S100P Protein Research Articles

Recognition of granulocyte-macrophage colony-stimulating factor by specific S100 proteins

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic myelopoietic growth factor and proinflammatory cytokine, clinically used for multiple indications and serving as a promising target for treatment of many disorders, including cancer, multiple sclerosis, rheumatoid arthritis, psoriasis, asthma, COVID-19. We have previously shown that dimeric Ca2+-bound forms of S100A6 and S100P proteins, members of the multifunctional S100 protein family, are specific to GM-CSF. To probe selectivity of these interactions, the affinity of recombinant human GM-CSF to dimeric Ca2+-loaded forms of 18 recombinant human S100 proteins was studied by surface plasmon resonance spectroscopy. Of them, only S100A4 protein specifically binds to GM-CSF with equilibrium dissociation constant, Kd, values of 0.3–2 μM, as confirmed by intrinsic fluorescence and chemical crosslinking data. Calcium removal prevents S100A4 binding to GM-CSF, whereas monomerization of S100A4/A6/P proteins disrupts S100A4/A6 interaction with GM-CSF and induces a slight decrease in S100P affinity for GM-CSF. Structural modelling indicates the presence in the GM-CSF molecule of a conserved S100A4/A6/P-binding site, consisting of the residues from its termini, helices I and III, some of which are involved in the interaction with GM-CSF receptors. The predicted involvement of the ‘hinge’ region and F89 residue of S100P in GM-CSF recognition was confirmed by mutagenesis. Examination of S100A4/A6/P ability to affect GM-CSF signaling showed that S100A4/A6 inhibit GM-CSF-induced suppression of viability of monocytic THP-1 cells. The ability of the S100 proteins to modulate GM-CSF activity is relevant to progression of various neoplasms and other diseases, according to bioinformatics analysis. The direct regulation of GM-CSF signaling by extracellular forms of the S100 proteins should be taken into account in the clinical use of GM-CSF and development of the therapeutic interventions targeting GM-CSF or its receptors.

Serum S100P and C4d proteins as markers for active proliferative lupus nephritis in Egyptian patients with systemic lupus erythematosus

Lupus nephritis (LN), occurring in approximately 50% of individuals with systemic lupus erythematosus (SLE), significantly influences therapy and prognosis. Proliferative LN, more prevalent than non-proliferative LN, is associated with a poorer prognosis. This study aimed to investigate the potential correlation between serum levels of the pro-inflammatory proteins human calcium-binding S100P (S100P) and Human complement Fragment 4 (C4d) and confirmed proliferative LN through biopsy. The objective was to evaluate their reliability as biomarkers for disease activity in proliferative LN.
 Methods. Serum S100P and C4d were measured using enzyme-linked immunosorbent assay in 100 female patients with established SLE. Among these, 50 had confirmed proliferative LN through biopsy, and 50 did not. A control group of 50 healthy female donors' plasma samples was included.
 Results. SLE patients with proliferative LN exhibited significantly higher serum levels of S100P (P < 0.001) and C4d (P < 0.001) compared to SLE patients without LN and controls. Serum S100P demonstrated a significantly higher positive correlation with the activity index in renal biopsies (r = 0.784) compared to serum C4d (r = 0.416). Additionally, serum S100P had a significantly higher positive correlation with SLEDAI-R than serum C4d (r = 0.651 vs. r = 0.257). Both serum S100P and C4d showed promise as reliable biomarkers for the diagnosis of active proliferative LN, as indicated by the ROC curve and AUC assessments.
 Conclusion. Serum concentrations of S100P and C4d emerge as useful indicators for identifying SLE patients with active proliferative LN.

Interaction of S100A6 Protein with the Four-Helical Cytokines.

S100 is a family of over 20 structurally homologous, but functionally diverse regulatory (calcium/zinc)-binding proteins of vertebrates. The involvement of S100 proteins in numerous vital (patho)physiological processes is mediated by their interaction with various (intra/extra)cellular protein partners, including cell surface receptors. Furthermore, recent studies have revealed the ability of specific S100 proteins to modulate cell signaling via direct interaction with cytokines. Previously, we revealed the binding of ca. 71% of the four-helical cytokines via the S100P protein, due to the presence in its molecule of a cytokine-binding site overlapping with the binding site for the S100P receptor. Here, we show that another S100 protein, S100A6 (that has a pairwise sequence identity with S100P of 35%), specifically binds numerous four-helical cytokines. We have studied the affinity of the recombinant forms of 35 human four-helical cytokines from all structural families of this fold to Ca2+-loaded recombinant human S100A6, using surface plasmon resonance spectroscopy. S100A6 recognizes 26 of the cytokines from all families of this fold, with equilibrium dissociation constants from 0.3 nM to 12 µM. Overall, S100A6 interacts with ca. 73% of the four-helical cytokines studied to date, with a selectivity equivalent to that for the S100P protein, with the differences limited to the binding of interleukin-2 and oncostatin M. The molecular docking study evidences the presence in the S100A6 molecule of a cytokine-binding site, analogous to that found in S100P. The findings argue the presence in some of the promiscuous members of the S100 family of a site specific to a wide range of four-helical cytokines. This unique feature of the S100 proteins potentially allows them to modulate the activity of the numerous four-helical cytokines in the disorders accompanied by an excessive release of the cytokines.

The Potent G-Quadruplex-Binding Compound QN-302 Downregulates S100P Gene Expression in Cells and in an In Vivo Model of Pancreatic Cancer

The naphthalene diimide compound QN-302, designed to bind to G-quadruplex DNA sequences within the promoter regions of cancer-related genes, has high anti-proliferative activity in pancreatic cancer cell lines and anti-tumor activity in several experimental models for the disease. We show here that QN-302 also causes downregulation of the expression of the S100P gene and the S100P protein in cells and in vivo. This protein is well established as being involved in key proliferation and motility pathways in several human cancers and has been identified as a potential biomarker in pancreatic cancer. The S100P gene contains 60 putative quadruplex-forming sequences, one of which is in the promoter region, 48 nucleotides upstream from the transcription start site. We report biophysical and molecular modeling studies showing that this sequence forms a highly stable G-quadruplex in vitro, which is further stabilized by QN-302. We also report transcriptome analyses showing that S100P expression is highly upregulated in tissues from human pancreatic cancer tumors, compared to normal pancreas material. The extent of upregulation is dependent on the degree of differentiation of tumor cells, with the most poorly differentiated, from more advanced disease, having the highest level of S100P expression. The experimental drug QN-302 is currently in pre-IND development (as of Q1 2023), and its ability to downregulate S100P protein expression supports a role for this protein as a marker of therapeutic response in pancreatic cancer. These results are also consistent with the hypothesis that the S100P promoter G-quadruplex is a potential therapeutic target in pancreatic cancer at the transcriptional level for QN-302.

Calcium-Bound S100P Protein Is a Promiscuous Binding Partner of the Four-Helical Cytokines.

S100 proteins are multifunctional calcium-binding proteins of vertebrates that act intracellularly, extracellularly, or both, and are engaged in the progression of many socially significant diseases. Their extracellular action is typically mediated by the recognition of specific receptor proteins. Recent studies indicate the ability of some S100 proteins to affect cytokine signaling through direct interaction with cytokines. S100P was shown to be the S100 protein most actively involved in interactions with some four-helical cytokines. To assess the selectivity of the S100P protein binding to four-helical cytokines, we have probed the interaction of Ca2+-bound recombinant human S100P with a panel of 32 four-helical human cytokines covering all structural families of this fold, using surface plasmon resonance spectroscopy. A total of 22 cytokines from all families of four-helical cytokines are S100P binders with the equilibrium dissociation constants, Kd, ranging from 1 nM to 3 µM (below the Kd value for the S100P complex with the V domain of its conventional receptor, receptor for advanced glycation end products, RAGE). Molecular docking and mutagenesis studies revealed the presence in the S100P molecule of a cytokine-binding site, which overlaps with the RAGE-binding site. Since S100 binding to four-helical cytokines inhibits their signaling in some cases, the revealed ability of the S100P protein to interact with ca. 71% of the four-helical cytokines indicates that S100P may serve as a poorly selective inhibitor of their action.

Interferon-β Activity Is Affected by S100B Protein.

Interferon-β (IFN-β) is a pleiotropic cytokine secreted in response to various pathological conditions and is clinically used for therapy of multiple sclerosis. Its application for treatment of cancer, infections and pulmonary diseases is limited by incomplete understanding of regulatory mechanisms of its functioning. Recently, we reported that IFN-β activity is affected by interactions with S100A1, S100A4, S100A6, and S100P proteins, which are members of the S100 protein family of multifunctional Ca2+-binding proteins possessing cytokine-like activities (Int J Mol Sci. 2020;21(24):9473). Here we show that IFN-β interacts with one more representative of the S100 protein family, the S100B protein, involved in numerous oncological and neurological diseases. The use of chemical crosslinking, intrinsic fluorescence, and surface plasmon resonance spectroscopy revealed IFN-β binding to Ca2+-loaded dimeric and monomeric forms of the S100B protein. Calcium depletion blocks the S100B–IFN-β interaction. S100B monomerization increases its affinity to IFN-β by 2.7 orders of magnitude (equilibrium dissociation constant of the complex reaches 47 pM). Crystal violet assay demonstrated that combined application of IFN-β and S100B (5–25 nM) eliminates their inhibitory effects on MCF-7 cell viability. Bioinformatics analysis showed that the direct modulation of IFN-β activity by the S100B protein described here could be relevant to progression of multiple oncological and neurological diseases.

Erythropoietin Interacts with Specific S100 Proteins.

Erythropoietin (EPO) is a clinically significant four-helical cytokine, exhibiting erythropoietic, cytoprotective, immunomodulatory, and cancer-promoting activities. Despite vast knowledge on its signaling pathways and physiological effects, extracellular factors regulating EPO activity remain underexplored. Here we show by surface plasmon resonance spectroscopy, that among eighteen members of Ca2+-binding proteins of the S100 protein family studied, only S100A2, S100A6 and S100P proteins specifically recognize EPO with equilibrium dissociation constants ranging from 81 nM to 0.5 µM. The interactions occur exclusively under calcium excess. Bioinformatics analysis showed that the EPO-S100 interactions could be relevant to progression of neoplastic diseases, including cancer, and other diseases. The detailed knowledge of distinct physiological effects of the EPO-S100 interactions could favor development of more efficient clinical implications of EPO. Summing up our data with previous findings, we conclude that S100 proteins are potentially able to directly affect functional activities of specific members of all families of four-helical cytokines, and cytokines of other structural superfamilies.

The Role of the C-Terminal Lysine of S100P in S100P-Induced Cell Migration and Metastasis.

S100P protein is a potent inducer of metastasis in a model system, and its presence in cancer cells of patients is strongly associated with their reduced survival times. A well-established Furth Wistar rat metastasis model system, methods for measuring cell migration, and specific inhibitors were used to study pathways of motility-driven metastasis. Cells expressing C-terminal mutant S100P proteins display markedly-reduced S100P-driven metastasis in vivo and cell migration in vitro. These cells fail to display the low focal adhesion numbers observed in cells expressing wild-type S100P, and the mutant S100P proteins exhibit reduced biochemical interaction with non-muscle myosin heavy chain isoform IIA in vitro. Extracellular inhibitors of the S100P-dependent plasminogen activation pathway reduce, but only in part, wild-type S100P-dependent cell migration; they are without effect on S100P-negative cells or cells expressing C-terminal mutant S100P proteins and have no effect on the numbers of focal adhesions. Recombinant wild-type S100P protein, added extracellularly to S100P-negative cells, stimulates cell migration, which is abolished by these inhibitors. The results identify at least two S100P-dependent pathways of migration, one cell surface and the other intracellularly-linked, and identify its C-terminal lysine as a target for inhibiting multiple migration-promoting activities of S100P protein and S100P-driven metastasis.

P–376 S100P in syncytiotrophoblast regulates lipid metabolism during early pregnancy

Abstract Study question Does S100P involve in the regulation of lipid metabolism in trophoblast syncytialization during the early stage of pregnancy? Summary answer S100P suppressed lipid droplets overloading in trophoblast syncytialization during the early stage of pregnancy. What is known already S100P is a 95-amino acid protein belongs to the large family of S100 calcium-binding proteins and is exclusively expressed in syncytiotrophoblast layer of placenta, enhancing trophoblast cells proliferation, motility and invasion. Recent studies indicated S100P was aberrantly expressed in various malignant tumors, promoting their proliferation, survival, the formation and invasion of vessels, and has a close relationship with their grade malignancy, hormone dependency and the drug-resistance. Syncytialization is trophoblast cells differentiated from cytotrophoblasts fused into multinucleated cell clusters with cell boundaries deficiency. Study design, size, duration Tissues were collected from elective first trimester pregnancy terminations of healthy people and patients with recurrent spontaneous abortion. Human trophoblast stem cells (hTSCs) were isolated from Week 6 to Week 8 of fresh placental tissues (n = 8) and use forskolin to induce syncytialization. Participants/materials, setting, methods Tissues were collected from elective first trimester surgical pregnancy terminations to determine localization, abundance and function of S100P. The level of S100P protein and gene was measured by Real-time quantitative PCR (RT-qPCR), western blot, ELISA and immunofluorescence in hTSC induced ST(2D)-TSCT. The lipid droplets were assessed by Oil red O stain, BODIPY and electron microscope. Serum S100P concentration in normal and recurrent miscarriage patients were measured by ELISA. Main results and the role of chance S100P was exclusively localized in syncytiotrophoblast layer of the placental villous from gestational weeks 6–8 (n = 4); S100p serum concentration was increased markedly as the gestation progressed (n = 40, p < 0.05); the protein abundance of S100P was increased during syncytialization of hTSCs; Lipid droplets increased during syncytialization and knockdown of S100P leads to trophoblast cell death with the overload of lipid droplet; The expression of S100P was down regulated and the amount of lipid droplets apparently accumulated in villus of recurrent miscarriage patients (n = 4). Limitations, reasons for caution The role of S100P on placenta lacks the evidence from animal models. Wider implications of the findings: S100P is an important factor facilitating in trophoblast syncytialization in the first trimester of pregnancy and could be a biomarker of the early pregnancy sustenance. Trial registration number Not applicable

S100 Calcium-Binding Protein P Secreted from Megakaryocytes Promotes Osteoclast Maturation.

Megakaryocytes (MKs) differentiate from hematopoietic stem cells and produce platelets at the final stage of differentiation. MKs directly interact with bone cells during bone remodeling. However, whether MKs are involved in regulating bone metabolism through indirect regulatory effects on bone cells is unclear. Here, we observed increased osteoclast differentiation of bone marrow-derived macrophages (BMMs) cultured in MK-cultured conditioned medium (MK CM), suggesting that this medium contains factors secreted from MKs that affect osteoclastogenesis. To identify the MK-secreted factor, DNA microarray analysis of the human leukemia cell line K562 and MKs was performed, and S100 calcium-binding protein P (S100P) was selected as a candidate gene affecting osteoclast differentiation. S100P was more highly expressed in MKs than in K562 cells, and showed higher levels in MK CM than in K562-cultured conditioned medium. In BMMs cultured in the presence of recombinant human S100P protein, osteoclast differentiation was promoted and marker gene expression was increased. The resorption area was significantly larger in S100P protein-treated osteoclasts, demonstrating enhanced resorption activity. Overall, S100P secreted from MKs promotes osteoclast differentiation and resorption activity, suggesting that MKs indirectly regulate osteoclast differentiation and activity through the paracrine action of S100P.

Proof-of-concept study investigating the role of S100P-RAGE in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is an epithelial carcinoma that arises from the lining of the nasopharyngeal mucosa. The efficacy of radiation therapy is limited due to radiation resistance, particularly in the advanced stages of NPC. The S100P protein is a small isoform of the S100 protein family, which is involved in the regulation of various intracellular and extracellular processes, including proliferation, differentiation and intracellular signaling. The aim of the current study was to investigate the significance of the S100P-RAGE axis in NPC progression. The expression levels of S100P and receptor for activated glycation end-products (RAGE) in NPC specimens were determined by western blotting. In addition, the effect of the S100P-RAGE axis on NPC was evaluated in vitro by proliferation and migration assays using C666-1 cells treated with S100P or the RAGE inhibitor FPS-ZM1. The underlying mechanism was also investigated by western blotting. The expression of S100P and RAGE was detected in clinical specimens from 15 patients with NPC and 15 patients with benign nasopharyngeal inflammation, and was observed to be higher in NPC tissues compared with inflamed tissues. Furthermore, the interaction of S100P with RAGE increased the proliferation and migration potential of C666-1 cells, and activated mitogen-activated protein kinase and NF-κB signaling. These results indicate that the S100P-RAGE axis exerts a promoting effect on the progression of NPC. Therefore therapeutic strategies targeting S100P-RAGE merit further exploration for the treatment of NPC.

Tissue mRNA for S100A4, S100A6, S100A8, S100A9, S100A11 and S100P Proteins in Colorectal Neoplasia: A Pilot Study.

S100 proteins are involved in the pathogenesis of sporadic colorectal carcinoma through different mechanisms. The aim of our study was to assess tissue mRNA encoding S100 proteins in patients with non-advanced and advanced colorectal adenoma. Mucosal biopsies were taken from the caecum, transverse colon and rectum during diagnostic and/or therapeutic colonoscopy. Another biopsy was obtained from adenomatous tissue in the advanced adenoma group. The tissue mRNA for each S100 protein (S100A4, S100A6, S100A8, S100A9, S100A11 and S100P) was investigated. Eighteen biopsies were obtained from the healthy mucosa in controls and the non-advanced adenoma group (six individuals in each group) and thirty biopsies in the advanced adenoma group (ten patients). Nine biopsies were obtained from advanced adenoma tissue (9/10 patients). Significant differences in mRNA investigated in the healthy mucosa were identified between (1) controls and the advanced adenoma group for S100A6 (p = 0.012), (2) controls and the non-advanced adenoma group for S100A8 (p = 0.033) and (3) controls and the advanced adenoma group for S100A11 (p = 0.005). In the advanced adenoma group, differences between the healthy mucosa and adenomatous tissue were found in S100A6 (p = 0.002), S100A8 (p = 0.002), S100A9 (p = 0.021) and S100A11 (p = 0.029). Abnormal mRNA expression for different S100 proteins was identified in the pathological adenomatous tissue as well as in the morphologically normal large intestinal mucosa.

Relationship Between the Expression of S100P in Gastric Cancer Tissue and Clinical Pathology

Purpose: Discuss the diagnosis value and action mechanism of S100P for gastric cancer and the influence of S100P in tumor metastasis. Method: Collect 100 cases of gastric cancer tissues diagnosed as per histopathology by our hospital, apply immunohistochemistry method, test the positive expression condition of S100P protein, compare with 100 cases of control tissue specimens, and analyze the relationship between S100 protein expression condition in gastric cancer tissue and clinical pathology. Result: the positive rate (51.0%) of S100P in gastric cancer tissue is significantly lower than chronic atrophic gastritis with atypical hyperplasia, superficial gastritis and normal gastric mucosa (P<0.001 or P<0.05), while the difference between gastric cancer group and group of chronic atrophic gastritis with atypical hyperplasia has no statistic significance (P>0.05). The difference of positive rate of S100P in gastric cancer tissue with gender, age, tumor size, tumor position and Lauren typing has no statistic significance. In well differentiated adenocarcinoma, moderately differentiated adenocarcinoma, poorly differentiated adenocarcinoma and adenocarcinoma anaplastic, the positive rate (80.56%) of S100P of the three groups has significant difference (χ²=13.912, P=0.001). The positive rate of S100P which doesn’t invade serosa is significantly higher than the positive rate (34.38%, χ²=19.662, P<0.001) of S100P invading serosa and invading beyond the serosa, and the difference of positive rate of S100P of Phase I, Phase II, Phase III and Phase IV also has statistic difference (χ²=13.347, P=0.004), the positive rate (73.17%) of localized S100P protein is significantly higher than the positive rate (35.59%, χ²=13.669, P<0.001) of invasive type; the positive rate (74.42%) of S100P without lymph node metastasis is significantly higher than the positive rate (33.33%, χ²=15.020, P<0.001) with lymph node metastasis. Conclusion: the positive rate of S100P protein in gastric cancer tissue is significantly lower than the para-carcinoma tissue; the lower the differentiation degree is, the deeper the invasion is; the patients who have later TNM staging and Borrmanntyping, and lymph node metastasis have lower positive rate of S100P protein, which indicates that the low expression of S100P protein participates in the occurrence, development, invasion and metastasis of gastric cancer, and is an independent dangerous factor affecting the patients’ prognosis.

A Novel DNA Aptamer Targeting S100P Induces Antitumor Effects in Colorectal Cancer Cells.

Colorectal cancer (CRC) is a prevalent malignancy with poor prognosis and survival. As a Ca2+ binding protein, S100P plays a role in calcium-dependent signal transduction pathways that involve in diverse biological processes. Our previous studies have shown that S100P is overexpressed in CRC tissues and regulates cell growth, invasion, and metastasis in CRC. Therefore, S100P is expected to be an effective target for CRC therapy. Aptamers are short single-stranded oligonucleotides that could serve as specific and high-affinity probes to a wide range of target molecules for therapeutic purposes. In this study, we generated a novel DNA aptamer against S100P (AptS100P-1) by way of the SELEX process and high-throughput sequencing. The binding assay showed that AptS100P-1 had a high affinity for S100P protein. Further experiments indicated that AptS100P-1 is relatively stable in a cell culture system and could be used in flow cytometry analysis, dot blot assay, and fluorescence microscopy analysis to detect S100P. Moreover, AptS100P-1 was capable of binding to cells and had an inhibitory effect on CRC cell growth in vitro and in vivo. Also, AptS100P-1 inhibited the migration and epithelial-mesenchymal transition of CRC cells expressing S100P. These results indicate a novel DNA aptamer targeting S100P, which might be a potential therapeutic strategy for targeting S100P against S100P-expressing CRC.

Calcium-Binding Protein S100P Promotes Tumor Progression but Enhances Chemosensitivity in Breast Cancer.

BackgroundChemoresistance remains one of the obstacles to overcome in the treatment of breast cancer. S100 calcium-binding protein P (S100P) has been observed to be overexpressed in several cancers and has been associated with drug resistance, metastasis, and prognosis. However, the role of S100P in chemoresistance in breast cancer has not been thoroughly determined.MethodsImmunohistochemistry was used to evaluate the expression level of S100P protein in 22 pairs (pre-chemo and post-chemo) of breast cancer tissue from patients who underwent neoadjuvant chemotherapy. The influence of S100P on the biological behavior and chemosensitivity of breast cancer cells was then investigated.ResultsThe protein level of S100P in breast cancer tissue was significantly higher than in benign fibroadenoma (p < 0.001). The S100P expression level was shown to be decreased by 46.55% after neoadjuvant chemotherapy (p = 0.015). Subgroup analysis revealed that S100P reduction (57.58%) was mainly observed in the HER2+ tumors (p = 0.027). Our in vitro experiments showed that the knockdown of S100P suppressed the proliferation, adhesion, migrative and invasive abilities of T47D and SK-BR-3 breast cancer cells. We further demonstrated that this knockdown increased the chemoresistance to paclitaxel and cisplatin in SK-BR-3 cells. We found S100P exerted its function by upregulating NF-κB, CCND1 and Vimentin, but downregulating E-cadherin.ConclusionS100P promotes the aggressive properties of breast cancer cells and may be considered as a promising therapeutic target. Moreover, S100P can be used to predict the therapeutic effect of chemotherapy in HER2+ breast cancer patients.

Effect of dexmedetomidine on brain function and hemodynamics in patients undergoing lung cancer resection

Effect of dexmedetomidine on the brain function and hemodynamics in patients undergoing lung cancer resection were explored. Eighty-seven patients with lung cancer undergoing lung cancer resection in Weifang People's Hospital from January 2014 to June 2018 were enrolled in this study. Patients conventionally anesthetized by propofol, midazolam, sufentanil, or cisatracurium besilate (41 cases) were assigned to the control group and those anesthetized by conventional anesthetic and dexmedetomidine (46 cases) were assigned to the research group. The hemodynamic parameters, neuron-specific enolase (NSE), and astrocyte S-100p protein (S-100β) were compared between the two groups before induction (T0), 5 min after induction (T1), at the end of surgery (T2), time of extubation (T3), and 5 min after extubation (T4). The cognitive function of patients was graded by the mini-mental state examination (MMSE) after patients recovered from anesthesia. In both the control group and the research group, the levels of mean arterial pressure (MAP), heart rate (HR), and central venous pressure (CVP). were statistically higher at T2 and T3 than those at T0 (all P<0.05). The levels of MAP, HR, and CVP were statistically lower in the research group than those in the control group at T2 and T3 (P<0.05). The levels of serum NSE and S100β protein in the research group and the control group increased at T2, T3, and T4, the control group was higher than the research group at each time point, and the difference was statistically significant (P<0.05). Comparison of the MMSE score and the total case number of adverse reactions between the two groups showed no statistical difference (both P>0.05). The MMSE score was positively correlated with the serum levels of NSE and S100β in the two groups (r-values were 0.661 and 0.585, P<0.05). Dexmedetomidine can effectively protect patients' perioperative brain function with small impacts on perioperative hemodynamics, so it is worthy of clinical application.

Direct interaction of metastasis-inducing S100P protein with tubulin causes enhanced cell migration without changes in cell adhesion.

Overexpression of S100P promotes breast cancer metastasis in animals and elevated levels in primary breast cancers are associated with poor patient outcomes. S100P can differentially interact with nonmuscle myosin (NM) isoforms (IIA > IIC > IIB) leading to the redistribution of actomyosin filaments to enhance cell migration. Using COS-7 cells which do not naturally express NMIIA, S100P is now shown to interact directly with α,β-tubulin in vitro and in vivo with an equilibrium Kd of 2–3 × 10−7 M. The overexpressed S100P is located mainly in nuclei and microtubule organising centres (MTOC) and it significantly reduces their number, slows down tubulin polymerisation and enhances cell migration in S100P-induced COS-7 or HeLa cells. It fails, however, to significantly reduce cell adhesion, in contrast with NMIIA-containing S100P-inducible HeLa cells. When taxol is used to stabilise MTs or colchicine to dissociate MTs, S100P's stimulation of migration is abolished. Affinity-chromatography of tryptic digests of α and β-tubulin on S100P-bound beads identifies multiple S100P-binding sites consistent with S100P binding to all four half molecules in gel-overlay assays. When screened by NMR and ITC for interacting with S100P, four chemically synthesised peptides show interactions with low micromolar dissociation constants. The two highest affinity peptides significantly inhibit binding of S100P to α,β-tubulin and, when tagged for cellular entry, also inhibit S100P-induced reduction in tubulin polymerisation and S100P-enhancement of COS-7 or HeLa cell migration. A third peptide incapable of interacting with S100P also fails in this respect. Thus S100P can interact directly with two different cytoskeletal filaments to independently enhance cell migration, the most important step in the metastatic cascade.

Highly specific interaction of monomeric S100P protein with interferon beta

S100 proteins are EF-hand calcium-binding proteins of vertebrates exerting numerous intra- and extracellular actions and involved into multiple diseases. Some of S100 proteins serve as extracellular damage signals via interaction with receptors. Although several S100 proteins directly bind specific cytokines, this phenomenon remains underexplored. Using chemical crosslinking, intrinsic fluorescence and surface plasmon resonance spectroscopies, we show that S100P protein interacts with interferon beta (IFN-β) depending on calcium level and oligomeric state of S100P. Dimeric Ca2+-loaded S100P binds IFN-β with equilibrium dissociation constants, Kd, of 0.05–0.6 μM. S100P monomerization favors this interaction decreasing Kd values down to 0.3–2 nM. Calcium depletion drastically lowers S100P affinity to IFN-β. Other related EF-hand proteins studied (calmodulin, α-parvalbumin and S100G) do not bind IFN-β, thereby confirming selectivity of the S100P - IFN-β interaction. Crystal violet assay reveals that the S100P binding suppresses IFN-β cytotoxicity to MCF-7 breast cancer cells. Since several cancers (breast, colon, lung, liver, etc.) exhibit dysregulated functioning of S100P and IFN-β, their interaction could be relevant to the cancer progression and directed therapeutic interventions.

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.

Analyzing the structural and functional roles of residues from the ‘black’ and ‘gray’ clusters of human S100P protein

Two highly conserved structural motifs observed in members of the EF-hand family of calcium binding proteins. The motifs provide a supporting scaffold for the Ca2+ binding loops and contribute to the hydrophobic core of the EF-hand domain. Each structural motif represents a cluster of three amino acids called cluster I (‘black’ cluster) and cluster II (‘grey’ cluster). Cluster I is more conserved and mostly incorporates aromatic amino acids. In contrast, cluster II is noticeably less conserved and includes a mix of aromatic, hydrophobic, and polar amino acids of different sizes. In the human calcium binding S100 P protein, these ‘black’ and ‘gray’ clusters include residues F15, F71, and F74 and L33, L58, and K30, respectively. To evaluate the effects of these clusters on structure and functionality of human S100 P, we have performed Ala scanning. The resulting mutants were studied by a multiparametric approach that included circular dichroism, scanning calorimetry, dynamic light scattering, chemical crosslinking, and fluorescent probes. Spectrofluorimetric Ca2+-titration of wild type S100 P showed that S100 P dimer has 1–2 strong calcium binding sites (K1 = 4 × 106 M−1) and two cooperative low affinity (K2 = 4 × 104 M−1) binding sites. Similarly, the S100 P mutants possess two types of calcium binding sites. This analysis revealed that the alanine substitutions in the clusters I and II caused comparable changes in the S100 P functional properties. However, analysis of heat- or GuHCl-induced unfolding of these proteins showed that the alanine substitutions in the cluster I caused notably more pronounced decrease in the protein stability compared to the changes caused by alanine substitutions in the cluster II. Opposite to literature data, the F15 A substitution did not cause the S100 P dimer dissociation, indicating that F15 is not crucial for dimer stability. Overall, similar to parvalbumins, the S100 P cluster I is more important for protein conformational stability than the cluster II.