Human S100 Research Articles

Covalent Inhibitors of S100A4 Block the Formation of a Pro-Metastasis Non-Muscle Myosin 2A Complex.

The S100 protein family functions as protein-protein interaction adaptors regulated by Ca2+ binding. Formation of various S100 complexes plays a central role in cell functions, from calcium homeostasis to cell signaling, and is implicated in cell growth, migration, and tumorigenesis. We established a suite of biochemical and cellular assays for small molecule screening based on known S100 protein-protein interactions. From 25 human S100 proteins, we focused our attention on S100A4 because of its well-established role in cancer progression and metastasizes by interacting with nonmuscle myosin II (NMII). We identified several potent and selective inhibitors of this interaction and established the covalent nature of binding, confirmed by mass spectrometry and crystal structures. 5b showed on-target activity in cells and inhibition of cancer cell migration. The identified S100A4 inhibitors can serve as a basis for the discovery of new cancer drugs operating via a novel mode of action.

Malignant Melanoma of the Oral Cavity: An Aggressive and Rare Entity.

It is exceptionally unusual to see oral malignant melanoma, which is highly aggressive with dismal prognosis. A 61-year-old female patient presented with hyperpigmented and hemorrhagic areas in the anterior mandible. Microscopically, positive Immunohistochemistry staining with monoclonal antibody Human Melanoma Black (HMB45) and S100 protein, confirmed the diagnosis as malignant melanoma.

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.

The Role of Serum Calgranulin C as a Non-Invasive Marker for Inflammatory Bowel Disease

Abstract Background IBD has been recognized as a systemic inflammatory entity and as such, it is anticipated to induce changes exceeding the boundaries of bowel mucosa, being reflected in a broader spectrum of tissues, including blood. Calgranulin c is a calcium binding protein with proinflammatory properties. It has been associated with different diseases of inflammatory origin, including inflammatory bowel disease (IBD). In our present study, the utility of serum calgranulin c as marker for inflammatory bowel disease was tested. Objective is to evaluate the role of serum calgranulin c as a diagnostic and prognostic marker in IBD patients and to correlate it's level with different biochemical, endoscopic and clinical parameters. Patients and Methods Calgranulin c serum levels were determined in 50 patients with ulcerative colitis (UC), 20 patients with Crohn’s disease (CD) and 73 with functional bowel disorder (IBS). Calgranulin c serum concentrations were analysed using double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) to assay the level of Human S100 Calcium Binding Protein A12(S100A12) in samples. Results In the present study we found that the mean serum calgranulin c were 723.640±529.055 ng/ml (range: 63-2000) in UC, 759.233±797.963 ng/ml (range: 110-2000) in CD and 80.850±24.416 ng/mL (range: 52-135) in control group. UC and CD patients had significantly higher serum calgranulin c levels compared to IBS patients with p < 0.001 for both. the best cut-off for serum calgranulin c levels in prediction of IBD was >110 ng/Ml with sensitivity 96.67%, specificity 85.00%, and test accuracy was 97.8%. best cut-off for serum calgranulin c levels in prediction of UC was >135 ng/mL. the best cut-off for serum calgranulin c levels in prediction of CD was >110 ng/Ml. Moreover, the best cut-off for serum calgranulin c levels of > 435 ng/mL could predict activity in UC group depend on UCEIS, depend on ACGUCAI and Erosions and ulcers in endoscopy. another cut off level of Serum calgranulin c in prediction of luminal bleeding in endoscopy in UC patients was >625 ng/ml. the best cut off level of Serum calgranulin c in prediction of activity among CD patients depend on CDAI was >437 ng/ml. Conclusion Serum calgranulin c was found to be higher in patients with IBD than with functional bowel disorded (IBS). Levels of serum calgranulin c varied significantly with disease severity in patients with UC and CD. Serum calgranulin c was found to be higher among patients with active CD and UC than those in remission. Serum calgranulin c can be used as a non invasive marker of inflammatory bowel disease. It can distinguish IBD from IBS and can be considered as a maker of activity in IBD patients.

Occurrence of Human Defensins and S100 Proteins in Head and Neck Basal Cell Carcinoma (BCC) Entities: hBD3 and S100A4 as Potential Biomarkers to Evaluate Successful Surgical Therapy

Background: The goal of this study is the identification of potential marker molecules for characterizing different basal cell carcinoma entities, to help improve clinical decisions for surgical resection therapy. Methods: Three different entities, sclerodermiform, solid and superficial basal cell carcinomas, were subjected to immunohistochemical microscopy and histomorphometric analyses for human α- (DEFA1/3; DEFA4) and β-defensins (hBD1/2/3) and special S100 proteins (S100A4/7/8/9). Thirty specimens of the three entities were evaluated. Analyses were performed by comparing tissue and cellular localization and staining intensities of tumorous with non-tumorous areas. Staining intensities were semiquantitatively examined by using an RGB-based model. Results: Human defensins are present in all three entities of basal cell carcinomas. They all show cytoplasmic immunostaining in cells of the epithelium, stroma and tumor. Notably, human β-defensin3 is accumulated in the cell nuclei of sclerodermiform and superficial basal cell carcinomas. S100A4 and A7 are undetectable in tumor regions. However, S100A4 occurs in cancer-associated stroma cells with nuclear staining in superficial basal cell carcinomas. Conclusion: Two candidates, namely hBD3 and S100A4, might be used as potential clinical tools for evaluating successful surgical resection therapy to avoid aesthetic and functional facial deformation.

S100A12 promotes Mn(II) binding to pneumococcal PsaA and staphylococcal MntC by Zn(II) sequestration

Human S100A12 (calgranulin C, EN-RAGE) is a Zn(II)-sequestering host-defense protein that contributes to the metal-withholding innate immune response against microbial pathogens. S100A12 coordinates Zn(II) ions at two His3Asp sites with high affinity. A similar His3Asp site found in calprotectin (S100A8/S100A9, calgranulin A/B), a closely related human S100 protein, can sequester divalent metal ions from the solute-binding proteins (SBPs) pneumococcal PsaA (pneumococcal surface protein A) and staphylococcal MntC (manganese transport protein C). Both SBPs are components of Mn(II) transporters and capture extracellular Mn(II) ions for subsequent delivery into the bacterial cytosol. Nevertheless, PsaA and MntC exhibit a thermodynamic preference for Zn(II) over Mn(II), and Zn(II) binding can interfere with Mn(II) acquisition. In this work, we have used a biotinylated variant of S100A12 to show that S100A12 can sequester Zn(II) ions from PsaA and MntC. Moreover, electron paramagnetic resonance (EPR) spectroscopy indicates that by sequestering Zn(II) from Zn(II)-bound PsaA and MntC, S100A12 promotes Mn(II) binding to the SBPs. These results inform the function of S100A12 in Zn(II) sequestration, and further suggest that Zn(II)-sequestering S100 proteins may inadvertently protect bacterial pathogens during infection.

Promiscuity mapping of the S100 protein family using a high-throughput holdup assay

S100 proteins are small, typically homodimeric, vertebrate-specific EF-hand proteins that establish Ca2+-dependent protein–protein interactions in the intra- and extracellular environment and are overexpressed in various pathologies. There are about 20 distinct human S100 proteins with numerous potential partner proteins. Here, we used a quantitative holdup assay to measure affinity profiles of most members of the S100 protein family against a library of chemically synthetized foldamers. The profiles allowed us to quantitatively map the binding promiscuity of each member towards the foldamer library. Since the library was designed to systematically contain most binary natural amino acid side chain combinations, the data also provide insight into the promiscuity of each S100 protein towards all potential naturally occurring S100 partners in the human proteome. Such information will be precious for future drug design to interfere with S100 related pathologies.

Zinc sequestration by human calprotectin facilitates manganese binding to the bacterial solute-binding proteins PsaA and MntC.

Zinc is an essential transition metal nutrient for bacterial survival and growth but may become toxic when present at elevated levels. The Gram-positive bacterial pathogen Streptococcus pneumoniae is sensitive to zinc poisoning, which results in growth inhibition and lower resistance to oxidative stress. Streptococcus pneumoniae has a relatively high manganese requirement, and zinc toxicity in this pathogen has been attributed to the coordination of Zn(II) at the Mn(II) site of the solute-binding protein (SBP) PsaA, which prevents Mn(II) uptake by the PsaABC transport system. In this work, we investigate the Zn(II)-binding properties of pneumococcal PsaA and staphylococcal MntC, a related SBP expressed by another Gram-positive bacterial pathogen, Staphylococcus aureus, which contributes to Mn(II) uptake. X-ray absorption spectroscopic studies demonstrate that both SBPs harbor Zn(II) sites best described as five-coordinate, and metal-binding studies in solution show that both SBPs bind Zn(II) reversibly with sub-nanomolar affinities. Moreover, both SBPs exhibit a strong thermodynamic preference for Zn(II) ions, which readily displace bound Mn(II) ions from these proteins. We also evaluate the Zn(II) competition between these SBPs and the human S100 protein calprotectin (CP, S100A8/S100A9 oligomer), an abundant host-defense protein that is involved in the metal-withholding innate immune response. CP can sequester Zn(II) from PsaA and MntC, which facilitates Mn(II) binding to the SBPs. These results demonstrate that CP can inhibit Zn(II) poisoning of the SBPs and provide molecular insight into how S100 proteins may inadvertently benefit bacterial pathogens rather than the host.

Novel Lysine-Rich Delivery Peptides of Plant Origin ERD and Human S100: The Effect of Carboxyfluorescein Conjugation, Influence of Aromatic and Proline Residues, Cellular Internalization, and Penetration Ability

The need for novel drug delivery peptides is an important issue of the modern pharmaceutical research. Here, we test K-rich peptides from plant dehydrin ERD14 (ERD-A, ERD-B, and ERD-C) and the C-terminal CPP-resembling region of S100A4 (S100) using the 5(6)-carboxyfluorescein (Cf) tag at the N-terminus. Via a combined pH-dependent NMR and fluorescence study, we analyze the effect of the Cf conjugation/modification on the structural behavior, separately investigating the (5)-Cf and (6)-Cf forms. Flow cytometry results show that all peptides internalize; however, there is a slight difference between the cellular internalization of (5)- and (6)-Cf-peptides. We indicate the possible importance of residues with an aromatic sidechain and proline. We prove that ERD-A localizes mostly in the cytosol, ERD-B and S100 have partial colocalization with lysosomal staining, and ERD-C mainly localizes within vesicle-like compartments, while the uptake mechanism mainly occurs through energy-dependent paths.

The calcium binding protein S100\u03b2 marks hedgehog-responsive resident vascular stem cells within vascular lesions

A hallmark of subclinical atherosclerosis is the accumulation of vascular smooth muscle cell (SMC)-like cells leading to intimal thickening. While medial SMCs contribute, the participation of hedgehog-responsive resident vascular stem cells (vSCs) to lesion formation remains unclear. Using transgenic eGFP mice and genetic lineage tracing of S100β vSCs in vivo, we identified S100β/Sca1 cells derived from a S100β non-SMC parent population within lesions that co-localise with smooth muscle α-actin (SMA) cells following iatrogenic flow restriction, an effect attenuated following hedgehog inhibition with the smoothened inhibitor, cyclopamine. In vitro, S100β/Sca1 cells isolated from atheroprone regions of the mouse aorta expressed hedgehog signalling components, acquired the di-methylation of histone 3 lysine 4 (H3K4me2) stable SMC epigenetic mark at the Myh11 locus and underwent myogenic differentiation in response to recombinant sonic hedgehog (SHh). Both S100β and PTCH1 cells were present in human vessels while S100β cells were enriched in arteriosclerotic lesions. Recombinant SHh promoted myogenic differentiation of human induced pluripotent stem cell-derived S100β neuroectoderm progenitors in vitro. We conclude that hedgehog-responsive S100β vSCs contribute to lesion formation and support targeting hedgehog signalling to treat subclinical arteriosclerosis.

Meningeal melanocytoma in the cerebellopontine angle: A rare case report and review of the literature

Abstract Primary meningeal melanocytoma (MM) in the cerebellopontine angle (CPA) region is an extremely rare neoplasm that originates from the melanocytes in the leptomeninges. These lesions are usually misdiagnosed as they mimic other common CPA lesions through their nonspecific presenting symptoms, signs, and radiological characteristics. Here, we report a 47-year-old Chinese female patient who presented with a 1-month history of the right-sided tongue numbness and 1-week history of the right-sided face numbness that had been worsening for 2 days. The tumor, in the right CPA region, showed a slight isointensity on T1-weighted image and mixed signal intensity on T2-weighted image. The clinical presentation, surgical treatment, and pathologic characteristics were determined. The tumor was microsurgically resected and gross-total resection was achieved. The tumor revealed a solid, capsulated, brown-black lesion. Immunohistochemistry showed that the tumor cells were positive for human melanoma black-45 (HMB-45), melanoma antigen (MelanA), S100, SOX10, and BRAF, confirming the final diagnosis of meningeal melanocytoma. Ultimately, no signs of radiological local recurrence were observed during the two-year follow-up. Collectively, meningeal melanocytoma is difficult to distinguish from common tumors in the CPA region before operation due to the lack of specificity in imaging and symptoms. Complete surgical resection is the best therapeutic option for this tumor. Although the tumor is commonly considered as a benign lesion, recurrence and metastasis are common, and pathogenesis remains unclear.

Serum Levels of Soluble Triggering Receptor Expressed on Myeloid Cells-1 Associated with the Severity and Outcome of Acute Ischemic Stroke.

Stroke is a neurological emergency, where the mechanism of the blood supply to the brain is impaired, resulting in brain cell ischemia and death. Neuroinflammation is a key component in the ischemic cascade that results in cell damage and death after cerebral ischemia. The triggering receptor expressed on myeloid cells-1 (TREM-1) modulates neuroinflammation after acute ischemic stroke. In the present study, 60 patients with acute ischemic stroke, who had been subjected to neurological examinations and National Institutes of Health Stroke Scale (NIHSS) and brain magnetic resonance imaging studies, were enrolled in the emergency room of Kaohsiung Chang Gung Memorial Hospital. Twenty-four healthy volunteers were recruited as controls. The serum levels of soluble TREM-1 (sTREM-1), human S100 calcium-binding protein B (S100B), and proinflammatory cytokines and chemokines, including tumor necrosis α (TNF-α), interleukin 1β, interleukin 6 (IL-6), interleukin 8, and interferon-γ were measured immediately after acute ischemic stroke. The serum levels of sTREM-1, TNFα, IL-6, and S100B were correlated with the stroke volume and NIHSS, after acute ischemic stroke. Additionally, the serum levels of sTREM-1 were significantly positively correlated with S100B. The functional outcomes were evaluated 6 months after ischemic stroke by the Barthel index, which was correlated with the age and levels of sTREM-1 and S100B. We suggest that acute ischemic stroke induces neuroinflammation by the activation of the TREM-1 signaling pathway and the downstream inflammatory machinery that modulates the inflammatory response and ischemic neuronal cell death. From a translational perspective, our results may allow for the development of a new therapeutic strategy for acute ischemic stroke by targeting the TREM-1 signaling pathway.

Structural properties of target binding by profilaggrin A and B domains and other S100 fused-type calcium-binding proteins

BackgroundProfilaggrin belongs to the S100 fused-type protein family expressed in keratinocytes and is important for skin barrier integrity. Its N-terminus contains an S100 (“A”) domain and a unique “B” domain with a nuclear localization sequence. ObjectiveTo determine whether profilaggrin B domain cooperates with the S100 domain to bind macromolecules. To characterize the biochemical and structural properties of the profilaggrin N-terminal “AB” domain and compare it to other S100 fused-type proteins. MethodsWe used biochemical (protease protection, light scattering, fluorescence spectroscopy, pull-down assays) and computational techniques (sequence analysis, molecular modeling with crystallographic structures) to examine human profilaggrin and S100 fused-type proteins. ResultsComparing profilaggrin S100 crystal structure with models of the other S100 fused-type proteins demonstrated each has a unique chemical composition of solvent accessible surface around the hydrophobic binding pocket. S100 fused-type proteins exhibit higher pocket hydrophobicity than soluble S100 proteins. The inter-EF-hand linker in S100 fused-type proteins contains conserved hydrophobic residues involved in binding substrates. Profilaggrin B domain cooperates with the S100 domain to bind annexin II and keratin intermediate filaments in a calcium-dependent manner using exposed cationic surface. Using molecular modeling we demonstrate profilaggrin B domain likely interacts with annexin II domains I and II. Steric clash analysis shows annexin II N-terminal peptide is favored to bind profilaggrin among S100 fused-type proteins. ConclusionThe N-terminal S100 and B domains of profilaggrin cooperate to bind substrate molecules in granular layer keratinocytes to provide epidermal barrier functions.

Zooming into the Dark Side of Human Annexin-S100 Complexes: Dynamic Alliance of Flexible Partners.

Annexins and S100 proteins form two large families of Ca2+-binding proteins. They are quite different both structurally and functionally, with S100 proteins being small (10–12 kDa) acidic regulatory proteins from the EF-hand superfamily of Ca2+-binding proteins, and with annexins being at least three-fold larger (329 ± 12 versus 98 ± 7 residues) and using non-EF-hand-based mechanism for calcium binding. Members of both families have multiple biological roles, being able to bind to a large cohort of partners and possessing a multitude of functions. Furthermore, annexins and S100 proteins can interact with each other in either a Ca2+-dependent or Ca2+-independent manner, forming functional annexin-S100 complexes. Such functional polymorphism and binding indiscrimination are rather unexpected, since structural information is available for many annexins and S100 proteins, which therefore are considered as ordered proteins that should follow the classical “one protein–one structure–one function” model. On the other hand, the ability to be engaged in a wide range of interactions with multiple, often unrelated, binding partners and possess multiple functions represent characteristic features of intrinsically disordered proteins (IDPs) and intrinsically disordered protein regions (IDPRs); i.e., functional proteins or protein regions lacking unique tertiary structures. The aim of this paper is to provide an overview of the functional roles of human annexins and S100 proteins, and to use the protein intrinsic disorder perspective to explain their exceptional multifunctionality and binding promiscuity.

Potential role of the compound Eucommia bone tonic granules in patients with osteoarthritis and osteonecrosis: A retrospective study.

Osteoarthritis is a major source of pain, disability, and socioeconomic cost worldwide. Osteonecrosis is a disabling disorder that frequently occurs in the younger population aged from 20-50 years. The compound Eucommia bone tonic granules, a traditional Chinese medicine, can alleviate the damage of osteoarthritis and osteonecrosis. To investigate the potential role of the compound Eucommia bone tonic granules (Eucommia) in the treatment of patients with osteoarthritis and osteonecrosis. One-hundred forty osteoarthritis and osteonecrosis cases admitted to our hospital from January 2013 to December 2017 were selected. Patients were divided into two groups: Eucommia-meloxicam group and meloxicam group. Clinical efficacy and the Western Ontario and McMaster Universities Arthritis Index (WOMAC) score were evaluated according to the evaluation criteria of orthopedic diseases. The levels of bone-GLA protein, interleukin-17, recombinant human S100 calcium binding protein A12, sphingosine 1-phosphate, cystatin C, creatinine, and hemoglobin in peripheral blood were determined. The total effective rate in the two osteoarthritis groups was not different, but the total effective rate in the two osteonecrosis groups was significantly different. The overall efficacy of Eucommia-meloxicam group was superior to that of the meloxicam group. WOMAC showed that pain, stiffness, and dysfunction in the two groups of osteoarthritis and osteonecrosis before and after treatment were significantly different. The concentration of recombinant human S100 calcium binding protein A12, sphingosine 1-phosphate, cystatin C, creatinine, and hemoglobin before and after treatment in the Eucommia-meloxicam group and meloxicam group of osteoarthritis and osteonecrosis were significantly different, and the two treatment groups were significantly different from each other for osteoarthritis. Our findings indicate that Eucommia can effectively enhance the curative effect of meloxicam, and the combination of Eucommia and meloxicam is superior to meloxicam alone.

S100 A9 is expressed and secreted by the oviduct epithelium, interacts with gametes and affects parameters of human sperm capacitation in vitro.

Our previous findings demonstrate that some oviductal secretion proteins bind to gametes and affect sperm physiology and gamete interaction. One of these proteins possesses an estimated molecular weight of 14 kDa. The objective of this study was to isolate and identify this 14 kDa protein, to localize it in the human oviduct, to detect gamete binding sites for the protein, and to evaluate its effects on sperm capacitation parameters and gamete interaction. Explants from the human oviductal tissues of premenopausal women were cultured in the presence of [35 S]-Methionine-proteins ([35S]-Met-proteins). De novo synthesized secreted [35 S]-Met-proteins were isolated from the culture media by affinity chromatography using their sperm membrane binding ability and analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using liquid chromatography-tandem mass spectrometry peptide sequencing, human S100 A9 was identified as one of the isolated proteins from the 14 kDa protein band. S100 A9 was detected in oviduct epithelium and oviduct secretion using immunohistochemistry and a Western blot. S100 A9 binding to human oocytes and spermatozoa was assessed by indirect immunofluorescence. The acrosome reaction (AR) affected S100 A9 ability to bind sperm cells. The presence of S100 A9 significantly increased both the induced AR and the sperm protein tyrosine phosphorylation, with respect to controls. However, the protein did not affect sperm-zona pellucida interaction. Results indicate that S100 A9 is present in the human oviduct and that it modulates parameters of sperm capacitation in vitro. Hence, the protein might contribute to the regulation of the reproductive process in the oviductal microenvironment.

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.

Clear Cell Sugar Tumour

A 56-year-old female smoker with a history of right breast cancer (T1N0M0) had an incidental finding of an 8-mm right lower lobe solitary pulmonary nodule on computed tomography scan. The lesion was not positron emission tomography avid; however, it slowly progressed in size on surveillance computed tomography to 11 mm. She proceeded to thoracotomy and right lower lobectomy with lymph node sampling. Histology confirmed a clear cell sugar tumour. Clear cell sugar tumour of the lung is a remarkably rare benign pulmonary neoplasm of perivascular epithelioid origin, and a member of the PEComatous group of tumours. PEComas of the lung are divided into lymphangioleiomyomatosis, benign PEComas (including clear cell sugar tumour), and malignant PEComas. PEComas can be found in other sites, including the female genital and gastrointestinal tracts. The tumours characteristically contain a large amount of glycogen; therefore, leading differentials to consider are metastatic renal cell carcinoma and clear cell pulmonary carcinomas. Diagnosis is established by demonstrating immunoreactivity for human melanoma black (HMB-45) and S-100 protein, with no reactivity for cytokeratin 7 or epithelial membrane antigen. The general recommendation is for surgical resection without adjuvant chemotherapy.

Isolation and Characterization of S100 Protein-Protein Complexes.

S100 proteins are small, mostly dimeric, EF-hand Ca2+-binding proteins. Upon Ca2+ binding, a conformational change occurs resulting in the exposure of a shallow hydrophobic binding groove in each subunit. Interestingly, S100 proteins can interact with their partners in two ways: symmetrically, when the two partners identically bind into each groove, or asymmetrically, when only one partner binds to the S100 dimer occupying both binding pockets. Here we present a heterologous expression and purification protocol for all known human S100 proteins as well as for their partner peptides. Moreover, we provide a detailed description of three in vitro methods to determine the affinity, stoichiometry, and kinetics of S100 protein-protein interactions.

Blocking the interaction between S100A9 protein and RAGE V domain using S100A12 protein.

The proteins S100A9 and S100A12 are associated with the human S100 calcium-binding protein family. These proteins promote interaction with target proteins and alter their conformation when they bind to calcium ions in EF-hand motifs. The V domain of RAGE (Receptor for Advanced Glycation End products) is crucial for S100A9 binding. The binding of RAGE with S100 family proteins aids in cell proliferation. In this report, we demonstrate that S100A12 protein hinders the binding of S100A9 with the RAGE V-domain. We used fluorescence and NMR spectroscopy to analyze the interaction of S100A9 with S100A12. The binary complex models of S100A9-S100A12 were developed using data obtained from 1H-15N HSQC NMR titrations and the HADDOCK program. We overlaid the complex models of S100A9-S100A12 with the same orientation of S100A9 and the RAGE V-domain. This complex showed that S100A12 protein blocks the interaction between S100A9 and the RAGE V-domain. It means S100A12 may be used as an antagonist for S100A9. The results could be favorable for developing anti-cancer drugs based on S100 family proteins.