Cloning, characterization, and expression of calcyphosine 2, a novel human gene encoding an EF-hand Ca(2+)-binding protein

Calcyphosine is a calcium-binding protein containing four EF-hand domains, initially identified as thyroid protein p24. It was first cloned and its counterparts in rabbit, human, and mouse, crayfish and lobster of invertebrate were also cloned. Here we describe the cloning and characterization of a novel human calcyphosine gene. The 3829-bp cDNA encodes a EF-hand Ca(2+)-binding protein homologous to the dog calcyphosine. It also contains two EF-hand Ca(2+)-binding motif. It is abundantly expressed in many tissues including by RT-PCR analysis and believed to play important role in calcium signaling. It was mapped to human genome 12q15.     

The crystal structure at 2A resolution of the Ca2+ -binding protein S100P

S100P is a small calcium-binding protein of the S100 EF-hand-containing family of proteins. Elevated levels of its mRNA are reported to be associated with the progression to hormone independence and metastasis of prostate cancer and to be associated with loss of senescence in human breast epithelial cells in vitro. The first structure of human recombinant S100P in calcium-bound form is now reported at 2.0A resolution by X-ray diffraction. A flexible linker connects the two EF-hand motifs. The protein exists as a homodimer formed by non-covalent interactions between large hydrophobic areas on monomeric S100P. Experiments with an optical biosensor to study binding parameters of the S100P monomer interaction showed that the association rate constant was faster in the presence of calcium than in their absence, whereas the dissociation rate constant was independent of calcium. The K(d) values were 64(+/-24)nM and 2.5(+/-0.8) microM in the presence and in the absence of calcium ions, respectively. Dimerization of S100P is demonstrated in vivo using the yeast two-hybrid system. The effect of mutation of specific amino acids suggests that dimerization in vivo can be affected by amino acids on the dimer interface and in the hydrophobic core.     

New perspectives on S100 proteins: a multi-functional Ca 2+ -, Zn 2+ - and Cu 2+ -binding protein family

S100 proteins (16 members) show a very divergent pattern of cell- and tissue-specific expression, of subcel-lular localizations and relocations, of post-translational modifications, and of affinities for Ca 2+ , Zn 2+ , and Cu 2+ , consistent with their pleiotropic intra- and extracellular functions. Up to 40 target proteins are reported to interact with S100 proteins and for S100A1 alone 15 target proteins are presently known. Therefore it is not surprising that many functional roles have been proposed and that several human disorders such as cancer, neurodegenerative diseases, cardiomyopathies, inflammations, diabetes, and allergies are associated with an altered expression of S100 proteins. It is not unlikely that their biological activity in some cases is regulated by Zn 2+ and Cu 2+ , rather than by Ca 2+ Despite the numerous putative functions of S100 proteins, their three-dimensional structures of, e.g., S100B, S100A6, and S100A7 are surprisingly similar. They contain a compact dimerization domain whose conformation is rather insensitive to Ca 2+ binding and two lateral a-helices III and III, which project outward of each subunit when Ca 2+ is bound. Target docking depends on the two hydrophobic patches in front of the paired EF-hand generated by the binding of Ca 2+. The selec-tivity in target binding is assured by the central linker between the two EF-hands and the C-terminal tail. It appears that the S100-binding domain in some target proteins contains a basic amphiphilic a-helix and that the mode of interaction and activation bears structural similarity to that of calmodulin.© Kluwer Academic Publishers     

Characterization, evolution and tissue-specific expression of AmphiCalbin, a novel gene encoding EF-hand calcium-binding protein in amphioxus Branchiostoma belcheri

An amphioxus full-length cDNA, AmphiCalbin, encoding a novel EF-hand calcium-binding protein （EFCaBP）, was isolated from the gut cDNA library of amphioxus Branchiostoma belcheri. It consists of 1321 bp with a 636 bp open reading frame encoding a protein of 211 amino acids with a molecular mass of approximately 24.5 kDa. The phylogenetic analysis offers two interesting inferences. First, AmphiCalbin clusters with a group of unnamed EFCaBPs that are differentiated from other identified EFCaBPs. Second, AmphiCalbin falls at the base of the vertebrate unnamed EFCaBPs clade, probably representing their prototype. This is also corroborated by the fact that AmphiCalbin has an exon-intron organization identical to that of vertebrate unnamed EFCaBP genes. Both tissue-section in situ hybridization and whole-mount in situ hybridization prove a tissue-specific expression pattern of AmphiCalbin, with high levels of expression in the digestive system and gonads. It is proposed that AmphiCalbin might play a role in the digestive system and gonads. These observations lay the foundation for further understanding of the function of the unnamed EFCaBPs.     

Calcium-dependent tetramer formation of S100A8 and S100A9 is essential for biological activity

S100 proteins comprise the largest family of calcium-binding proteins. Members of this family usually form homo- or heterodimers, which may associate to higher-order oligomers in a calcium-dependent manner. The heterodimers of S100A8 and S100A9 represent the major calcium-binding proteins in phagocytes. Both proteins regulate migration of these cells via modulation of tubulin polymerization. Calcium binding induces formation of (S100A8/S100A9)2 tetramers. The functional relevance of these higher-order oligomers of S100 proteins, however, is not yet clear. To investigate the importance of higher-order oligomerization for S100 proteins, we created a set of mutations within S100A9 (N69A, E78A, N69A+E78A) destroying the high-affinity C-terminal calcium-binding site (EF-hand II). Mutations in EF-hand II did not interfere with formation of the S100A8/S100A9 heterodimer as demonstrated by yeast two-hybrid experiments and pull-down assays. In contrast, mass spectrometric analysis and density gradient centrifugation revealed that calcium-induced association of (S100A8/S100A9)2 tetramers was strictly dependent on a functional EF-hand II in S100A9. Failure of tetramer formation was associated with a lack of functional activity of S100A8/S100A9 complexes in promoting the formation of microtubules. Thus, our data demonstrate that calcium-dependent formation of (S100A8/S100A9)2 tetramers is an essential prerequisite for biological function. This is the first report showing a functional relevance of calcium-induced higher-order oligomerization in the S100 family.     

Structures of EF-hand Ca 2+-binding proteins: Diversity in the organization, packing and response to Ca 2+ Binding

The growing database of three-dimensional structures of EF-hand calcium-binding proteins is revealing a previously unrecognized variability in the coformations and organizations of EF-hand binding motifs. The structures of twelve different EF-hand proteins for which coordinates are publicly available are discussed and related to their respective biological and biophysical properties. The classical picture of calcium sensors and calcium signal modulators is presented, along with variants on the basic theme and new structural paradigms.© Kluwer Academic Publishers     

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.     

Monomeric state of S100P protein: Experimental and molecular dynamics study

S100 proteins constitute a large subfamily of the EF-hand superfamily of calcium binding proteins. They possess one classical EF-hand Ca²⁺-binding domain and an atypical EF-hand domain. Most of the S100 proteins form stable symmetric homodimers. An analysis of literature data on S100 proteins showed that their physiological concentrations could be much lower than dissociation constants of their dimeric forms. It means that just monomeric forms of these proteins are important for their functioning. In the present work, thermal denaturation of apo-S100P protein monitored by intrinsic tyrosine fluorescence has been studied at various protein concentrations within the region from 0.04–10 μM. A transition from the dimeric to monomeric form results in a decrease in protein thermal stability shifting the mid-transition temperature from 85 to 75 °C. Monomeric S100P immobilized on the surface of a sensor chip of a surface plasmon resonance instrument forms calcium dependent 1 to 1 complexes with human interleukin-11 (equilibrium dissociation constant 1.2 nM). In contrast, immobilized interleukin-11 binds two molecules of dimeric S100P with dissociation constants of 32 nM and 288 nM. Since effective dissociation constant of dimeric S100P protein is very low (0.5 μM as evaluated from our data) the sensitivity of the existing physical methods does not allow carrying out a detailed study of S100P monomer properties. For this reason, we have used molecular dynamics methods to evaluate structural changes in S100P upon its transition from the dimeric to monomeric state. 80-ns molecular dynamics simulations of kinetics of formation of S100P, S100B and S100A11 monomers from the corresponding dimers have been carried out. It was found that during the transition from the homo-dimer to monomer form, the three S100 monomer structures undergo the following changes: (1) the helices in the four-helix bundles within each monomer rotate in order to shield the exposed non-polar residues; (2) almost all lost contacts at the dimer interface are substituted with equivalent and newly formed interactions inside each monomer, and new stabilizing interactions are formed; and (3) all monomers recreate functional hydrophobic cores. The results of the present study show that both dimeric and monomeric forms of S100 proteins can be functional.     

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).     

Oligomerization state of S100B at nanomolar concentration determined by large-zone analytical gel filtration chromatography.

S100B is a Ca(2+)-binding protein known to be a non-covalently associated dimer, S100B(beta beta), at high concentrations (0.2-3.0 mM) under reducing conditions. The solution structure of apo-S100B (beta beta) shows that the subunits associate in an antiparallel manner to form a tightly packed hydrophobic core at the dimer interface involving six of eight helices and the C-terminal loop (Drohat AC, Amburgey JC, Abildgaard F, Starich MR, Baldisseri D, Weber DJ. 1996. Solution structure of rat apo-S100B (beta beta) as determined by NMR spectroscopy. Biochemistry 35:11577-11588). The C-terminal loop, however, is also known to participate in the binding of S100B to target proteins, so its participation in the dimer interface raises questions as to the physiological relevance of dimeric S100B (beta beta). Therefore, we investigated the oligomerization state of S100B at low concentrations (1-10,000 nM) using large-zone analytical gel filtration chromatography with 35S-labeled S100B. We found that S100B exists (> 99%) as a non-covalently associated dimer, S100B (beta beta), at 1 nM subunit concentration (500 pM dimer) in the presence or absence of saturating levels of Ca2+, which implies a dissociation constant in the picomolar range or lower. These results demonstrate for the first time that in reducing environments and at physiological concentrations, S100B exists as dimeric S100B (beta beta) in the presence or absence of Ca2+, and that the non-covalent dimer is most likely the form of S100B presented to target proteins.     

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.     

Ca2+ -dependent interaction of S100A1 with the sarcoplasmic reticulum Ca2+ -ATPase2a and phospholamban in the human heart

The Ca(2+)-binding S100A1 protein displays a specific and high expression level in the human myocardium and is considered to be an important regulator of heart contractility. Diminished protein levels detected in dilated cardiomyopathy possibly contribute to impaired Ca(2+) handling and contractility in heart failure. To elucidate the S100A1 signaling pathway in the human heart, we searched for S100A1 target proteins by applying S100A1-specific affinity chromatography and immunoprecipitation techniques. We detected the formation of a Ca(2+)-dependent complex of S100A1 with SERCA2a and PLB in the human myocardium. Using confocal laser scanning microscopy, we showed that all three proteins co-localize at the level of the SR in primary mouse cardiomyocytes and confirmed these results by immunoelectron microscopy in human biopsies. Our results support a regulatory role of S100A1 in the contraction-relaxation cycle in the human heart.     

S100 and S100 fused-type protein families in epidermal maturation with special focus on S100A3 in mammalian hair cuticles

Epithelial Ca²⁺-regulation, which governs cornified envelope formation in the skin epidermis and hair follicles, closely coincides with the expression of S100A3, filaggrin and trichohyalin, and the post-translational modification of these proteins by Ca²⁺-dependent peptidylarginine deiminases. This review summarizes the current nomenclature and evolutional aspects of S100 Ca²⁺-binding proteins and S100 fused-type proteins (SFTPs) classified as a separate protein family with special reference to the molecular structure and function of S100A3 dominantly expressed in hair cuticular cells. Both S100 and SFTP family members are identified by two distinct types of Ca²⁺-binding loops in an N-terminal pseudo EF-hand motif followed by a canonical EF-hand motif. Seventeen members of the S100 protein family including S100A3 are clustered with seven related genes encoding SFTPs on human chromosome 1q21, implicating their association with epidermal maturation and diseases. Human S100A3 is characterized by two disulphide bridges and a preformed Zn²⁺-pocket, and may transfer Ca²⁺ ions to peptidylarginine deiminases after its citrullination-mediated tetramerization. Phylogenetic analysis utilizing current genome databases suggests that divergence of the S100A3 gene coincided with the emergence of hair, a defining feature of mammals, and that the involvement of S100A3 in epithelial Ca²⁺-cycling occurred as a result of a skin adaptation in terrestrial mammals.     

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.     

Ca2+-Binding S100 Proteins in the Central Nervous System

A number of neurodegenerative disorders have been attributed to abberrations of intracellular Ca²⁺ homeostasis regulated by Ca²⁺-binding proteins. This chapter will focus on the S100B and S100A6 proteins, which are highly expressed in the central nervous system. Their protein structures, localizations, and association with brain pathology as well as their potential use in clinical diagnostics will be discussed.     

活动期溃疡性结肠炎血清S100A2、S100A12与炎症因子的相关性分析及对复发的预测价值

摘要 目的：探讨活动期溃疡性结肠炎（UC）血清S100钙结合蛋白（S100）A2、S100A12与炎症因子的相关性分析及对复发的预测价值。方法：选取2019年1月～2021年1月我院收治的102例活动期UC患者为活动期组,根据2年后是否复发分为复发组和未复发组,另选取同期来院复查的50名缓解期UC患者为缓解期组,50名体检健康者为对照组。检测并比较三组血清S100A2、S100A12与炎症因子[C反应蛋白（CRP）、白细胞介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）]水平。采用Pearson/Spearman相关性分析活动期UC患者血清S100A2、S100A12与炎症因子水平的相关性,单因素及多因素Logistic回归分析活动期UC复发的影响因素,受试者工作特征曲线分析血清S100A2、S100A12水平对活动期UC复发的预测价值。结果：对照组、缓解期组、活动期组血清S100A2、S100A12、CRP、IL-6、TNF-α水平依次升高（P＜0.05）。活动期UC患者血清S100A2、S100A12与CRP、IL-6、TNF-α水平均呈正相关（P均＜0.001）。随访2年,102例活动期UC患者复发率为56.86%（58/102）。单因素分析显示,病情严重程度、CRP、IL-6、TNF-α、S100A2、S100A12为活动期UC患者复发的影响因素（P＜0.05）。多因素Logistic回归分析显示,病情严重程度为重度和CRP、IL-6、TNF-α、S100A2、S100A12升高为活动期UC患者复发的独立危险因素（P＜0.05）。血清S100A2、S100A12水平单独和联合预测活动期UC复发的曲线下面积分别为0.782、0.784、0.876,两指标联合预测活动期UC患者复发的预测价值大于各指标单独预测。结论：活动期UC患者血清S100A2、S100A12与炎症因子水平和复发密切相关,血清S100A2、S100A12联合预测活动期UC患者复发的价值较高。     

Identification of the binding site on S100B protein for the actin capping protein CapZ.

The calcium-binding protein S100B binds to several potential target proteins, but there is no detailed information showing the location of the binding site for any target protein on S100B. We have made backbone assignments of the calcium-bound form of S100B and used chemical-shift changes in spectra of 15N-labeled protein to locate the site that binds a peptide corresponding to residues 265-276 from CapZ alpha, the actin capping protein. The largest chemical-shift changes are observed for resonances arising from residues around the C terminus of the C-terminal helix of S100B and residues Val-8 to Asp-12 of the N-terminal helix. These residues are close to but not identical to residues that have been identified by mutational analysis to be important in other S100 protein-protein interactions. They make up a patch across the S100B dimer interface and include some residues that are quite buried in the structure of calcium-free S100B. We believe we may have identified a binding site that could be common to many S100 protein-protein interactions.     

Caleosins: Ca2+-binding proteins associated with lipid bodies

We have previously identified a rice gene encoding a 27 kDa protein with a single Ca²⁺-binding EF-hand and a putative membrane anchor. We report here similar genes termed caleosins, CLO, in other plants and fungi; they comprise a multigene family of at least five members in Arabidopsis (AtClo1–5). Northern hybridization demonstrated that AtClo2–4 mRNAs levels were low in various tissues, while AtClo1 mRNA levels were high in developing embryos and mature seeds. Analysis of transgenic Arabidopsis plants expressing the GUS reporter under control of the AtClo1 promoter showed strong levels of expression in developing embryos and also in root tip cells. Antibodies raised against AtCLO1 were used to detect caleosin in cellular fractions of Arabidopsis and rapeseed. This indicated that caleosins are a novel class of lipid body proteins, which may also be associated with an ER subdomain.