Member Of S100 Protein Family Research Articles

High Expression of the Ca2+-Binding Proteins S100A8 and S100A9 Cause Glucocorticoid Resistance in MLL-Rearranged Infant Acute Lymphoblastic Leukemia.

Abstract 729MLL-rearranged Acute Lymphoblastic Leukemia (ALL) in infants (i.e. children <1 year of age) represents an aggressive and difficult to treat type of leukemia, displaying cellular resistance to several chemotherapeutics, especially to glucocorticoids like prednisolone. As prednisolone response is highly predictive for clinical outcome, it is of utmost importance to unravel the mechanism underlying resistance to this drug.To gain insights in the prednisolone resistance mechanism, we compared gene expression profiles (Affymetrix HU133plus2) from prednisolone-resistant and prednisolone-sensitive MLL-rearranged infant ALL patients. This gene signature revealed that multiple genes involved in calcium signaling were up-regulated in prednisolone-resistant samples. Most pronounced up-regulation was observed for the S100 protein family members S100A8 and S100A9, which are calcium-binding proteins, that function in a complex. Quantitative RT-PCR (TaqMan) analyses confirmed that S100A8 and S100A9 mRNA expression was ∼100-fold higher in prednisolone-resistant cells compared to patients sensitive to this drug (p=0.008).Glucocorticoids are known to induce apoptosis by releasing Ca2+ from the endoplasmic reticulum (ER) into the cytosol and towards the mitochondria. Within mitochondria, elevated Ca2+ levels induce cytochrome c release, triggering apoptosis. Since S100A8/A9 are capable of binding free cytosolic Ca2+, we hypothesized that over-representation of these cytosolic proteins may prevent Ca2+ to reach the mitochondria and forestall apoptosis.To test our hypothesis, we first co-incubated prednisolone-sensitive MLL-rearranged ALL cells with prednisolone and EGTA or BAPTA-AM. Both agents represent Ca2+chelators and scavenge free cytosolic Ca2+, thereby mimicking the Ca2+binding by S100A8/A9. Flow cytometry analyses showed that both EGTA and BAPTA-AM inhibited the free cytosolic calcium released by prednisolone. Cytotoxicity tests demonstrated that these prednisolone-sensitive cells became more resistant to prednisolone after co-incubation with either EGTA or BAPTA-AM. Next we asked whether enforced over-expression (using retroviral expression vectors) of S100A8 and/or S100A9 in prednisolone-sensitive MLL-rearranged ALL cells could also inhibit free cytosolic Ca2+and induce prednisolone resistance. Indeed, both S100A8 and S100A9 were capable of binding free cytosolic Ca2+ released by prednisolone, accompanied by a 30-40% increase in cell survival when either S100A8 or S100A9 were over-expressed alone. Simultaneous over-expression of both S100A8 and S100A9 almost completely reversed the prednisolone-sensitive into a prednisolone-resistance phenotype.In conclusion, these findings implicate that high expression of S100A8 and S100A9 contributes to prednisolone resistance in MLL-rearranged infant ALL. Disclosures:No relevant conflicts of interest to declare.

Epidermal growth factor receptor ligands as new extracellular targets for the metastasis‐promoting S100A4 protein

The function of S100A4, a member of the calcium-binding S100 protein family, has been associated with tumor invasion and metastasis. Although an essential pro-metastatic role of extracellular S100A4 in tumor progression has been demonstrated, the identification of the precise underlying mechanisms and protein partners (receptors) has remained elusive. To identify putative targets for extracellular S100A4, we screened a phage display peptide library using S100A4 as bait. We identified three independent peptide motifs with varying affinities for the S100A4 protein. Sequence analyses indicated that the most abundant peptide mimicked the F/YCC motif present in the epidermal growth factor domain of ErbB receptor ligands. S100A4 selectively interacted with a number of epidermal growth factor receptor (EGFR) ligands, demonstrating highest affinity for amphiregulin. Importantly, we found that S100A4 stimulated EGFR/ErbB2 receptor signaling and enhanced the amphiregulin-mediated proliferation of mouse embryonic fibroblasts. S100A4-neutralizing antibodies, as well as EGFR- and ErbB2 receptor-specific tyrosine kinase inhibitors, blocked these effects. The present results suggest that extracellular S100A4 regulates tumor progression by interacting with EGFR ligands, thereby enhancing EGFR/ErbB2 receptor signaling and cell proliferation. Structured digital abstract: * MINT-7256556: EGF (uniprotkb:P01133) binds (MI:0407) to S100A4 (uniprotkb:P26447) by far western blotting (MI:0047) * MINT-7256512: BC (uniprotkb:P35070) binds (MI:0407) to S100A4 (uniprotkb:P26447) by far western blotting (MI:0047) * MINT-7256485, MINT-7256618, MINT-7256636: AR (uniprotkb:P15514) binds (MI:0407) to S100A4 (uniprotkb:P26447) by far western blotting (MI:0047) * MINT-7256494: HB-EGF (uniprotkb:Q99075) binds (MI:0407) to S100A4 (uniprotkb:P26447) by far western blotting (MI:0047) * MINT-7256502: P53 (uniprotkb:P04637) binds (MI:0407) to S100A4 (uniprotkb:P26447) by far western blotting (MI:0047) * MINT-7256654: S100A2 (uniprotkb:P29034) binds (MI:0407) to AR (uniprotkb:P15514) by far western blotting (MI:0047) * MINT-7256693: S100A5 (uniprotkb:P33763) binds (MI:0407) to AR (uniprotkb:P15514) by far western blotting (MI:0047) * MINT-7256593: S100A4 (uniprotkb:P26447) binds (MI:0407) to BC (uniprotkb:P35070) by pull down (MI:0096) * MINT-7256567: S100A4 (uniprotkb:P26447) binds (MI:0407) to AR (uniprotkb:P15514) by pull down (MI:0096).

S100A11: Diverse Function and Pathology Corresponding to Different Target Proteins

S100A11, as a member of S100 protein family, while featuring the common identities as the other EF-hand Ca(2+)-binding family members, has its own individual characteristics. S100A11 is widely expressed in multiple tissues, and is located in cytoplasm, nucleus, and even cell periphery. S100A11 exists as a non-covalent homodimer with an antiparallel conformation. Ca(2+) binding to S100A11 would trigger conformational changes which would expose the hydrophobic cleft of S100A11 and facilitate its interaction with target proteins. Since S100A11 appears to lack enzymatic activity, in this article, corresponding to a variety of its target proteins, we systematically describe the biological roles of S100A11 and its possible mechanism in the processes of inflammation, regulation of enzyme activity, and cell growth regulation. As a dual cell growth mediator, S100A11 acts as either a tumor suppressor or promoter in many different types of tumors and would play respective roles in influencing the proliferation of the cancer cells. We intend to illustrate the biological function of the S100 protein, and shed light on the further research, which will provide us with a better understanding of it.

Annexin I and Annexin II N-Terminal Peptides Binding to S100 Protein Family Members: Specificity and Thermodynamic Characterization

The S100 proteins make up a family of dimeric calcium binding proteins that function in response to changing calcium levels. Several S100 binding proteins have been identified; however, the exact biological functions of the S100 proteins are largely unknown as there are several factors which modulate their functions. To address these issues, the specificity of binding of representative members of the human S100 proteins to short N-terminal peptides of annexin I (AnI) and annexin II (AnII) was investigated under controlled experimental conditions. AnI and AnII have been shown previously to interact with S100A11 and S100A10, respectively. This provided a unique opportunity to determine their binding specificity with the other members of the human S100 protein family. It was found that AnI binds S100A6 or S100A11 while AnII binds S100A10 or S100A11. This is the first report of the interaction between S100A6 and AnI. The fact that AnI and AnII bind to selected members of the S100 protein family shows that these interactions are specific and that the mode of binding is different from that of calmodulin, as it was found not to bind AnI or AnII. From the analysis of the thermodynamics of interactions, the binding seems to be entropically driven. It was found that both AnI and AnII undergo a coil-to-helix transition upon binding to their respective binding partners. The observation that there is an overlap in functionality is not surprising due to considerable sequence homology between S100 protein family members. In fact, the functional overlap can explain previous failures of S100 knockout constructs to show any detectable changes in phenotype despite numerous implications of these proteins in important cellular processes.

Crosstalk Between Calcium, Amyloid β and the Receptor for Advanced Glycation Endproducts in Alzheimer's Disease

Hallmarks of Alzheimer's disease (AD) include the accumulation of amyloid beta peptide (Abeta), hyperphosphorylation of tau protein, and increased inflammatory activity in the hippocampus and cerebral cortex. The receptor for advanced glycation endproducts (RAGE) has been shown to interact with Abeta and to modulate Abeta transport across the blood-brain barrier. Furthermore, RAGE is upregulated at sites of inflammation and its activation results in distinct intracellular signaling cascades in respect to Abeta conformers. Besides Abeta, RAGE interacts with several members of the calcium binding S100 protein family, amphoterin and advanced glycation endproducts. Mounting evidence suggests that RAGE is a key player in the signaling pathways triggered by Abeta and S100 proteins in AD. In this review, we discuss recent discoveries about the crosstalk between RAGE, Abeta and S100 proteins in the pathophysiology of AD.

S100A1 and Calmodulin Compete for the Same Binding Site on Ryanodine Receptor

In heart and skeletal muscle an S100 protein family member, S100A1, binds to the ryanodine receptor (RyR) and promotes Ca(2+) release. Using competition binding assays, we further characterized this system in skeletal muscle and showed that Ca(2+)-S100A1 competes with Ca(2+)-calmodulin (CaM) for the same binding site on RyR1. In addition, the NMR structure was determined for Ca(2+)-S100A1 bound to a peptide derived from this CaM/S100A1 binding domain, a region conserved in RyR1 and RyR2 and termed RyRP12 (residues 3616-3627 in human RyR1). Examination of the S100A1-RyRP12 complex revealed residues of the helical RyRP12 peptide (Lys-3616, Trp-3620, Lys-3622, Leu-3623, Leu-3624, and Lys-3626) that are involved in favorable hydrophobic and electrostatic interactions with Ca(2+)-S100A1. These same residues were shown previously to be important for RyR1 binding to Ca(2+)-CaM. A model for regulating muscle contraction is presented in which Ca(2+)-S100A1 and Ca(2+)-CaM compete directly for the same binding site on the ryanodine receptor.

Analysis of the structure of human apo‐S100B at low temperature indicates a unimodal conformational distribution is adopted by calcium‐free S100 proteins

S100B is one of the best-characterized members of the calcium-signaling S100 protein family. Most S100 proteins are dimeric, with each monomer containing two EF-hand calcium-binding sites (EF1, EF2). S100B and other S100 proteins respond to calcium increases in the cell by coordinating calcium and undergoing a conformational change that allows them to interact with a variety of cellular targets. Although several three dimensional structures of S100 proteins are available in the calcium-free (apo-) state it has been observed that these structures appear to adopt a wide range of conformations in the EF2 site with respect to the positioning of helix III, the helix that undergoes the most dramatic calcium-induced conformational change. In this work, we have determined the structure of human apo-S100B at 10 degrees C to examine whether temperature might be responsible for these structural differences. Further, we have used this data, and other available apo-S100 structures, to show that despite the range of interhelical angles adopted in the apo-S100 structures, normal Gaussian distributions about the mean angles found in the structure of human apo-S100B are observed. This finding, only obvious from the analysis of all available apo-S100 proteins, provides direct structural evidence that helix III is a loosely packed helix. This is likely a necessary functional property of the S100 proteins that facilitates the calcium-induced conformational change of helix III. In contrast, the calcium-bound structures of the S100 proteins show significantly smaller variability in the interhelical angles. This shows that calcium binding to the S100 proteins causes not only a conformational change but results in a tighter distribution of helices within the EF2 calcium binding site required for target protein interactions.

Proteins Associated with Cisplatin Resistance in Ovarian Cancer Cells Identified by Quantitative Proteomic Technology and Integrated with mRNA Expression Levels

Nearly all women diagnosed with ovarian cancer receive combination chemotherapy including cis- or carboplatin. Despite high initial response rates, resistance to cisplatin develops in roughly one-third of women during primary treatment and in all women treated for recurrent disease. ICAT coupled with tandem MS is a quantitative proteomic technique for high throughput protein expression profiling of complex protein mixtures. Using ICAT/MS/MS we profiled the nuclear, cytosolic, and microsomal fractions obtained from IGROV-1 [corrected] (cisplatin-sensitive) and IGROV-1/CP [corrected] (cisplatin-resistant) ovarian cancer cell lines. The proteomes of cisplatin-sensitive and -resistant ovarian cancer cells were compared, and protein expression was correlated with mRNA expression profiles. A total of 1117 proteins were identified and quantified. The relative expression of 121 of these varied between the two cell lines. Sixty-three proteins were overexpressed in cisplatin-sensitive, and 58 were over expressed in cisplatin-resistant cells. Examples of proteins at least 5-fold overexpressed in resistant cells and with biological relevance to cancer include cell recognition molecule CASPR3 (13.3-fold), S100 protein family members (8.7-fold), junction adhesion molecule Claudin 4 (7.2-fold), and CDC42-binding protein kinase beta (5.4-fold). Examples of cancer-related proteins at least 5-fold overexpressed in sensitive cells include hepatocyte growth factor inhibitor 1B (13.3-fold) and programmed cell death 6-interacting protein (12.7-fold). The direction of changes in expression levels between proteins and mRNAs were not always in the same direction, possibly reflecting posttranscriptional control of protein expression. We identified proteins whose expression profiles correlate with cisplatin resistance in ovarian cancer cells. Several proteins may be involved in modulating response to cisplatin and have potential as markers of treatment response or treatment targets.

Identification of Human Hornerin and Its Expression in Regenerating and Psoriatic Skin

We previously isolated a new member of the fused-type S100 protein family (hornerin) from the mouse (Makino, T., Takaishi, M., Morohashi, M., and Huh, N.-h. (2001) J. Biol. Chem. 276, 47445-47452). Mouse hornerin shares structural features, expression profiles, and intracellular localization with profilaggrin, indicating possible involvement of hornerin in cornification. In this study, we identified and partially characterized a human ortholog of mouse hornerin. The human hornerin gene was mapped between trichohyalin and filaggrin on chromosome 1q21.3, the region being completely syntenic with the counterpart of the mouse. The deduced amino acid sequence of 2850 residues shows typical structural features of "fused-type" S100 protein family members. Mature transcripts and protein from human hornerin were not detected in normal stratified epithelium, including the trunk epidermis, tongue, and esophagus. After screening of various normal and pathological human tissues, we found that human hornerin was expressed in psoriatic skin. Hornerin protein was present in the keratinizing region, although at a lower level and in fewer cells compared with filaggrin. Mature transcripts and protein from hornerin were also detected in regenerating human skin after wounding. Hornerin mRNA was induced 5 days after wounding. The mRNA level remained almost constant until 15 days and declined at 30 days after wounding. Hornerin protein was detected in the proximal epidermis (but not in the distal epidermis) at 15 days after wounding. These results indicate that hornerin has a function similar to but distinct from that of filaggrin in cornification.

Hyperzincaemia and hypercalprotectinaemia: a new disorder of zinc metabolism

Calprotectin (complex of S100A8 and S100A9) is the major calcium and zinc-binding protein of phagocytes. We report a new syndrome with recurrent infections, inflammation, and hyperzincaemia associated with excessively high plasma concentrations of calprotectin. We measured calprotectin in plasma and protein fractions by ELISA assay and zinc by atomic absorption spectrometry. Plasma proteins were fractionated by size exclusion chromatography and electrophoresis. Mass spectra of purified proteins were determined by MALDI-TOFMS. We assessed five patients, two of whom are related. All patients had much the same biochemical findings of hyperzincaemia (77-200 micromol/L, reference range 11-18 micromol/L) and raised plasma calprotectin concentrations (1.4-6.5 g/L, reference range <1 mg/L). All patients presented with recurrent infections, hepatosplenomegaly, anaemia, and evidence of systemic inflammation. Three patients had cutaneous inflammation and three presented in infancy with severe growth failure. Size exclusion chromatography showed that zinc and calprotectin were associated in a broad fraction with molecular weight range 100-300 kDa. Analysis by electrophoresis and mass spectrometry showed that the patients' protein contained normal S100A8 and S100A9 subunits. Dysregulation of zinc metabolism associated with accumulation in plasma of S100A8 and S100A9 defines a new disease, which encompasses a pathological role for dysregulation of two members of the large S100 protein family.

Expression of members of the calcium-binding S-100 protein family in a rat model of cerebral basilar artery vasospasm.

The aim of this study was to investigate the role of S-100 proteins in the onset of vasospasm induced by subarachnoid hemorrhage (SAH), which leads to severe neurological morbidity and death. It has recently been argued that modifications in the levels of expression of some intracellular signaling elements controlling the organization of the actin cytoskeleton (including the rho A small guanosine triphosphatase and its related kinases) play significant roles in the induction of smooth-muscle cell contraction, a calcium-dependent process that is pathognomonic of SAH-induced vasospasm at the molecular level. Several members of the calcium-binding S-100 protein family are known to exercise significant control over the organization of the actin cytoskeleton. The levels of expression of S-100 proteins in SAH-induced vasospasm have never been investigated. The authors therefore used a double-hemorrhage rat model of SAH-induced vasospasm to determine whether the levels of expression of S-100B, S-100A1, S-100A2, S-100A4, and S-100A6 proteins on immunohistochemical studies were significantly modified in this pathological condition. Quantitative determination of immunohistochemically confirmed expression of S-100 proteins (accomplished with the aid of computer-assisted microscopy) revealed that SAH-induced vasospasm is accompanied by a very significant increase in S-100B, S-100A2, and, to a lesser extent, in S-100A4 and S-100A6 expression, whereas this condition is not accompanied by significant modifications to S-100A1 expression. Such significant modifications in the levels of expression of different members of the S-100 protein family in SAH-induced vasospasm could relate to the various roles played by this specific class of calcium-binding proteins at the level of actin cytoskeleton organization. These modifications in S-100 protein expression seem relatively specific to SAH-induced vasospasm, because heparin-induced epilepsy-like symptoms were accompanied by dramatically distinct profiles of S-100 protein expression.

Regulation of Calcyclin (S100A6) Binding by Alternative Splicing in the N-terminal Regulatory Domain of Annexin XI Isoforms

Annexin XI is a Ca2+/phospholipid-binding protein that interacts with a member of S100 protein family, calcyclin (S100A6), in a Ca2+-dependent manner. There are two isoforms of annexin XI, annexin XI-A and -B, generated by alternative splicing in the N-terminal regulatory domain. To determine the role of the alternative splicing region in the calcyclin-binding, we identified and characterized its calcyclin binding site. Experiments with glutathione S-transferase fusion proteins with N-terminal sites of annexin XI-A showed the calcyclin binding site to be in residues Gln49-Thr62 of rabbit annexin XI-A, which contains part of the splicing region. A synthesized peptide corresponding to Tyr43-Thr62 of annexin XI-A inhibited the interaction of annexin XI with calcyclin in liposome co-pelleting assay. The calcyclin binding site possesses a hydrophobic residue cluster conserved among S100 binding sites of annexin I and II. Recombinant annexin XI isoforms were expressed in Sf9 cells using a baculovirus expression system. In contrast to annexin XI-A, it was found that annexin XI-B protein could not bind to calcyclin by the liposome co-pelleting assay. In Sf9 cells coexpressing calcyclin with annexin XI isoforms, the calcyclin binding was observed only for annexin XI-A isoform. These results indicate that the calcyclin binding ability of annexin XI is an annexin XI-A isoform-specific character, suggesting that annexin XI isoforms might play distinct roles in cells through each alternative splicing regions.

Interference of BAD (Bcl-xL/Bcl-2-associated death promoter)-induced apoptosis in mammalian cells by 14-3-3 isoforms and P11.

Apoptosis and survival of diverse cell types are under hormonal control, but intracellular mechanisms regulating cell death are unclear. The Bcl-2/Ced-9 family of proteins contains conserved Bcl-2 homology regions that mediate the formation of homo- or heterodimers important for enhancing or suppressing apoptosis. Unlike most other members of the Bcl-2 family, BAD (Bcl-xL/Bcl-2 associated death promoter), a death enhancer, has no C-terminal transmembrane domain for targeting to the outer mitochondrial membrane and nuclear envelope. We hypothesized that BAD, in addition to binding Bcl-xL and Bcl-2, may interact with proteins outside the Bcl-2 family. Using the yeast two-hybrid system to search for BAD-binding proteins in an ovarian fusion cDNA library, we identified multiple cDNA clones encoding different isoforms of 14-3-3, a group of evolutionally conserved proteins essential for signal transduction and cell cycle progression. Point mutation of BAD in one (S137A), but not the other (S113A), putative binding site found in diverse 14-3-3 interacting proteins abolished the interaction between BAD and 14-3-3 without affecting interactions between BAD and Bcl-2. Because the S137A BAD mutant presumably resembles an underphosphorylated form of BAD, we used this mutant to screen for additional BAD-interacting proteins in the yeast two-hybrid system. P11, a nerve growth factor-induced neurite extension factor and member of the calcium-binding S-100 protein family, interacted strongly with the mutant BAD but less effectively with the wild type protein. In Chinese hamster ovary (CHO) cells, transient expression of wild type BAD or its mutants increased apoptotic cell death, which was blocked by cotransfection with the baculovirus-derived cysteine protease inhibitor, P35. Cotransfection with 14-3-3 suppressed apoptosis induced by wild type or the S113A mutant BAD but not by the S137A mutant incapable of binding 14-3-3. Furthermore, cotransfection with P11 attenuated the proapoptotic effect of both wild type BAD and the S137A mutant. For both 14-3-3 and P11, direct binding to BAD was also demonstrated in vitro. These results suggest that both 14-3-3 and P11 may function as BAD-binding proteins to dampen its apoptotic activity. Because the 14-3-3 family of proteins could interact with key signaling proteins including Raf-1 kinase, protein kinase C, and phosphatidyl inositol 3 kinase, whereas P11 is an early response gene induced by the neuronal survival factor, nerve growth factor, the present findings suggest that BAD plays an important role in mediating communication between different signal transduction pathways regulated by hormonal signals and the apoptotic mechanism controlled by Bcl-2 family members.

Spectral studies on the calcium-binding properties of Mts1 protein and its interaction with target protein

Two calcium-binding sites of the Mts1 protein, a member of S-100 protein family, were distinguished with the Fluo-3 fluorescent technique. The geometric mean of the apparent dissociation constant ( K ̄ d ) for these two sites is 2.6 μM; the Hill coefficient ( n H) is 0.98. In the presence of a novel target protein p37, isolated from the mouse adenocarcinoma cell line CSML-100, Mts1 binds Ca 2+ ions with higher affinity and with strong positive cooperativity ( K ̄ d =0.2 μM, n H=1.91). Interaction of Mts1 with p37 is confirmed by the fluorescent probe 2- p-toluidinylnaphthalene-6-sulfonate (TNS). Reaction with TNS shows that p37 interacts with the hydrophobic site of Mts1 which is exposed due to the binding of Ca 2+ ions.

O-274 - Subcellular Distribution and Cell Cycle-Associated Expression of New Member of S100 Protein Family (S100C)

O-274 - Subcellular Distribution and Cell Cycle-Associated Expression of New Member of S100 Protein Family (S100C)

O-561 - Molecular structure and expression of a new member of S100 protein family (S100C) from porcine cardiac muscle

O-561 - Molecular structure and expression of a new member of S100 protein family (S100C) from porcine cardiac muscle