Solution NMR structure of S100B bound to the high-affinity target peptide TRTK-12

The solution NMR structure is reported for Ca(2+)-loaded S100B bound to a 12-residue peptide, TRTK-12, from the actin capping protein CapZ (alpha1 or alpha2 subunit, residues 265-276: TRTKIDWNKILS). This peptide was discovered by Dimlich and co-workers by screening a bacteriophage random peptide display library, and it matches exactly the consensus S100B binding sequence ((K/R)(L/I)XWXXIL). As with other S100B target proteins, a calcium-dependent conformational change in S100B is required for TRTK-12 binding. The TRTK-12 peptide is an amphipathic helix (residues W7 to S12) in the S100B-TRTK complex, and helix 4 of S100B is extended by three or four residues upon peptide binding. However, helical TRTK-12 in the S100B-peptide complex is uniquely oriented when compared to the three-dimensional structures of other S100-peptide complexes. The three-dimensional structure of the S100B-TRTK peptide complex illustrates that residues in the S100B binding consensus sequence (K4, I5, W7, I10, L11) are all involved in the S100B-peptide interface, which can explain its orientation in the S100B binding pocket and its relatively high binding affinity. A comparison of the S100B-TRTK peptide structure to the structures of apo- and Ca(2+)-bound S100B illustrates that the binding site of TRTK-12 is buried in apo-S100B, but is exposed in Ca(2+)-bound S100B as necessary to bind the TRTK-12 peptide.     

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-Mediated Inhibition of the Phosphorylation of GFAP Is Prevented by TRTK-12

S100B belongs to a family of calcium-binding proteins involved in cell cycle and cytoskeleton regulation. We observed an inhibitory effect of S100B on glial fibrillary acidic protein (GFAP) phosphorylation, when stimulated by cAMP or Ca2+/calmodulin, in a cytoskeletal fraction from primary astrocyte cultures. We found that S100B has no direct effect on CaM KII activity, the major kinase in this cytoskeletal fraction able to phosphorylate GFAP. The inhibition of GFAP phosphorylation is most likely due to the binding of S100B to the phosphorylation sites on this protein and blocking the access of these sites to the protein kinases. This inhibition was dependent on Ca2+. However, Zn2+ could substitute for Ca2+. The inhibitory effect of S100B was prevented by TRTK-12, a peptide that blocks S100B interaction with several target proteins including glial fibrillary acidic protein. These data suggest a role for S100B in the assembly of intermediate filaments in astrocytes.     

The S100B Protein Inhibits Phosphorylation of GFAP and Vimentin in a Cytoskeletal Fraction from Immature Rat Hippocampus

The S100B protein belongs to a family of small Ca²⁺-binding proteins involved in several functions including cytoskeletal reorganization. The effect of S100B on protein phosphorylation was investigated in a cytoskeletal fraction prepared from immature rat hippocampus. An inhibitory effect of 5 M S100B on total protein phosphorylation, ranging from 25% to 40%, was observed in the presence of Ca²⁺ alone, Ca²⁺ plus calmodulin or Ca²⁺ plus cAMP. Analysis by two dimensional electrophoresis revealed a Ca²⁺/calmodulin-dependent and a Ca²⁺/cAMP-dependent inhibitory effect of S100B, ranging from 62% to 67% of control, on the phosphorylation of the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin. The fact that S100B binds to the N-terminal domain of GFAP and that the two proteins are co-localized in astrocytes suggests a potential in vivo role for S100B in modulating the phosphorylation of intermediate filament proteins in glia.     

Glutamate Uptake Is Stimulated by Extracellular S100B in Hippocampal Astrocytes

Summary  

AHNAK interaction with the annexin 2/S100A10 complex regulates cell membrane cytoarchitecture

Remodelling of the plasma membrane cytoarchitecture is crucial for the regulation of epithelial cell adhesion and permeability. In Madin-Darby canine kidney cells, the protein AHNAK relocates from the cytosol to the cytosolic surface of the plasma membrane during the formation of cell-cell contacts and the development of epithelial polarity. This targeting is reversible and regulated by Ca(2+)-dependent cell-cell adhesion. At the plasma membrane, AHNAK associates as a multimeric complex with actin and the annexin 2/S100A10 complex. The S100A10 subunit serves to mediate the interaction between annexin 2 and the COOH-terminal regulatory domain of AHNAK. Down-regulation of both annexin 2 and S100A10 using an annexin 2-specific small interfering RNA inhibits the association of AHNAK with plasma membrane. In Madin-Darby canine kidney cells, down-regulation of AHNAK using AHNAK-specific small interfering RNA prevents cortical actin cytoskeleton reorganization required to support cell height. We propose that the interaction of AHNAK with the annexin 2/S100A10 regulates cortical actin cytoskeleton organization and cell membrane cytoarchitecture.     

Resveratrol Increases Glutamate Uptake, Glutathione Content, and S100B Secretion in Cortical Astrocyte Cultures

Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a polyphenol present in grapes and red wine, which has antioxidant properties and a wide range of other biological effects. In this study, we investigated the effect of resveratrol, in a concentration range of 10–250 μM, on primary cortical astrocytes; evaluating cell morphology, parameters of glutamate metabolism such as glutamate uptake, glutamine synthetase activity and glutathione total content, and S100B secretion. Astrocyte cultures were prepared of cerebral cortex from neonate Wistar rats. Morphology was evaluated by phase-contrast microscopy and immunocytochemistry for glial fibrillary acidic protein (GFAP). Glutamate uptake was measured using l-[2,3-³H]glutamate. Glutamine synthetase and content of glutathione were measured by enzymatic colorimetric assays. S100B content was determined by ELISA. Typical polygonal morphology becomes stellated when astrocyte cultures were exposed to 250 μM resveratrol for 24 h. At concentration of 25 μM, resveratrol was able to increase glutamate uptake and glutathione content. Conversely, at 250 μM, resveratrol decreased glutamate uptake. Unexpectedly, resveratrol at this high concentration increased glutamine synthetase activity. Extracellular S100B increased from 50 μM upwards. Our findings reinforce the protective role of this compound in some brain disorders, particularly those involving glutamate toxicity. However, the underlying mechanisms of these changes are not clear at the moment and it is necessary caution with its administration because elevated levels of this compound could contribute to aggravate these conditions.     

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.     

Assignment and secondary structure of calcium-bound human S100B

The NMR assignments of backbone ¹H, ¹³C,and ¹⁵N resonances for calcium-bound human S100B werecompleted via heteronuclear multidimensional NMR spectroscopic techniques.NOE correlations, amide exchange, ³J_HN_Hcoupling constants, and CSI analysis were used to identify the secondarystructure for Ca-S100B. The protein is comprised of four helices (helix I,Glu²-;Arg²⁰; helix II,Glu³¹-;Asn³⁸; helix III,Gln⁵⁰-;Thr⁵⁹; helix IV,Phe⁷⁰-;Phe⁸⁷), three loops (loop I,Glu²¹-;His²⁵; loop II,Glu³⁹-;Glu⁴⁹; loop III,Leu⁶⁰-;Gly⁶⁶), and two -strands(strand I, Lys²⁶>-;Lys²⁸; strand II,Glu⁶⁷-;Asp⁶⁹) which form a shortantiparallel -sheet. Helix IV is extended by approximately one turnwhen compared to the secondary structures of apo-rat [Drohat et al. (1996)Biochemistry, 35, 11577-;11588] and bovine S100B [Kilby et al. (1996)Structure, 4, 1041-;1052]. In addition, several residues outside thecalcium-binding loops in S100B undergo significant backbone chemical shiftchanges upon binding calcium which are not observed in the related proteincalbindin D_9k. Together these observations support previoussite-directed mutagenesis, absorption spectroscopy, and cysteine chemicalreactivity experiments, suggesting that the C-terminus in Ca-S100B isimportant for interactions with other proteins.     

Structural stability and reversible unfolding of recombinant porcine S100A12

Porcine S100A12 is a member of the S100 proteins, family of small acidic calcium-binding proteins characterized by the presence of two EF-hand motifs. These proteins are involved in many cellular events such as the regulation of protein phosphorylation, enzymatic activity, protein-protein interaction, Ca2+ homeostasis, inflammatory processes and intermediate filament polymerization. In addition, members of this family bind Zn2+ or Ca2+ with cooperative effect on binding. In this study, the gene sequence encoding porcine S100A12 was obtained by the synthetic gene approach using E. coli codon bias. Additionally, we report a thermodynamic study of the recombinant S100A12 using circular dichroism, fluorescence and isothermal titration calorimetry. The results of urea and temperature induced unfolding and refolding processes indicated a reversible two-state process. Also, the ANS fluorescence studies showed that in presence of divalent ions the protein exposes hydrophobic sites which could facilitate the interaction with other proteins and trigger the physiological responses.     

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.     

急性非ST段抬高型心肌梗死患者血清S100A4、S100A12与PCI术后预后的关系

摘要 目的：探讨急性非ST段抬高型心肌梗死（ANSTEMI）患者血清S100钙结合蛋白A4（S100A4）、S100钙结合蛋白A12（S100A12）与经皮冠状动脉介入治疗（PCI）术后预后的关系。方法：选取2020年1月～2021年7月上海交通大学医学院附属瑞金医院急诊科收治的224例ANSTEMI患者为ANSTEMI组,PCI术后随访1年,根据预后情况分为预后不良组和预后良好组,另选取同期67名健康体检者为对照组。采用酶联免疫吸附法检测血清S100A4、S100A12水平。采用多因素Logistic回归分析 ANSTEMI患者PCI术后预后不良的影响因素,采用受试者工作特征（ROC）曲线分析血清S100A4、S100A12水平对ANSTEMI患者PCI术后预后不良的预测价值。结果：ANSTEMI组血清S100A4、S100A12水平高于对照组（P＜0.05）。随访1年,224例STEMI患者PCI术后预后不良发生率为16.07%（36/224）。多因素Logistic回归分析显示,年龄偏大、KILLIP分级≥Ⅱ级、S100A4、S100A12水平升高为ANSTEMI患者PCI术后预后不良的独立危险因素,左心室射血分数（LVEF）升高为其独立保护因素（P＜0.05）。ROC曲线分析显示,血清S100A4、S100A12水平联合预测ANSTEMI患者PCI术后预后不良的曲线下面积（AUC）大于S100A4、S100A12单独预测。结论：血清S100A4、S100A12水平升高与ANSTEMI患者PCI术后预后不良密切相关,血清S100A4、S100A12水平联合预测ANSTEMI患者PCI术后预后不良的价值较高。     

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。     

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.     

体外培养过程中许旺细胞S100表达规律研究

目的：目前研究发现,周围神经中许旺细胞的标志物有很多,S100是其中之一。S100在体内表达的变化规律已有比较深入的研究,但是其在体外培养的许旺细胞的表达规律尚不清楚。因此,本课题研究小鼠许旺细胞在体外培养过程中S100蛋白的表达变化规律。方法：取新生（出生5-7 d）C57BL/6小鼠的坐骨神经,酶消化分离获取细胞后,培养纯化扩增3次。用S100免疫荧光法及RT-PCR技术研究许旺细胞在体外培养过程中S100的表达规律。结果：从坐骨神经消化所得到的许旺细胞,早期并不都表达S100,阳性率约为43.48%,随着培养时间延长（培养8天）,所有许旺细胞均表达S100,能够达到阳性率95.66%。结论：体外培养的许旺细胞,其标志物S100阳性率表达随培养时间延长而增加。并且我们发现,S100并不能作为一个可靠的标志物来单独应用鉴定体外培养早期的许旺细胞。     

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.     

Isolated Light Chain of Tetanus Toxin Inhibits Exocytosis: Studies in Digitonin-Permeabilized Cells

Previous work indicates that the heavy chain of tetanus toxin is responsible for the binding of the toxin to the neuronal membrane and its subsequent internalization. In the present study, the light chain of tetanus toxin mimicked the holotoxin in inhibiting Ca2+-dependent secretion of [3H]norepinephrine from digitonin-permeabilized adrenal chromaffin cells. Preincubation of tetanus toxin with monoclonal antibodies to the light chain prevented the inhibition by tetanus toxin. Preincubation of tetanus toxin with nonimmune ascites fluid or with monoclonal antibodies directed against the C fragment (the C-terminal of the heavy chain) or the heavy-chain portion of the B fragment did not prevent inhibition by tetanus toxin. The data indicate that the light chain is responsible for the intracellular blockade of exocytosis.     

S100B protein is released from rat neonatal neurons, astrocytes, and microglia by in vitro trauma and anti-S100 increases trauma-induced delayed neuronal injury and negates the protective effect of exogenous S100B on neurons

S100B protein is found in brain, has been used as a marker for brain injury and is neurotrophic. Using a well-characterized in vitro model of brain cell trauma, we have previously shown that strain injury causes S100B release from neonatal rat neuronal plus glial cultures and that exogenous S100B reduces delayed post-traumatic neuronal damage even when given at 6 or 24 h post-trauma. The purpose of the current studies was to measure post-traumatic S100B release by specific brain cell types and to examine the effect of an antibody to S100 on post-traumatic delayed (48 h) neuronal injury and the protective effect of exogenous S100B. Neonatal rat cortical cells grown on a deformable elastic membrane were subjected to a strain (stretch) injury produced by a 50 ms displacement of the membrane. S100B was measured with an ELISA kit. Trauma released S100B from pure cultures of astrocytes, microglia, and neurons. Anti-S100 reduced released S100B to below detectable levels, increased delayed neuronal injury in traumatized cells and negated the protective effect of exogenous S100B on injured cells. Heat denatured anti-S100 did not exacerbate injury. These studies provide further evidence for a protective role for S100B following neuronal trauma.