S100P, a novel Ca(2+)-binding protein from human placenta. cDNA cloning, recombinant protein expression and Ca2+ binding properties.

A novel member of the S100 protein family, present in human placenta, has been characterized by protein sequencing, cDNA cloning, and analysis of Ca(2+)-binding properties. Since the placenta protein of 95 amino acid residues shares about 50% sequence identity with the brain S100 proteins alpha and beta, we proposed the name S100P. The cDNA was expressed in Escherichia coli and recombinant S100P was purified in high yield. S100P is a homodimer and has two functional EF hands/polypeptide chain. The low-affinity site (Kd = 800 microM), which, in analogy to S100 beta, seems to involve the N-terminal EF hand, can be followed by the Ca(2+)-dependent decrease in tyrosine fluorescence. The high-affinity site, provided by the C-terminal EF hand, influences the reactivity of the sole cysteine which is located in the C-terminal extension (Cys85). Binding to the high-affinity site (Kd = 1.6 microM) can be monitored by fluorescence spectroscopy of S100P labelled at Cys85 with 6-proprionyl-2-dimethylaminonaphthalene (Prodan). The Prodan fluorescence shows a Ca(2+)-dependent red shift of the maximum emission wavelength from 485 nm to 502 nm, which is accompanied by an approximately twofold loss in integrated fluorescence intensity. This indicates that Cys85 becomes more exposed to the solvent in Ca(2+)-bound S100P, making this region of the molecule, the so-called C-terminal extension, an ideal candidate for a putative Ca(2+)-dependent interaction with a cellular target. In p11, a different member of the S100 family, the C-terminal extension which contains a corresponding cysteine (Cys82 in p11), is involved in a Ca(2+)-independent complex formation with the protein ligand annexin II. The combined results support the hypothesis that S100 proteins interact in general with their targets after a Ca(2+)-dependent conformational change which involves hydrophobic residues of the C-terminal extension.     

Identification of a novel interaction between the Ca(2+)-binding protein S100A11 and the Ca(2+)- and phospholipid-binding protein annexin A6

S100A11 is a member of the S100 family of EF-hand Ca²⁺-binding proteins, which is expressed in smooth muscle and other tissues. Ca²⁺ binding to S100A11 induces a conformational change that exposes a hydrophobic surface for interaction with target proteins. Affinity chromatography with immobilized S100A11 was used to isolate a 70-kDa protein from smooth muscle that bound to S100A11 in a Ca²⁺-dependent manner and was identified by mass spectrometry as annexin A6. Direct Ca²⁺-dependent interaction between S100A11 and annexin A6 was confirmed by affinity chromatography of the purified bacterially expressed proteins, by gel overlay of annexin A6 with purified S100A11, by chemical cross-linking, and by coprecipitation of S100A11 with annexin A6 bound to liposomes. The expression of S100A11 and annexin A6 in the same cell type was verified by RT-PCR and immunocytochemistry of isolated vascular smooth muscle cells. The site of binding of S100A11 on annexin A6 was investigated by partial tryptic digestion and deletion mutagenesis. The unique NH₂ terminal head region of annexin A6 was not required for S100A11 binding, but binding sites were identified in both NH₂- and COOH-terminal halves of the molecule. We hypothesize that an agonist-induced increase in cytosolic free [Ca²⁺] leads to formation of a complex of S100A11 and annexin A6, which forms a physical connection between the plasma membrane and the cytoskeleton, or plays a role in the formation of signaling complexes at the level of the sarcolemma. smooth muscle; protein-protein interaction     

S100B(betabeta) inhibits the protein kinase C-dependent phosphorylation of a peptide derived from p53 in a Ca2+-dependent manner.

S100B(betabeta) is a dimeric Ca2+-binding protein that is known to inhibit the protein kinase C (PKC)-dependent phosphorylation of several proteins. To further characterize this inhibition, we synthesized peptides based on the PKC phosphorylation domains of p53 (residues 367-388), neuromodulin (residues 37-53), and the regulatory domain of PKC (residues 19-31), and tested them as substrates for PKC. All three peptides were shown to be good substrates for the catalytic domain of PKC. As for full-length p53 (Baudier J, Delphin C, Grunwald D, Khochbin S, Lawrence JJ. 1992. Proc Natl Acad Sci USA 89:11627-11631), S100B(betabeta) binds the p53 peptide and inhibits its PKC-dependent phosphorylation (IC50 = 10 +/- 7 microM) in a Ca2+-dependent manner. Similarly, phosphorylation of the neuromodulin peptide and the PKC regulatory domain peptide were inhibited by S100B(betabeta) in the presence of Ca2+ (IC50 = 17 +/- 5 microM; IC50 = 1 +/- 0.5 microM, respectively). At a minimum, the C-terminal EF-hand Ca2+-binding domain (residues 61-72) of each S100beta subunit must be saturated to inhibit phosphorylation of the p53 peptide as determined by comparing the Ca2+ dependence of inhibition ([Ca]IC50 = 29.3 +/- 17.6 microM) to the dissociation of Ca2+ from the C-terminal EF-hand Ca2+-binding domain of S100B(betabeta).     

Molecular modeling of single polypeptide chain of calcium-binding protein p26olf from dimeric S100B(betabeta).

P26olf from olfactory tissue of frog, which may be involved in olfactory transduction or adaptation, is a Ca2+-binding protein with 217 amino acids. The p26olf molecule contains two homologous parts consisting of the N-terminal half with amino acids 1-109 and the C-terminal half with amino acids 110-217. Each half resembles S100 protein with about 100 amino acids and contains two helix-loop-helix Ca2+-binding structural motifs known as EF-hands: a normal EF-hand at the C-terminus and a pseudo EF-hand at the N-terminus. Multiple alignment of the two S100-like domains of p26olf with 18 S100 proteins indicated that the C-terminal putative EF-hand of each domain contains a four-residue insertion when compared with the typical EF-hand motifs in the S100 protein, while the N-terminal EF-hand is homologous to its pseudo EF-hand. We constructed a three-dimensional model of the p26olf molecule based on results of the multiple alignment and NMR structures of dimeric S100B(betabeta) in the Ca2+-free state. The predicted structure of the p26olf single polypeptide chain satisfactorily adopts a folding pattern remarkably similar to dimeric S100B(betabeta). Each domain of p26olf consists of a unicornate-type four-helix bundle and they interact with each other in an antiparallel manner forming an X-type four-helix bundle between the two domains. The two S100-like domains of p26olf are linked by a loop with no steric hindrance, suggesting that this loop might play an important role in the function of p26olf. The circular dichroism spectral data support the predicted structure of p26olf and indicate that Ca2+-dependent conformational changes occur. Since the C-terminal putative EF-hand of each domain fully keeps the helix-loop-helix motif having a longer Ca2+-binding loop, regardless of the four-residue insertion, we propose that it is a new, novel EF-hand, although it is unclear whether this EF-hand binds Ca2+. P26olf is a new member of the S100 protein family.     

S100-annexin complexes--biology of conditional association

S100 proteins and annexins both constitute groups of Ca2+-binding proteins, each of which comprises more than 10 members. S100 proteins are small, dimeric, EF-hand-type Ca2+-binding proteins that exert both intracellular and extracellular functions. Within the cells, S100 proteins regulate various reactions, including phosphorylation, in response to changes in the intracellular Ca2+ concentration. Although S100 proteins are known to be associated with many diseases, exact pathological contributions have not been proven in detail. Annexins are non-EF-hand-type Ca2+-binding proteins that exhibit Ca2+-dependent binding to phospholipids and membranes in various tissues. Annexins bring different membranes into proximity and assist them to fuse, and therefore are believed to play a role in membrane trafficking and organization. Several S100 proteins and annexins are known to interact with each other in either a Ca2+-dependent or Ca2+-independent manner, and form complexes that exhibit biological activities. This review focuses on the interaction between S100 proteins and annexins, and the possible biological roles of these complexes. Recent studies have shown that S100-annexin complexes have a role in the differentiation of gonad cells and neurological disorders, such as depression. These complexes regulate the organization of membranes and vesicles, and thereby may participate in the appropriate disposition of membrane-associated proteins, including ion channels and/or receptors.     

Comprehensive interaction of dicalcin with annexins in frog olfactory and respiratory cilia

Dicalcin (renamed from p26olf) is a dimer form of S100 proteins found in frog olfactory epithelium. S100 proteins form a group of EF-hand Ca(2+)-binding proteins, and are known to interact with many kinds of target protein to modify their activities. To determine the role of dicalcin in the olfactory epithelium, we identified its binding proteins. Several proteins in frog olfactory epithelium were found to bind to dicalcin in a Ca(2+)-dependent manner. Among them, 38 kDa and 35 kDa proteins were most abundant. Our analysis showed that these were a mixture of annexin A1, annexin A2 and annexin A5. Immunohistochemical analysis showed that dicalcin and all of these three subtypes of annexin colocalize in the olfactory cilia. Dicalcin was found to be present in a quantity almost sufficient to bind all of these annexins. Colocalization of dicalcin and the three subtypes of annexin was also observed in the frog respiratory cilia. Dicalcin facilitated Ca(2+)-dependent liposome aggregation caused by annexin A1 or annexin A2, and this facilitation was additive when both annexin A1 and annexin A2 were present. In this facilitation effect, the effective Ca(2+) concentrations were different between annexin A1 and annexin A2, and therefore the dicalcin-annexin system in frog olfactory and respiratory cilia can cover a wide range of Ca(2+) concentrations. These results suggested that this system is associated with abnormal increases in the Ca(2+) concentration in the olfactory and other motile cilia.     

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.     

Mapping of three unique Ca(2+)-binding sites in human annexin II.

Site-directed mutagenesis was employed to map and characterize Ca(2+)-binding sites in annexin II, a member of the annexin family of Ca(2+)- and phospholipid-binding proteins which serves as a major cellular substrate for the tyrosine kinase encoded by the src oncogene. Several single amino acid substitutions were introduced in the human annexin II and the various mutant proteins were scored for their affinity towards Ca2+ in different assays. The data support our previous finding [Thiel, C., Weber, K. and Gerke V. (1991) J. Biol. Chem. 266, 14,732-14,739] that a Ca(2+)-binding site is present in the third of the four repeat segments which comprise the 33-kDa protein core of annexin II. In addition to Gly206 and Thr207, which are localized in the highly conserved endonexin fold of the third repeat, Glu246 is involved in the formation of this site. Thus the architecture of this Ca(2+)-binding site in solution is very similar, if not identical, to that of Ca2+ sites identified recently in annexin V crystals [Huber, R., Schneider, M., Mayr, I., R?misch, J. and Paques, E.-P. (1990) FEBS Lett. 275, 15-21]. In addition to the site in repeat 3, we have mapped sites of presumably similar architecture in repeats 2 and 4 of annexin II. Again, an acidic amino acid which is located 40 residues C-terminal to the conserved glycine at position 4 of the endonexin fold is indispensable for high-affinity Ca2+ binding: Asp161 in the second and Asp321 in the fourth repeat. In contrast, repeat 1 does not contain an acidic amino acid at a corresponding position and also shows deviations from the other repeats in the sequence surrounding the conserved glycine. These results on annexin II together with the crystallographic information on annexin V reveal that annexins can differ in the position of the Ca2+ sites. Ca(2+)-binding sites of similar structure are present in repeats 2, 3, and 4 of annexin II while in annexin V they occur in repeats 1, 2, and 4. We also synthesized an annexin II derivative with mutations in all three Ca2+ sites. This molecule shows a greatly reduced affinity for the divalent cation. However, it is still able to bind Ca2+, indicating the presence of (an) additional Ca2+ site(s) of presumably different architecture.     

Insights into S100 target specificity examined by a new interaction between S100A11 and annexin A2

S100 proteins are a group of EF-hand calcium-signaling proteins, many of which interact with members of the calcium- and phospholipid-binding annexin family of proteins. This calcium-sensitive interaction enables two neighboring membrane surfaces, complexed to different annexin proteins, to be brought into close proximity for membrane reorganization, using the S100 protein as a bridging molecule. S100A11 and S100A10 are two members of the S100 family found to interact with the N-termini of annexins A1 and A2, respectively. Despite the high degree of structural similarity between these two complexes and the sequences of the peptides, earlier studies have shown that there is little or no cross-reactivity between these two S100s and the annexin peptides. In the current work the specificity and the affinity of the interaction of the N-terminal sequences of annexins A1 and A2 with Ca2+-S100A11 were investigated. Through the use of alanine-scanning peptide array experiments and NMR spectroscopy, an approximate 5-fold tighter interaction was identified between Ca2+-S100A11 and annexin A2 (approximately 3 microM) compared to annexin A1 (approximately 15 microM). Chemical shift mapping revealed that the binding site for annexin A2 on S100A11 was similar to that observed for the annexin A1 but with distinct differences involving the C-terminus of the annexin A2 peptide. In addition, kinetic measurements based on NMR titration data showed that annexin A2 binding to Ca2+-S100A11 occurs at a comparable rate (approximately 120 s(-1)) to that observed for membrane fusion processes such as endo- and exocytosis.     

CacyBP/SIP,a calcyclin and Siah-1-interacting protein,binds EF-hand proteins of the S100 family

Recently, a human ortholog of mouse calcyclin (S100A6)-binding protein (CacyBP) called SIP (Siah-1-interacting protein) was shown to be a component of a novel ubiquitinylation pathway regulating beta-catenin degradation (Matsuzawa, S., and Reed, J. C. (2001) Mol. Cell 7, 915-926). In murine brain, CacyBP/SIP is expressed at a high level, but S100A6 is expressed at a very low level. Consequently we carried out experiments to determine if CacyBP/SIP binds to other S100 proteins in this tissue. Using CacyBP/SIP affinity chromatography, we found that S100B from the brain extract binds to CacyBP/SIP in a Ca2+-dependent manner. Using a nitrocellulose overlay assay with 125I-CacyBP/SIP and CacyBP/SIP affinity chromatography, we found that this protein binds purified S100A1, S100A6, S100A12, S100B, and S100P but not S100A4, calbindin D(9k), parvalbumin, and calmodulin. The interaction of S100 proteins with CacyBP/SIP occurs via its C-terminal fragment (residues 155-229). Co-immunoprecipitation of CacyBP/SIP with S100B from brain and with S100A6 from Ehrlich ascites tumor cells suggests that these interactions are physiologically relevant and that the ubiquitinylation complex involving CacyBP/SIP might be regulated by S100 proteins.     

Calcium- and cell cycle-dependent association of annexin 11 with the nuclear envelope

Annexin 11 is a widely expressed calcium- and phospholipid-binding protein that resides in the nucleoplasm in many cultured cell lines. This is in contrast to its most extensively characterized in vitro ligand, the small calcium-binding protein S100A6 (calcyclin), which is concentrated in the nuclear envelope. Here we have examined the significance of the association of annexin 11 and S100A6 by asking whether circumstances exist in which the two proteins occupy the same subcellular localization. First, we show that in both A431 and vascular smooth muscle cells, elevation of intracellular Ca2+ leads to translocation of annexin 11 from the nucleus to the nuclear envelope where it co-localizes with S100A6. We also demonstrate, using fusions of annexin 11 with green fluorescent protein, that whereas the C-terminal core domain of annexin 11 is essential for Ca2+ sensitivity, the N-terminal domain is required for targeting to the nuclear envelope. Second, we show that annexin 11 relocalizes to the nuclear envelope as A431 cells transit from early to mid-prophase. In late prophase, at the time of nuclear envelope breakdown, annexin 11 and S100A6 become intensely localized with lamina-associated polypeptide 2 to folds in the nuclear envelope. From metaphase to telophase S100A6 is degraded, but in late telophase annexin 11 associates with the reforming nuclear envelope before resuming a nucleoplasmic location in interphase. These results show that co-localization of annexin 11 and S100A6 at the nuclear envelope may be regulated either by elevation of intracellular Ca2+ or by cell cycle progression and provide the first evidence that these proteins may associate in vivo.     

Structural changes in the C-terminus of Ca2+-bound rat S100B (beta beta) upon binding to a peptide derived from the C-terminal regulatory domain of p53.

S100B(beta beta) is a dimeric Ca2+-binding protein that interacts with p53, inhibits its phosphorylation by protein kinase C (PKC) and promotes disassembly of the p53 tetramer. Likewise, a 22 residue peptide derived from the C-terminal regulatory domain of p53 has been shown to interact with S100B(beta beta) in a Ca2+-dependent manner and inhibits its phosphorylation by PKC. Hence, structural studies of Ca2+-loaded S100B(beta beta) bound to the p53 peptide were initiated to characterize this interaction. Analysis of nuclear Overhauser effect (NOE) correlations, amide proton exchange rates, 3J(NH-H alpha) coupling constants, and chemical shift index data show that, like apo- and Ca2+-bound S100B(beta beta), S100B remains a dimer in the p53 peptide complex, and each subunit has four helices (helix 1, Glu2-Arg20; helix 2, Lys29-Asn38; helix 3, Gln50-Asp61; helix 4, Phe70-Phe87), four loops (loop 1, Glu21-His25; loop 2, Glu39-Glu49; loop 3, Glu62-Gly66; loop 4, Phe88-Glu91), and two beta-strands (beta-strand 1, Lys26-Lys28; beta-strand 2, Glu67-Asp69), which forms a short antiparallel beta-sheet. However, in the presence of the p53 peptide helix 4 is longer by five residues than in apo- or Ca2+-bound S100B(beta beta). Furthermore, the amide proton exchange rates in helix 3 (K55, V56, E58, T59, L60, D61) are significantly slower than those of Ca2+-bound S100B(beta beta). Together, these observations plus intermolecular NOE correlations between the p53 peptide and S100B(beta beta) support the notion that the p53 peptide binds in a region of S100B(beta beta), which includes residues in helix 2, helix 3, loop 2, and the C-terminal loop, and that binding of the p53 peptide interacts with and induces the extension of helix 4.     

Involvement of the recoverin C-terminal segment in recognition of the target enzyme rhodopsin kinase

NCS (neuronal Ca2+ sensor) proteins belong to a family of calmodulin-related EF-hand Ca2+-binding proteins which, in spite of a high degree of structural similarity, are able to selectively recognize and regulate individual effector enzymes in a Ca2+-dependent manner. NCS proteins vary at their C-termini, which could therefore serve as structural control elements providing specific functions such as target recognition or Ca2+ sensitivity. Recoverin, an NCS protein operating in vision, regulates the activity of rhodopsin kinase, GRK1, in a Ca2+-dependent manner. In the present study, we investigated a series of recoverin forms that were mutated at the C-terminus. Using pull-down assays, surface plasmon resonance spectroscopy and rhodopsin phosphorylation assays, we demonstrated that truncation of recoverin at the C-terminus significantly reduced the affinity of recoverin for rhodopsin kinase. Site-directed mutagenesis of single amino acids in combination with structural analysis and computational modelling of the recoverin-kinase complex provided insight into the protein-protein interface between the kinase and the C-terminus of recoverin. Based on these results we suggest that Phe3 from the N-terminal helix of rhodopsin kinase and Lys192 from the C-terminal segment of recoverin form a cation-π interaction pair which is essential for target recognition by recoverin. Taken together, the results of the present study reveal a novel rhodopsin-kinase-binding site within the C-terminal region of recoverin, and highlights its significance for target recognition and regulation.     

Interaction of S100 proteins with the antiallergic drugs, olopatadine, amlexanox, and cromolyn: identification of putative drug binding sites on S100A1 protein

S100 proteins are a multigenic family of low-molecular-weight Ca(2+)-binding proteins comprising 19 members. These proteins undergo a conformational change by Ca(2+)-binding and consequently interact with their target proteins. Recently, we reported that two antiallergic drugs, Amlexanox and Cromolyn, bind to S100A12 and S100A13 of the S100 protein family. In the present study, we used a newly developed antiallergic drug, Olopatadine, as a ligand for affinity chromatography and examined binding specificity of the drug to S100 protein family. Olopatadine binds specifically to S100 proteins, such as S100A1, S100B, S100L, S100A12, and S100A13, in a Ca(2+)-dependent manner but not to calmodulin. Mutagenesis study showed that amino acid residues 76-85 in S100A1 are necessary for its binding to Olopatadine. In contrast, residues 89-94 were identified as an Amlexanox-binding site in S100A1. Moreover, Olopatadine did not competitively inhibit S100A1-binding site of Amlexanox. Furthermore, we showed that Olopatadine inhibited the binding of S100A1 target protein's binding site peptides to S100A1. These results indicate that C-terminal region of S100A1 is important for antiallergic drug binding, although the drug binding sites are different according to each antiallergic drug. Differences in the binding sites of S100A1 to antiallergic drugs suggest that the regulatory functions of S100 proteins may exist in several regions. Therefore, these drugs may serve as useful tools for evaluating the physiological significance of S100 protein family.     

Binding of annexins to lung lamellar bodies and the PMA-stimulated secretion of annexin V from alveolar type II cells

To identify lung lamellar body (LB)-binding proteins, the fractions binding to LB-Sepharose 4B in a Ca(2+)-dependent manner from the lung soluble fractions were analyzed with Mono Q column. Four annexins (annexins III, IV, V, and VIII) were identified by partial amino acid sequence analyses as the LB-binding proteins in the lung soluble fractions. A control experiment using phospholipid (phosphatidylserine/phosphatidylglycerol/phosphtidylcholine) liposome-Sepharose 4B revealed that annexins III, IV and V were the Ca(2+)-dependent proteins binding to the column in the lung soluble fractions, while annexin VIII was not detected. Thus, annexin VIII might preferentially bind to LB. On the other hand, the only Ca(2+)-dependent LB-binding protein identified in the bronchoalveolar lavage fluids was annexin V. It was further demonstrated that annexin V was secreted by isolated alveolar type II cells from rats and that the secretion was stimulated by the addition of phorbol ester (PMA), a potent stimulator of surfactant secretion. The PMA-dependent stimulation of annexin V was attenuated by preincubation with surfactant protein-A (SP-A), a potent inhibitor of surfactant secretion. As LB is thought to be an intracellular store of pulmonary surfactant, which is secreted by alveolar type II cells, annexin V is likely to be secreted together with the lamellar body.     

Structure of Ca2+-bound S100A4 and its interaction with peptides derived from nonmuscle myosin-IIA

S100A4, also known as mts1, is a member of the S100 family of Ca2+-binding proteins that is directly involved in tumor invasion and metastasis via interactions with specific protein targets, including nonmuscle myosin-IIA (MIIA). Human S100A4 binds two Ca2+ ions with the typical EF-hand exhibiting an affinity that is nearly 1 order of magnitude tighter than that of the pseudo-EF-hand. To examine how Ca2+ modifies the overall organization and structure of the protein, we determined the 1.7 A crystal structure of the human Ca2+-S100A4. Ca2+ binding induces a large reorientation of helix 3 in the typical EF-hand. This reorganization exposes a hydrophobic cleft that is comprised of residues from the hinge region,helix 3, and helix 4, which afford specific target recognition and binding. The Ca2+-dependent conformational change is required for S100A4 to bind peptide sequences derived from the C-terminal portion of the MIIA rod with submicromolar affinity. In addition, the level of binding of Ca2+ to both EF-hands increases by 1 order of magnitude in the presence of MIIA. NMR spectroscopy studies demonstrate that following titration with a MIIA peptide, the largest chemical shift perturbations and exchange broadening effects occur for residues in the hydrophobic pocket of Ca2+-S100A4. Most of these residues are not exposed in apo-S100A4 and explain the Ca2+ dependence of formation of theS100A4-MIIA complex. These studies provide the foundation for understanding S100A4 target recognition and may support the development of reagents that interfere with S100A4 function.     

S100A8 and S100A9 in human arterial wall. Implications for atherogenesis

Atherogenesis is a complex process involving inflammation. S100A8 and S100A9, the Ca2+-binding neutrophil cytosolic proteins, are associated with innate immunity and regulate processes leading to leukocyte adhesion and transmigration. In neutrophils and monocytes the S100A8-S100A9 complex regulates phosphorylation, NADPH-oxidase activity, and fatty acid transport. The proteins have anti-microbial properties, and S100A8 may play a role in oxidant defense in inflammation. Murine S100A8 is regulated by inflammatory mediators and recruits macrophages with a proatherogenic phenotype. S100A9 but not S100A8 was found in macrophages in ApoE-/- murine atherosclerotic lesions, whereas both proteins are expressed in human giant cell arteritis. Here we demonstrate S100A8 and S100A9 protein and mRNA in macrophages, foam cells, and neovessels in human atheroma. Monomeric and complexed forms were detected in plaque extracts. S100A9 was strongly expressed in calcifying areas and the surrounding extracellular matrix. Vascular matrix vesicles contain high levels of Ca2+-binding proteins and phospholipids that regulate calcification. Matrix vesicles characterized by electron microscopy, x-ray microanalysis, nucleoside triphosphate pyrophosphohydrolase assay and cholesterol/phospholipid analysis contained predominantly S100A9. We propose that S100A9 associated with lipid structures in matrix vesicles may influence phospholipid-Ca2+ binding properties to promote dystrophic calcification. S100A8 and S100A9 were more sensitive to hypochlorite oxidation than albumin or low density lipoprotein and immunoaffinity confirmed S100A8-S100A9 complexes; some were resistant to reduction, suggesting that hypochlorite may contribute to protein cross-linking. S100A8 and S100A9 in atherosclerotic plaque and calcifying matrix vesicles may significantly influence redox- and Ca2+-dependent processes during atherogenesis and its chronic complications, particularly dystrophic calcification.     

Purification and properties of a novel nucleotide-hydrolysing enzyme (5''-nucleotidase) from Boophilus microplus.

Conformational changes in the beta-subunit of the bovine brain Ca2+-binding protein S100b (S100-beta) accompanying Ca2+ binding were investigated by analysis of the spectroscopic properties of the single tyrosine residue (Tyr17 beta) and flow-dialysis binding experiments. S100-beta binds Ca2+ sequentially at two sites to change the conformation of the protein. The first Ca2+ ion binds to site II beta, a typical Ca2+-binding site in the C-terminal region, and it does not significantly perturb the proximal environment of Tyr17 beta. After the first site is occupied, another Ca2+ ion binds to the N-terminal Ca2+-binding site, I beta, and strengthens a hydrogen bond between Tyr17 beta and a neighbouring carboxylate acceptor group, which results in a large increase in the Tyr17 beta fluorescence spectrum half-width and a positive absorption and c.d. signal between 290 and 275 nm. Ca2+ binding to the S100b.Zn2+6 complex, studied by flow-dialysis and fluorescence measurements showed that, although Zn2+ ions increase the affinity of S100b protein for Ca2+, the Ca2+-binding sequence was not changed. Tb3+ (terbium ion) binding studies on the S100b.Zn2+6 complex proved that Tb3+ antagonizes only Ca2+ binding site II beta and confirmed the sequential occupation of Ca2+-binding sites on the S100b.Zn2+6 complex.     

Ca2+-dependent interaction of the inhibitory region of troponin I with acidic residues in the N-terminal domain of troponin C

Ca2+ regulation of vertebrate striated muscle contraction is initiated by conformational changes in the N-terminal, regulatory domain of the Ca2+-binding protein troponin C (TnC), altering the interaction of TnC with the other subunits of troponin complex, TnI and TnT. We have investigated the role of acidic amino acid residues in the N-terminal, regulatory domain of TnC in binding to the inhibitory region (residues 96-116) of TnI. We constructed three double mutants of TnC (E53A/E54A, E60A/E61A and E85A/D86A), in which pairs of acidic amino acid residues were replaced by neutral alanines, and measured their affinities for synthetic inhibitory peptides. These peptides had the same amino acid sequence as TnI segments 95-116, 95-119 or 95-124, except that the natural Phe-100 of TnI was replaced by a tryptophan residue. Significant Ca2+-dependent increases in the affinities of the two longer peptides, but not the shortest one, to TnC could be detected by changes in Trp fluorescence. In the presence of Ca2+, all the mutant TnCs showed about the same affinity as wild-type TnC for the inhibitory peptides. In the presence of Mg2+ and EGTA, the N-terminal, regulatory Ca2+-binding sites of TnC are unoccupied. Under these conditions, the affinity of TnC(E85A/D86A) for inhibitory peptides was about half that of wild-type TnC, while the other two mutants had about the same affinity. These results imply a Ca2+-dependent change in the interaction of TnC Glu-85 and/or Asp-86 with residues (117-124) on the C-terminal side of the inhibitory region of TnI. Since Glu-85 and/or Asp-86 of TnC have also been demonstrated to be involved in Ca2+-dependent regulation through interaction with TnT, this region of TnC must be critical for troponin function.     

S100A1 binds to the calmodulin-binding site of ryanodine receptor and modulates skeletal muscle excitation-contraction coupling

S100A1, a 21-kDa dimeric Ca2+-binding protein, is an enhancer of cardiac Ca2+ release and contractility and a potential therapeutic agent for the treatment of cardiomyopathy. The role of S100A1 in skeletal muscle has been less well defined. Additionally, the precise molecular mechanism underlying S100A1 modulation of sarcoplasmic reticulum Ca2+ release in striated muscle has not been fully elucidated. Here, utilizing a genetic approach to knock out S100A1, we demonstrate a direct physiological role of S100A1 in excitation-contraction coupling in skeletal muscle. We show that the absence of S100A1 leads to decreased global myoplasmic Ca2+ transients following electrical excitation. Using high speed confocal microscopy, we demonstrate with high temporal resolution depressed activation of sarcoplasmic reticulum Ca2+ release in S100A1-/- muscle fibers. Through competition assays with sarcoplasmic reticulum vesicles and through tryptophan fluorescence experiments, we also identify a novel S100A1-binding site on the cytoplasmic face of the intact ryanodine receptor that is conserved throughout striated muscle and corresponds to a previously identified calmodulin-binding site. Using a 12-mer peptide of this putative binding domain, we demonstrate low micromolar binding affinity to S100A1. NMR spectroscopy reveals this peptide binds within the Ca2+-dependent hydrophobic pocket of S100A1. Taken together, these data suggest that S100A1 plays a significant role in skeletal muscle excitation-contraction coupling, primarily through specific interactions with a conserved binding domain of the ryanodine receptor. This warrants further investigation into the use of S100A1 as a therapeutic target for the treatment of both cardiac and skeletal myopathies.