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.     

Bovine Brain S100 Proteins: Separation and Characterization of a New S100 Protein Species

Three S100 protein species (S100a, S100b, S100a') have been purified from bovine brain using a modification of standard preparative methods. A higher yield for each protein was obtained at the last separation step. Characterization by urea/sodium dodecyl sulfate/polyacrylamide gel electrophoresis, UV absorption spectra, and fluorescence parameters provided evidence of a new tryptophan-containing S100 protein called S100a', which exhibits, as S100a and S100b, the properties of a Ca2+ binding protein.     

Mouse S100G protein exhibits properties characteristic of a calcium sensor

Bovine S100 G (calbindin D_9k, small Ca²⁺-binding protein of the EF-hand superfamily) is considered as a calcium buffer protein; i.e., the binding of Ca²⁺ practically does not change its general conformation. A set of experimental approaches has been used to study structural properties of apo- and Ca²⁺-loaded forms of mouse S100 G (81.4% identity in amino acid sequence with bovine S100 G). This analysis revealed that, in contrast to bovine S100 G, the removal of calcium ions increases α-helices content of mouse S100 G protein and enhances its accessibility to digestion by α-chymotrypsin. Furthermore, mouse apo-S100 G is characterized by a decreased surface hydrophobicity and reduced tendency for oligomerization. Such behavior is typical of calcium sensor proteins. Apo-state of mouse S100 G still has rather compact structure, which can be cooperatively unfolded by temperature and GdnHCl. Computational analysis of amino acid sequences of S100 G proteins shows that these proteins could be in a disordered state upon a removal of the bound calcium ions. The experimental data show that, although mouse apo-S100 G is flexible compared to the Ca²⁺-loaded state, the apo-form is not completely disordered and preserves some cooperatively meting structure. The origin of the unexpectedly high stability of mouse S100 G can be rationalized by an exceptionally strong association of its N- and C-terminal parts containing the EF-hands I and II, respectively.     

S100A8 and S100A9 activate MAP kinase and NF-kappaB signaling pathways and trigger translocation of RAGE in human prostate cancer cells

S100 proteins, a multigenic family of calcium-binding proteins, have been linked to human pathologies in recent years. Deregulated expression of S100 proteins, including S100A8 and S100A9, was reported in association with neoplastic disorders. In a previous study, we identified enhanced expression of S100A8 and S100A9 in human prostate cancer. To investigate potential functional implications of S100A8 and S100A9 in prostate cancer, we examined the influence of over-expressed and of purified recombinant S100A8 and S100A9 proteins in different prostate epithelial cell lines. S100A8 and S100A9 were secreted by prostate cancer cells, a finding which prompted us to analyze a possible function as extracellular ligands. S100A8/A9 induced the activation of NF-kappaB and an increased phosphorylation of p38 and p44/42 MAP kinases. In addition, extracellular S100A8/A9 stimulated migration of benign prostatic cells in vitro. Furthermore, in immunofluorescence experiments, we found a strong speckled co-localization of intracellular S100A8/A9 with RAGE after stimulating cells with recombinant S100A8/A9 protein or by increasing cytosolic Ca2+ levels. In summary, our findings show that S100A8 and S100A9 are linked to the activation of important features of prostate cancer cells.     

钙结合蛋白S100A14的结构预测及分析

钙结合蛋白S100A14是S100家族中的新成员,其空间结构与功能尚未阐明。采用服务器PredictProtein对人S100A14进行二级结构预测,利用同源建模法构建S100A14（序列12-102）的空间结构模型,经PROCHECK评估模型的可靠性,并将所构建的单体模型进行分子对接,预测S100A14形成同源二聚体的可能性及模式。结果显示,S100A14与S100A13的蛋白序列一致性最高,其C-端Ca2＋结合区存在多个变异,但Cu2＋和Zn2＋结合位点保守存在;helix I与helix IV较S100A13延伸长,而helix I、helix II和helix IV与S100A13的四个α螺旋一样具有两亲性的结构特征,并且在S100A13中扮演重要角色的W77在S100A14的helix IV（W85）中也保守存在。空间结构上,S100A14与S100A13具极大相似性;分子对接显示S100A14单体间可以通过疏水作用力形成＂X-型螺旋束＂同源二聚体。这些结构特征的分析将为S100A14的功能研究提供重要线索。     

Retinoic acid increases expression of the calcium-binding protein S100P in human gastric cancer cells

Retinoids mediate a wide spectrum of antitumor activities through induction of growth arrest, differentiation or apoptosis. To determine whether the effects of retinoids are mediated by specific gene activation or repression, one-day treatments of SC-M1 CL23 gastric cancer cells with vehicle alone or all-TRANS retinoic acid (tRA) (10 microM) were compared using differential display analysis. A 432-bp cDNA fragment from the tRA-treated cells was differentially amplified and its sequence analysis indicated homology with the calcium-binding protein S100P. Levels of S100P mRNA were increased 3.5-fold in SC-M1 CL23 gastric cancer cells treated with 10 microM tRA for 1 day, and the regulation was time- and concentration-dependent. Treatment with tRA (10 microM) also increased S100P mRNA levels in tRA-sensitive HtTA cells but not in inherent RA-resistant TMC-1 cells. However, the tRA-mediated increase in S100P expression was maintained in SC-M1/R cells that were established long-term in tRA-containing medium and had acquired partial RA resistance to tRA-induced growth suppression. In conclusion, tRA increases S100P expression, and the regulation remains intact in cells which develop acquired RA resistance.     

The hetero-oligomeric complex of the S100A8/S100A9 protein is extremely protease resistant

S100A8, S100A9 and S100A12 proteins are associated with inflammation and tissue remodelling, both processes known to be associated with high protease activity. Here, we report that homo-oligomeric forms of S100A8 and S100A9 are readily degraded by proteases, but that the preferred hetero-oligomeric S100A8/A9 complex displays a high resistance even against proteinase K degradation. S100A12 is not as protease resistant as the S100A8/A9 complex. Since specific functions have been assigned to the homo- and heterooligomeric forms of the S100A8 and A9 proteins, this finding may point to a post-translational level of regulation of the various functions of these proteins in inflammation and tissue remodelling.     

Characterization of the tissue-specific expression of the S100P gene which encodes an EF-hand Ca2+-binding protein*

S100 proteins are a calcium-binding protein family containing two EF-hand domains exclusively expressed in vertebrates and play roles in many cellular activities. Human S100P gene was first cloned as a 439 bp cDNA in placenta and it was found to be associated with human prostate cancer. Here we describe the cloning of the 1297 bp full-length cDNA, and the characterization of the tissue-specific expression of the human S100P gene. It is abundantly expressed in many tissues including placenta by Northern blot and RT-PCR analysis, unlike the expression pattern of other S100 family genes.     

Extensive analysis of the human platelet proteome by two-dimensional gel electrophoresis and mass spectrometry

Platelets play a key role in the control of bleeding and wound healing, contributing to the formation of vascular plugs. Under pathologic circumstances, they are involved in thrombotic disorders, including heart disease. Since platelets do not have a nucleus, proteomics offers a powerful alternative approach to provide data on protein expression in these cells, helping to address their biology. In this publication we extend the previously reported analysis of the pI 4-5 region of the human platelet proteome to the pI 5-11 region. By using narrow pI range two-dimensional electrophoresis (2-DE) for protein separation followed by high-throughput tandem mass spectrometry (MS/MS) for protein identification, we were able to identify 760 protein features, corresponding to 311 different genes, resulting in the annotation of 54% of the pI 5-11 range 2-DE proteome map. We evaluated the physicochemical properties and functions of the identified platelet proteome. Importantly, the main group of proteins identified is involved in intracellular signalling and regulation of the cytoskeleton. In addition, 11 hypothetical proteins are reported. In conclusion, this study provides a unique inventory of the platelet proteome, contributing to our understanding of platelet function and building the basis for the identification of new drug targets.     

Purification of the Ca-binding protein S100A1 from myocardium and recombinant Escherichia coli

S100A1 is a new regulatory protein of myocardial contractility that is differentially expressed in early and late stages of myocardial hypertrophy. In order to further investigate the multiple functions of S100A1 in various assay systems we developed a new strategy for isolating biologically active S100A1 protein. After EDTA extraction of myocardium or recombinant bacteria, S100A1 was purified by Octyl-Sepharose hydrophobic interaction chromatography and HiTrapQ anion-exchange chromatography yielding 1.4–2.0 mg/100 g wet tissue and 0.7–1.0 mg/100 ml bacterial culture. Native porcine as well as human recombinant S100A1 revealed biological activity in physiological and biochemical assays.     

Human S100b protein: formation of a tetramer from synthetic calcium-binding site peptides.

Human brain S100b protein is a unique calcium-binding protein comprised of two identical 91-amino acid polypeptide chains that each contain two proposed helix-loop-helix (EF-hand) calcium-binding sites. In order to probe the assembly of the four calcium-binding sites in S100b, a peptide comprised of the N-terminal 46 residues of S100b protein was synthesized and studied by CD and 1H NMR spectroscopies as a function of concentration and temperature. At relatively high peptide concentrations and in the absence of calcium, the peptide exhibited a significant proportion of alpha-helix (45%). Decreasing the peptide concentration led to a loss of alpha-helix as monitored by CD spectroscopy and coincident changes in the 1H NMR spectrum. These changes were also observed by 1H NMR spectroscopy as a function of temperature where it was observed that the Tm of the peptide was lowered approximately 14 degrees C with a 17-fold decrease in peptide concentration. Sedimentation equilibrium studies were used to determine that the peptide formed a tetramer in solution in the absence of calcium. It is proposed that this tetrameric fold also occurs in S100b and is a result of the interaction of portions of all four calcium-binding sites.     

Purification and partial characterization of canine S100A12

Canine S100A12 (cS100A12) is a calcium-binding protein of the S100 superfamily of EF-hand proteins, and its expression is restricted to neutrophils and monocytes. Interaction of S100A12 with the receptor for advanced glycation end products (RAGE) has been suggested to play a central role in inflammation. Moreover, S100A12 has been shown to represent a sensitive and specific marker for gastrointestinal inflammation in humans. Only human, porcine, bovine, and rabbit S100A12 have been purified to date, and an immunoassay for the quantification of S100A12 is available only for humans. Therefore, the aim of this study was to develop a protocol for the purification of S100A12 and to partially characterize this protein in the dog (Canis lupus familiaris) as a prelude to the development of an immunologic method for its detection and quantification in canine serum and fecal specimens. Leukocytes were isolated from canine whole blood by dextran sedimentation, and canine S100A12 was extracted from the cytosol fraction of these cells. Further purification of cS100A12 comprised of ammonium sulfate precipitation, hydrophobic interaction chromatography, and strong cation- and anion-exchange column chromatography. Canine S100A12 was successfully purified from canine whole blood. The relative molecular mass of the protein was estimated at 10,379.5 and isoelectric focusing revealed an isoelectric point of 6.0. The approximate specific absorbance of cS100A12 at 280 nm was determined to be 1.78 for a 1 mg/ml solution. The N-terminal AA sequence of the first 15 residues of cS100A12 was Thr-Lys-Leu-Glu-Asp-His-X-Glu-Gly-Ile-Val-Asp-Val-Phe-His, and revealed 100% identity with the predicted protein sequence available through the canine genome project. Sequence homology for the 14 N-terminal residues identified for cS100A12 with those of feline, bovine, porcine, and human S100A12 was 78.6%. We conclude that canine S100A12 can be successfully purified from canine whole blood using the described methods.     

The metastasis-promoting protein S100A4 regulates mammary branching morphogenesis

High levels of the S100 calcium binding protein S100A4 also called fibroblast specific protein 1 (FSP1) have been established as an inducer of metastasis and indicator of poor prognosis in breast cancer. The mechanism by which S100A4 leads to increased cancer aggressiveness has yet to be established; moreover, the function of this protein in normal mammary gland biology has not been investigated. To address the role of S100A4 in normal mammary gland, its spatial and temporal expression patterns and possible function in branching morphogenesis were investigated. We show that the protein is expressed mainly in cells of the stromal compartment of adult humans, and during active ductal development, in pregnancy and in involution of mouse mammary gland. In 3D culture models, topical addition of S100A4 induced a significant increase in the TGFα mediated branching phenotype and a concomitant increase in expression of a previously identified branching morphogen, metalloproteinase-3 (MMP-3). These events were found to be dependent on MEK activation. Downregulation of S100A4 using shRNA significantly reduced TGFα induced branching and altered E-cadherin localization. These findings provide evidence that S100A4 is developmentally regulated and that it plays a functional role in mammary gland development, in concert with TGFα by activating MMP-3, and increasing invasion into the fat pad during branching. We suggest that S100A4-mediated effects during branching morphogenesis provide a plausible mechanism for how it may function in breast cancer progression.     

钙结合蛋白S100A16对胰岛素抵抗的作用研究

目的探究钙结合蛋白S100A16在胰岛素抵抗中的作用。方法用S100A16抗体进行免疫沉淀,然后用蛋白质谱分析寻找与S100A16相互作用的蛋白。实验1转染Vector质粒的HepG2细胞作为对照,用转染shRNA质粒、S100A16过表达质粒干预作为处理组。实验2以慢性胰岛素刺激细胞构建胰岛素抵抗模型,采用转染shRNA质粒的细胞作为对照,用未转染和转染Vector质粒干预作为处理组。实验3以不做任何处理的细胞作为对照,在胰岛素抵抗模型中用吡格列酮干预作为处理组。Western blot检测相关蛋白的表达水平。组间比较采用成组t检验。结果与转染Vector质粒比较,转染S100A16过表达质粒中胎球蛋白A表达(1.39±0.54比2.85±0.25)水平上调(P<0.05);与转染Vector质粒比较,转染shRNA质粒胎球蛋白A蛋白表达(0.36±0.03比0.20±0.03)水平降低(P<0.01)。在胰岛素抵抗条件下,与转染shRNA质粒的细胞比较,未转染和转染Vector质粒的IRS-2蛋白表达(0.11±0.04比1.65±0.48)水平上调(P<0.01);与不做任何处理的细胞比较,用吡格列酮处理的细胞IRS-2表达(0.26±0.11比0.52±0.05)水平上升(P<0.01)。结论S100A16在HepG2细胞中通过胎球蛋白A促进胰岛素抵抗。     

S100A8、S100A9在幽门螺杆菌相关胃癌发生发展中的作用研究进展

S100家族是由20余个结构相似但功能各异的成员组成。该家族成员广泛参与感染、促炎、自身免疫等各种病理过程。近年来,越来越多学者发现S100家族成员在肿瘤的发展过程中也有不同程度的表达失调,且具有特异性。胃癌是我国常见的恶性肿瘤之一,国家癌症中心统计数据表明,2015年我国胃癌新发病率为679／10万,死亡率为498／10万[1],位居所有恶性肿瘤第2位。幽门螺杆菌（H. pylori）作为胃癌的Ⅰ类危险因子,目前其与胃癌的密切关系也得到了广大学者的认可。研究发现,S100家族成员——S100A8、S100A9在H. pylori感染相关胃炎、胃癌患者病理组织中表达显著上调,因此其在胃癌发生发展中的作用受到了学者的关注。本文主要就S100A8、S100A9在H. pylori相关胃癌发生发展中的作用作一综述。     

Substitution of methionine 63 or 83 in S100A9 and cysteine 42 in S100A8 abrogate the antifungal activities of S100A8/A9: potential role for oxidative regulation

S100A8 and S100A9 and their heterocomplex calprotectin (S100A8/A9) are abundant cytosolic constituents in human neutrophils previously shown to possess antifungal activity. This study was designed to investigate mechanisms involved in the modulation of the antifungal properties of S100A8/A9. S100A8, S100A9 and site-directed mutants of both proteins were tested for their antifungal effect against Candida albicans in microplate dilution assays. Whereas S100A8 alone did not inhibit fungal growth, S100A9 by itself had a moderate antifungal effect. Combining both proteins had the strongest effect. Supporting a potential role for oxidation in S100A8/A9, substitution of methionine 63 or 83 of S100A9 resulted in the loss of antifungal activity. Additionally, the substitution to alanine of cysteine 42 of S100A8 also caused a loss of S100A8's ability to enhance S100A9's antifungal effect. Overall, our data indicate that both S100A8 and S100A9 are required for their fully active antifungal effect and that oxidation regulates S100A8/A9 antifungal activity through mechanisms that remain to be elucidated and evaluated. Finally, together with our previous work describing the oxidation-sensitive anti-inflammatory effects of S100A8/A9, we propose that S100A8/A9 exerts an anti-inflammatory activity in healthy state and that conditions associated with oxidative stress activate the antifungal activity of S100A8/A9.     

A structural basis for S100 protein specificity derived from comparative analysis of apo and Ca(2+)-calcyclin

Calcyclin is a homodimeric protein belonging to the S100 subfamily of EF-hand Ca(2+)-binding proteins, which function in Ca(2+) signal transduction processes. A refined high-resolution solution structure of Ca(2+)-bound rabbit calcyclin has been determined by heteronuclear solution NMR. In order to understand the Ca(2+)-induced structural changes in S100 proteins, in-depth comparative structural analyses were used to compare the apo and Ca(2+)-bound states of calcyclin, the closely related S100B, and the prototypical Ca(2+)-sensor protein calmodulin. Upon Ca(2+) binding, the position and orientation of helix III in the second EF-hand is altered, whereas the rest of the protein, including the dimer interface, remains virtually unchanged. This Ca(2+)-induced structural change is much less drastic than the "opening" of the globular EF-hand domains that occurs in classical Ca(2+) sensors, such as calmodulin. Using homology models of calcyclin based on S100B, a binding site in calcyclin has been proposed for the N-terminal domain of annexin XI and the C-terminal domain of the neuronal calcyclin-binding protein. The structural basis for the specificity of S100 proteins is discussed in terms of the variation in sequence of critical contact residues in the common S100 target-binding site.     

S100A12 protein is a strong inducer of neurite outgrowth from primary hippocampal neurons

Several members of the S100 family of Ca(2+) binding proteins are at present known to be secreted and to have extracellular activities. We have investigated the neurite inducing potential of extracellularly added S100A12. Human recombinant S100A12 was found to dramatically induce neuritogenesis of hippocampal cells isolated from 17 to 19 days old rat embryos. The response to S100A12 was dependent on the dose in a bell-shaped manner. A 10-fold increase in neurite outgrowth was observed upon treatment with S100A12 in concentrations between 0.1 and 2.0 microM already after 24 h. Exposure to S100A12 for only 15 min was enough to induce neuritogenesis when measured after 24 h, but to obtain a maximal response, S100A12 had to be present in the culture for at least 4 h. The response to S100A12 was abolished by inhibitors of phospholipase C (PLC), protein kinase C (PKC), Ca(2+) flux, Ca(2+)/calmodulin dependent kinase II (CaMKII) or mitogen-activated protein kinase kinase (MEK). Therefore, we suggest that extracellular S100A12 triggers intracellular signal transduction in neurons, involving the classical mitogen-activated protein (MAP) kinase pathway and a phospholipase C-generated second messenger pathway leading to an increase in intracellular Ca(2+) and activation of PKC, ultimately resulting in neuronal differentiation.     

Genomic and phylogenetic analysis of the S100A7 (Psoriasin) gene duplications within the region of the S100 gene cluster on human chromosome 1q21

Abstract The human S100 gene family encodes the EF-hand superfamily of calcium-binding proteins, with at least 14 family members clustered relatively closely together on chromosome 1q21. We have analyzed the most recently available genomic sequence of the human S100 gene cluster for evidence of tandem gene duplications during primate evolutionary history. The sequences obtained from both GenBank and GoldenPath were analyzed in detail using various comparative sequence analysis tools. We found that of the S100A genes clustered relatively closely together within a genomic region of 260 kb, only the S100A7 (psoriasin) gene region showed evidence of recent duplications. The S100A7 gene duplicated region is composed of three distinct genomic regions, 33, 11, and 31 kb, respectively, that together harbor at least five identifiable S100A7-like genes. Regions 1 and 3 are in opposite orientation to each other, but each region carries two S100A7-like genes separated by an 11-kb intergenic region (region 2) that has only one S100A7-like gene, providing limited sequence resemblance to regions 1 and 3. The duplicated genomic regions 1 and 3 share a number of different retroelements including five Alu subfamily members that serve as molecular clocks. The shared (paralogous) Alu S insertions suggest that regions 1 and 3 were probably duplicated during or after the phase of AluS amplification some 30–40 mya. We used PCR to amplify an indel within intron 1 of the S100A7a and S100A7c genes that gave the same two expected product sizes using 40 human DNA samples and 1 chimpanzee sample, therefore confirming the presence of the region 1 and 3 duplication in these species. Comparative genomic analysis of the other S100 gene members shows no similarity between intergenic regions, suggesting that they diverged long before the emergence of the primates. This view was supported by the phylogenetic analysis of different human S100 proteins including the human S100A7 protein members. The S100A7 protein, also known as psoriasin, has important functions as a mediator and regulator in skin differentiation and disease (psoriasis), in breast cancer, and as a chemotactic factor for inflammatory cells. This is the first report of five copies of the S100A7 gene in the human genome, which may impact on our understanding of the possible dose effects of these genes in inflammation and normal skin development and pathogenesis.