proteins) in relation to the increasing levels of malignancy. While the S100A5 protein was significantly expressed in all the astrocytic tumors (but without any significant modifications in the levels of malignancy), the S100A2 protein was never expressed in these tumors. These data thus indicate that several S100 proteins play major biological roles in human astrocytic tumors.

Protein translocation between different subcellular compartments might play a significant role in various signal transduction pathways. The S100 family is comprised of the multifunctional, small, acidic proteins, some of which translocate in the form of vesicle-like structures upon increase in intracellular Ca(2+) levels. Previously, cells were fixed before and after calcium activation in order to examine the possible relocation of S100 proteins. In this study, we were able to track the real-time translocation. We compared the localization of endogenous S100A11 to that of the S100A11-green fluorescent protein. The application of thapsigargin, an agent increasing intracellular Ca(2+) levels, resulted in the relocation of the S100A11. In contrast, addition of EGTA, which specifically binds Ca(2+), either inhibited the ongoing process of translocation or prevented its induction. Since translocation was not affected by treatment with brefeldin A, it appears that S100A11 relocates in an endoplasmic reticulum-Golgi-independent pathway. Furthermore, the depolymerization of actin filaments by amlexanox did not affect the capacity of S100A11 to translocate. However, the time course treatment with demecolcine, which depolymerizes tubulin filaments, resulted in cease of translocation, suggesting that the tubulin network is required for this process.