S100A1 and S100B interactions with annexins

Members of the annexin protein family interact with members of the S100 protein family thereby forming heterotetramers in which an S100 homodimer crossbridges two copies of the pertinent annexin. Previous work has shown that S100A1 and S100B bind annexin VI in a Ca(2+)-dependent manner and that annexin VI, but not annexin V, blocks the inhibitory effect of S100A1 and S100B on intermediate filament assembly. We show here that both halves of annexin VI (i.e., the N-terminal half or annexin VI-a and the C-terminal half or annexin VI-b) bind individual S100s on unique sites and that annexin VI-b, but not annexin VI-a, blocks the ability of S100A1 and S100B to inhibit intermediate filament assembly. We also show that the C-terminal extension of S100A1 (and, by analogy, S100B), that was previously demonstrated to be critical for S100A1 and S100B binding to several target proteins including intermediate filament subunits, is not part of the S100 surface implicated in the recognition of annexin VI, annexin VI-a, or annexin VI-b. Evaluation of functional properties with a liposome stability and a calcium influx assay reveals the ability of both S100 proteins to permeabilize the membrane bilayer in a similar fashion like annexins. When tested in combinations with different annexin proteins both S100 proteins mostly lead to a decrease in the calcium influx activity although not all annexin/S100 combinations behave in the same manner. Latter observation supports the hypothesis that the S100-annexin interactions differ mechanistically depending on the particular protein partners.     

A logical sequence search for S100B target proteins

The EF-hand calcium-binding protein S100B has been shown to interact in vitro in a calcium-sensitive manner with many substrates. These potential S100B target proteins have been screened for the preservation of a previously identified consensus sequence across species. The results were compared to known structural and in vitro properties of the proteins to rationalize choices for potential binding partners. Our approach uncovered four oligomeric proteins tubulin (alpha and beta), glial fibrillary acidic protein (GFAP), desmin, and vimentin that have conserved regions matching the consensus sequence. In the type III intermediate filament proteins (GFAP, vimentin, and desmin), this region corresponds to a portion of a coiled-coil (helix 2A), the structural element responsible for their assembly. In tubulin, the sequence matches correspond to regions of alpha and beta tubulin found at the alpha beta tubulin interface. In both cases, these consensus sequence matches provide a logical explanation for in vitro observations that S100B is able to inhibit oligomerization of these proteins.     

S100B proteins that lack one or both cysteine residues can induce inflammatory responses in astrocytes and microglia

The astrocytic protein S100B stimulates neurite outgrowth and neuronal survival during CNS development. S100B can also stimulate glial activation, leading to induction of pro-inflammatory molecules like interleukin-1 beta (IL-1 beta) and inducible nitric oxide synthase (iNOS). Although it is known that S100B's neurotrophic activity requires a disulfide-linked dimeric form of the protein, the structural features of S100B that are important for glial activation have not been defined. As an initial step towards understanding the structural features of S100B required for its action on glia and to determine if these features are different from those required for its action on neurons, we tested two mutants of S100B for their ability to activate glia. The C68VC84S mutant lacks S100B's two cysteine residues (cys68, cys84) and lacks neurotrophic activity (Winningham-Major et al., 1989, J. Cell Biol. 109 3063-3071), and the truncation mutant S100B83stop lacks the C-terminal nine residues (including cys84) that have been shown to be important for some S100B:target protein interactions. We report here that both C68VC84S and S100B83stop stimulate glial activation, as determined by induction of iNOS and IL-1 beta in rat primary astrocyte and microglial cultures. C68VC84S showed activation profiles similar to those of wild-type S100B, demonstrating that a disulfide-linked dimer is not required for glial activation. S100B83stop also stimulated both iNOS and IL-1 beta, although S100B83stop was significantly less effective than wild-type S100B in inducing iNOS. These results indicate that the C-terminal region of S100B is not required for glial activation; however, its presence may influence the degree of activation by the protein. Altogether, these studies demonstrate that the structural features required for S100B's neurotrophic activity are distinct from those affecting its glial activation activity.     

The Zn2+ and Ca2+‐binding S100B and S100A1 proteins: beyond the myths

The S100 genes encode a conserved group of 21 vertebrate‐specific EF‐hand calcium‐binding proteins. Since their discovery in 1965, S100 proteins have remained enigmatic in terms of their cellular functions. In this review, we summarize the calcium‐ and zinc‐binding properties of the dimeric S100B and S100A1 proteins and highlight data that shed new light on the extracellular and intracellular regulation and functions of S100B. We point out that S100B and S100A1 homodimers are not functionally interchangeable and that in a S100A1/S100B heterodimer, S100A1 acts as a negative regulator for the ability of S100B to bind Zn²⁺. The Ca²⁺ and Zn²⁺‐dependent interactions of S100B with a wide array of proteins form the basis of its activities and have led to the derivation of some initial rules for S100B recognition of protein targets. However, recent findings have strongly suggested that these rules need to be revisited. Here, we describe a new consensus S100B binding motif present in intracellular and extracellular vertebrate‐specific proteins and propose a new model for stable interactions of S100B dimers with full‐length target proteins. A chaperone‐associated function for intracellular S100B in adaptive cellular stress responses is also discussed. This review may help guide future studies on the functions of S100 proteins in general.     

Calcium-regulated interaction of Sgt1 with S100A6 (calcyclin) and other S100 proteins

S100A6 (calcyclin), a small calcium-binding protein from the S100 family, interacts with several target proteins in a calcium-regulated manner. One target is Calcyclin-Binding Protein/Siah-1-Interacting Protein (CacyBP/SIP), a component of a novel pathway of beta-catenin ubiquitination. A recently discovered yeast homolog of CacyBP/SIP, Sgt1, associates with Skp1 and regulates its function in the Skp1/Cullin1/F-box complex ubiquitin ligase and in kinetochore complexes. S100A6-binding domain of CacyBP/SIP is in its C-terminal region, where the homology between CacyBP/SIP and Sgt1 is the greatest. Therefore, we hypothesized that Sgt1, through its C-terminal region, interacts with S100A6. We tested this hypothesis by performing affinity chromatography and chemical cross-linking experiments. Our results showed that Sgt1 binds to S100A6 in a calcium-regulated manner and that the S100A6-binding domain in Sgt1 is comprised of 71 C-terminal residues. Moreover, S100A6 does not influence Skp1-Sgt1 binding, a result suggesting that separate Sgt1 domains are responsible for interactions with S100A6 and Skp1. Sgt1 binds not only to S100A6 but also to S100B and S100P, other members of the S100 family. The interaction between S100A6 and Sgt1 is likely to be physiologically relevant because both proteins were co-immunoprecipitated from HEp-2 cell line extract using monoclonal anti-S100A6 antibody. Phosphorylation of the S100A6-binding domain of Sgt1 by casein kinase II was inhibited by S100A6, a result suggesting that the role of S100A6 binding is to regulate the phosphorylation of Sgt1. These findings suggest that protein ubiquitination via Sgt1-dependent pathway can be regulated by S100 proteins.     

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.     

Calcium-dependent interaction of S100B with the C-terminal domain of the tumor suppressor p53

In vitro, the S100B protein interacts with baculovirus recombinant p53 protein and protects p53 from thermal denaturation. This effect is isoform-specific and is not observed with S100A1, S100A6, or calmodulin. Using truncated p53 proteins in the N-terminal (p53(1-320)) and C-terminal (p53(73-393)) domains, we localized the S100B-binding region to the C-terminal region of p53. We have confirmed a calcium-dependent interaction of the S100B with a synthetic peptide corresponding to the C-terminal region of p53 (residues 319-393 in human p53) using plasmon resonance experiments on a BIAcore system. In the presence of calcium, the equilibrium affinity of the S100B for the C-terminal region of p53 immobilized on the sensor chip was 24 +/- 10 nM. To narrow down the region within p53 involved in S100B binding, two synthetic peptides, O1(357-381) (residues 357-381 in mouse p53) and YF-O2(320-346) (residues 320-346 in mouse p53), covering the C-terminal region of p53 were compared for their interaction with purified S100B. Only YF-O2 peptide interacts with S100B with high affinity. The YF-O2 motif is a critical determinant for the thermostability of p53 and also corresponds to a domain responsible for cytoplasmic sequestration of p53. Our results may explain the rescue of nuclear wild type p53 activities by S100B in fibroblast cell lines expressing the temperature-sensitive p53val135 mutant at the nonpermissive temperature.     

Modification of nuclear lamin proteins by a mevalonic acid derivative occurs in reticulocyte lysates and requires the cysteine residue of the C-terminal CXXM motif.

The C-terminus of nuclear lamins (CXXM) resembles a C-terminal motif (the CAAX box) of fungal mating factors and ras-related proteins. The CAAX box is subject to different types of post-translational modifications, including proteolytic processing, isoprenylation and carboxyl methylation. By peptide mapping we show that both chicken lamins A and B2 are processed proteolytically in vivo. However, whereas the entire CXXM motif is cleaved from lamin A, at most three C-terminal amino acids are removed from lamin B2. Following translation of cDNA-derived RNAs in reticulocyte lysates, lamin proteins specifically incorporate a derivative of [14C]mevalonic acid (MV), i.e. the precursor of a putative isoprenoid modification. Remarkably, no MV is incorporated into lamin B2 translated from a mutant cDNA encoding alanine instead of cysteine in the C-terminal CXXM motif. These results implicate this particular cysteine residue as the target for modification of lamin proteins by an isoprenoid MV derivative, and they indicate that isoprenylation is amenable to studies in cell-free systems. Moreover, our observations suggest that C-terminal processing of newly synthesized nuclear lamins is a multi-step process highly reminiscent of the pathway elaborated recently for ras-related proteins.     

Proinflammatory activities of S100: proteins S100A8, S100A9, and S100A8/A9 induce neutrophil chemotaxis and adhesion

S100A8 and S100A9 are small calcium-binding proteins that are highly expressed in neutrophil and monocyte cytosol and are found at high levels in the extracellular milieu during inflammatory conditions. Although reports have proposed a proinflammatory role for these proteins, their extracellular activity remains controversial. In this study, we report that S100A8, S100A9, and S100A8/A9 caused neutrophil chemotaxis at concentrations of 10(-12)-10(-9) M. S100A8, S100A9, and S100A8/A9 stimulated shedding of L-selectin, up-regulated and activated Mac-1, and induced neutrophil adhesion to fibrinogen in vitro. Neutralization with Ab showed that this adhesion was mediated by Mac-1. Neutrophil adhesion was also associated with an increase in intracellular calcium levels. However, neutrophil activation by S100A8, S100A9, and S100A8/A9 did not induce actin polymerization. Finally, injection of S100A8, S100A9, or S100A8/A9 into a murine air pouch model led to rapid, transient accumulation of neutrophils confirming their activities in vivo. These studies 1) show that S100A8, S100A9, and S100A8/A9 are potent stimulators of neutrophils and 2) strongly suggest that these proteins are involved in neutrophil migration to inflammatory sites.     

AutoMotif server: prediction of single residue post-translational modifications in proteins

The AutoMotif Server allows for identification of post-translational modification (PTM) sites in proteins based only on local sequence information. The local sequence preferences of short segments around PTM residues are described here as linear functional motifs (LFMs). Sequence models for all types of PTMs are trained by support vector machine on short-sequence fragments of proteins in the current release of Swiss-Prot database (phosphorylation by various protein kinases, sulfation, acetylation, methylation, amidation, etc.). The accuracy of the identification is estimated using the standard leave-one-out procedure. The sensitivities for all types of short LFMs are in the range of 70%. AVAILABILITY: The AutoMotif Server is available free for academic use at http://automotif.bioinfo.pl/     

Significance of C-terminal cysteine modifications to the biological activity of the Saccharomyces cerevisiae a-factor mating pheromone.

We have undertaken total synthesis of the Saccharomyces cerevisiae a-factor (NH2-YIIKGVFWDPAC[S-farnesyl]-COOCH3) and several Cys-12 analogs to determine the significance of S-farnesylation and carboxy-terminal methyl esterification to the biological activity of this lipopeptide mating pheromone. Replacement of either the farnesyl group or the carboxy-terminal methyl ester by a hydrogen atom resulted in marked reduction but not total loss of bioactivity as measured by a variety of assays. Moreover, both the farnesyl and methyl ester groups could be replaced by other substituents to produce biologically active analogs. The bioactivity of a-factor decreased as the number of prenyl units on the cysteine sulfur decreased from three to one, and an a-factor analog having the S-farnesyl group replaced by an S-hexadecanyl group was more active than an S-methyl a-factor analog. Thus, with two types of modifications, a-factor activity increased as the S-alkyl group became bulkier and more hydrophobic. MATa cells having deletions of the a-factor structural genes (mfal1 mfa2 mutants) were capable of mating with either sst2 or wild-type MAT alpha cells in the presence of exogenous a-factor, indicating that it is not absolutely essential for MATa cells to actively produce a-factor in order to mate. Various a-factor analogs were found to partially restore mating to these strains as well, and their relative activities in the mating restoration assay were similar to their activities in the other assays used in this study. Mating was not restored by addition of exogenous a-factor to a cross of a wild-type MAT alpha strain and a MATaste6 mutant, indicating a role of the STE6 gene product in mating in addition to its secretion of a-factor.     

The role of calcium in the conformational changes of the recombinant S100A8/S100A91

Calprotectin is a member of the EF-hand proteins, composed of two subunits, S100A8 (MRP8) and S100A9 (MRP14). These proteins are involved in important processes including cell signaling, regulation of inflammatory responses, cell cycle control, differentiation, regulation of ion channel activity and defense against microbial agents in a calcium dependent manner. In the present study, recombinant S100A8 and S100A9 were expressed in E. coli BL21 and then purified using Ni-NTA affinity chromatography. The structure of the S100A8/A9 complex in the presence and absence of calcium was assessed by circular dichroism and fluorescence spectroscopy. The intrinsic fluorescence emission spectra of the S100A8/A9 complex in the presence of calcium showed a reduction in fluorescence intensity, reflecting conformational changes within the protein with the exposure of aromatic residues to the protein surface. The far ultraviolet-circular dichroism spectra of the complex in the presence of calcium revealed minor changes in the regular secondary structure of the complex. Also, increased thermal stability of the S100A8/A9 complex in the presence of calcium was indicated.     

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 Ca(2+) binding through EF-hand motifs and binding of Zn(2+) and Cu(2+) at additional sites, usually at the homodimer interfaces. Ca(2+) 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.