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S100 proteins are distinct dimeric EF-hand Ca-binding proteins that can bind Zn, Mn, and other transition metals with high affinity at two sites in the dimer interface. Certain S100 proteins, including S100A7, S100A12, S100A8, and S100A9, play key roles in the innate immune response to pathogens. These proteins function via a "nutritional immunity" mechanism by depleting essential transition metals in the infection that are required for the invading organism to grow and thrive. They also act as damage-associated molecular pattern ligands, which activate pattern recognition receptors (e.g., Toll-like receptor 4, RAGE) that mediate inflammation. Here we present protocols for these S100 proteins for high-level production of recombinant protein, measurement of binding affinities using isothermal titration calorimetry, and an assay of antimicrobial activity.
We used mice lacking , a key component of the β-cell K-channel, to analyze the effects of a sustained elevation in the intracellular Ca concentration ([Ca]) on β-cell identity and gene expression. Lineage tracing analysis revealed the conversion of β-cells lacking into pancreatic polypeptide cells but not to α- or δ-cells. RNA-sequencing analysis of FACS-purified β-cells confirmed an increase in gene expression and revealed altered expression of more than 4,200 genes, many of which are involved in Ca signaling, the maintenance of β-cell identity, and cell adhesion. The expression of and , two highly upregulated genes, is closely correlated with membrane depolarization, suggesting their use as markers for an increase in [Ca] Moreover, a bioinformatics analysis predicts that many of the dysregulated genes are regulated by common transcription factors, one of which, , was confirmed to be directly controlled by Ca influx in β-cells. Interestingly, among the upregulated genes is , a putative marker of β-cell dedifferentiation, and other genes associated with β-cell failure. Taken together, our results suggest that chronically elevated β-cell [Ca] in islets contributes to the alteration of β-cell identity, islet cell numbers and morphology, and gene expression by disrupting a network of Ca-regulated genes.
© 2017 by the American Diabetes Association.
The S100 proteins are a unique class of EF-hand Ca(2+) binding proteins distributed in a cell-specific, tissue-specific, and cell cycle-specific manner in humans and other vertebrates. These proteins are distinguished by their distinctive homodimeric structure, both intracellular and extracellular functions, and the ability to bind transition metals at the dimer interface. Here we summarize current knowledge of S100 protein binding of Zn(2+), Cu(2+) and Mn(2+) ions, focusing on binding affinities, conformational changes that arise from metal binding, and the roles of transition metal binding in S100 protein function.
The S100 family of EF-hand calcium (Ca(2+))-binding proteins is essential for a wide range of cellular functions. During infection, certain S100 proteins act as damage-associated molecular patterns (DAMPs) and interact with pattern recognition receptors to modulate inflammatory responses. In addition, these inflammatory S100 proteins have potent antimicrobial properties and are essential components of the immune response to invading pathogens. In this review, we focus on S100 proteins that exhibit antimicrobial properties through the process of metal limitation, termed nutritional immunity, and discuss several recent advances in our understanding of S100 protein-mediated metal sequestration at the site of infection.
© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
During infectious processes, antimicrobial proteins are produced by both epithelial cells and innate immune cells. Some of these antimicrobial molecules function by targeting transition metals and sequestering these metals in a process referred to as "nutritional immunity." This chelation strategy ultimately starves invading pathogens, limiting their growth within the vertebrate host. Recent evidence suggests that these metal-binding antimicrobial molecules have the capacity to affect bacterial virulence, including toxin secretion systems. Our previous work showed that the S100A8/S100A9 heterodimer (calprotectin, or calgranulin A/B) binds zinc and represses the elaboration of the H. pylori cag type IV secretion system (T4SS). However, there are several other S100 proteins that are produced in response to infection. We hypothesized that the zinc-binding protein S100A12 (calgranulin C) is induced in response to H. pylori infection and also plays a role in controlling H. pylori growth and virulence. To test this, we analyzed gastric biopsy specimens from H. pylori-positive and -negative patients for S100A12 expression. These assays showed that S100A12 is induced in response to H. pylori infection and inhibits bacterial growth and viability in vitro by binding nutrient zinc. Furthermore, the data establish that the zinc-binding activity of the S100A12 protein represses the activity of the cag T4SS, as evidenced by the gastric cell "hummingbird" phenotype, interleukin 8 (IL-8) secretion, and CagA translocation assays. In addition, high-resolution field emission gun scanning electron microscopy (FEG-SEM) was used to demonstrate that S100A12 represses biogenesis of the cag T4SS. Together with our previous work, these data reveal that multiple S100 proteins can repress the elaboration of an oncogenic bacterial surface organelle.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Imaging mass spectrometry (IMS) was employed for the analysis of frozen skin biopsies to investigate the differences between stage IV pressure ulcers that remain stalled, stagnant, and unhealed versus those exhibiting clinical and histological signs of improvement. Our data reveal a rich diversity of proteins that are dynamically modulated, and we selectively highlight a family of calcium binding proteins (S-100 molecules) including calcyclin (S100-A6), calgranulins A (S100-A8) and B (S100-A9), and calgizzarin (S100-A11). IMS allowed us to target three discrete regions of interest: the wound bed, adjacent dermis, and hypertrophic epidermis. Plots derived using unsupervised principal component analysis of the global protein signatures within these three spatial niches indicate that these data from wound signatures have potential as a prognostic tool since they appear to delineate wounds that are favorably responding to therapeutic interventions versus those that remain stagnant or intractable in their healing status. Our discovery-based approach with IMS augments current knowledge of the molecular signatures within pressure ulcers while providing a rationale for a focused examination of the role of calcium modulators within the context of impaired wound healing.
Although the histologic features of alveolar soft part sarcoma and granular cell tumor are typically distinctive, occasional cases show a significant morphologic overlap. Differentiating these entities is crucial because granular cell tumor is almost always benign and alveolar soft part sarcoma is invariably malignant. We evaluated a panel of immunohistochemical stains (S-100 protein, inhibin, SOX10, nestin, calretinin, and TFE3) in 13 alveolar soft part sarcomas and 11 granular cell tumors. Tissue sections were also stained by the periodic acid-Schiff method after diastase digestion (PAS-D) and evaluated for coarse cytoplasmic granularity or crystalline cytoplasmic inclusions. S-100 protein, inhibin, SOX10, and nestin each distinguished granular cell tumor and alveolar soft part sarcoma with 100% sensitivity and specificity. PAS-D staining also distinguished cases with 100% accuracy, as granular cell tumor consistently demonstrated coarsely granular, PAS-D-positive cytoplasm and alveolar soft part sarcoma showed only focal intracytoplasmic crystalline inclusions. Although all granular cell tumors were calretinin positive, so were 46% of alveolar soft part sarcomas. TFE3 was positive in 91% of granular cell tumors and all alveolar soft part sarcomas. Together with PAS-D, immunohistochemical stains for S-100 protein, inhibin, SOX10, and nestin accurately identify alveolar soft part sarcoma and granular cell tumor. Although TFE3 has been reported as a relatively specific marker for alveolar soft part sarcoma, it should be recalled that it is also expressed in most granular cell tumors.
Copyright © 2014 Elsevier Inc. All rights reserved.
BACKGROUND - Pulmonary arterial hypertension (PAH) is a vasculopathy characterized by enhanced pulmonary artery smooth muscle cell (PASMC) proliferation and suppressed apoptosis. This results in both increase in pulmonary arterial pressure and pulmonary vascular resistance. Recent studies have shown the implication of the signal transducer and activator of transcription 3 (STAT3)/bone morphogenetic protein receptor 2 (BMPR2)/peroxisome proliferator-activated receptor gamma (PPARγ) in PAH. STAT3 activation induces BMPR2 downregulation, decreasing PPARγ, which both contribute to the proproliferative and antiapoptotic phenotype seen in PAH. In chondrocytes, activation of this axis has been attributed to the advanced glycation end-products receptor (RAGE). As RAGE is one of the most upregulated proteins in PAH patients' lungs and a strong STAT3 activator, we hypothesized that by activating STAT3, RAGE induces BMPR2 and PPARγ downregulation, promoting PAH-PASMC proliferation and resistance to apoptosis.
METHODS AND RESULTS - In vitro, using PASMCs isolated from PAH and healthy patients, we demonstrated that RAGE is overexpressed in PAH-PASMC (6-fold increase), thus inducing STAT3 activation (from 10% to 40% positive cells) and decrease in BMPR2 and PPARγ levels (>50% decrease). Pharmacological activation of RAGE in control cells by S100A4 recapitulates the PAH phenotype (increasing RAGE by 6-fold, thus activating STAT3 and decreasing BMPR2 and PPARγ). In both conditions, this phenotype is totally reversed on RAGE inhibition. In vivo, RAGE inhibition in monocrotaline- and Sugen-induced PAH demonstrates therapeutic effects characterized by PA pressure and right ventricular hypertrophy decrease (control rats have an mPAP around 15 mm Hg, PAH rats have an mPAP >40 mm Hg, and with RAGE inhibition, mPAP decreases to 20 and 28 mm Hg, respectively, in MCT and Sugen models). This was associated with significant improvement in lung perfusion and vascular remodeling due to decrease in proliferation (>50% decrease) and BMPR2/PPARγ axis restoration (increased by ≥60%).
CONCLUSION - We have demonstrated the implications of RAGE in PAH etiology. Thus, RAGE constitutes a new attractive therapeutic target for PAH.
The adult mammalian heart possesses little regenerative potential following injury. Fibrosis due to activation of cardiac fibroblasts impedes cardiac regeneration and contributes to loss of contractile function, pathological remodelling and susceptibility to arrhythmias. Cardiac fibroblasts account for a majority of cells in the heart and represent a potential cellular source for restoration of cardiac function following injury through phenotypic reprogramming to a myocardial cell fate. Here we show that four transcription factors, GATA4, HAND2, MEF2C and TBX5, can cooperatively reprogram adult mouse tail-tip and cardiac fibroblasts into beating cardiac-like myocytes in vitro. Forced expression of these factors in dividing non-cardiomyocytes in mice reprograms these cells into functional cardiac-like myocytes, improves cardiac function and reduces adverse ventricular remodelling following myocardial infarction. Our results suggest a strategy for cardiac repair through reprogramming fibroblasts resident in the heart with cardiogenic transcription factors or other molecules.