S100P is a novel interaction partner and regulator of IQGAP1.

Ca(2+)-binding proteins of the S100 family participate in intracellular Ca(2+) signaling by binding to and regulating specific cellular targets in their Ca(2+)-loaded conformation. Because the information on specific cellular targets of different S100 proteins is still limited, we developed an affinity approach that selects for protein targets only binding to the physiologically active dimer of an S100 protein. Using this approach, we here identify IQGAP1 as a novel and dimer-specific target of S100P, a member of the S100 family enriched in the cortical cytoskeleton. The interaction between S100P and IQGAP1 is strictly Ca(2+)-dependent and characterized by a dissociation constant of 0.2 µM. Binding occurs primarily through the IQ domain of IQGAP1 and the first EF hand loop of S100P, thus representing a novel structural principle of S100-target protein interactions. Upon cell stimulation, S100P and IQGAP1 co-localize at or in close proximity to the plasma membrane, and complex formation can be linked to altered signal transduction properties of IQGAP1. Specifically, the EGF-induced tyrosine phosphorylation of IQGAP1 that is thought to function in assembling signaling intermediates at IQGAP1 scaffolds in the subplasmalemmal region is markedly reduced in cells overexpressing S100P but not in cells expressing an S100P mutant deficient in IQGAP1 binding. Furthermore, B-Raf binding to IQGAP1 and MEK1/2 activation occurring downstream of IQGAP1 in EGF-triggered signaling cascades are compromised at elevated S100P levels. Thus, S100P is a novel Ca(2+)-dependent regulator of IQGAP1 that can down-regulate the function of IQGAP1 as a signaling intermediate by direct interaction.

Quantification of periodontal pathogens by paper point sampling from the coronal and apical aspect of periodontal lesions by real-time PCR.

The present study compared the recovery of six periodontal pathogens by paper point samples from two different aspects of periodontal lesions by quantitative real-time polymerase chain reaction (PCR). Twenty patients with untreated chronic periodontitis were randomized into two groups. Before subgingival instrumentation and after 10 weeks samples in group A were taken first with a paper point half length (HP) of the probing depth, then with a paper point full length (FP) at the same site. In group B sampling sequence was reversed. Analysis by real-time PCR enabled quantification of six bacteria as well as total bacterial count (TBC). Statistical analysis included t test, Kappa, and Spearman's correlations. Higher TBC could be harvested by use of FP than by HP (mean of differences of ln-transformed counts before therapy: -0.791, CI [-1.515, -0.068], SD 0.770, p = 0.034; after therapy: -0.563, CI [-1.151, 0.024], SD 0.625, p = 0.059). The plaque composition regarding total target pathogens was similar for both samples. Both, for TBC as well as for single target bacteria a strong positive correlation was found between HP and FP (Kappa, Spearman correlation: Aggregatibacter actinomycetemcomitans 0.807, 0.778; Fusobacterium nucleatum 0.573, 0.772; Porphyromonas gingivalis 0.733, 0.824; Prevotella intermedia 0.480, 0.756; Treponema denticola 0.807, 0.814; and Tannerella forsythia 0.692, 0.695). The recovery of target pathogens was similar following sampling at various depths of the periodontal lesion.

Comparison of curet and paper point sampling of subgingival bacteria as analyzed by real-time polymerase chain reaction.

BACKGROUND: The outcome of microbiological diagnostics may depend on the sampling technique. It was the aim of the present study to compare two widely used sampling techniques for subgingival bacteria using quantitative real-time polymerase chain reaction. METHODS: Twenty patients with chronic periodontitis were randomized into two groups. In group A, samples were taken first with a paper point and then with a curet at the same site (single-rooted teeth with probing depth >5 mm) before scaling and root planing and after 10 weeks. The sampling sequence was reversed in group B. The analysis enabled the quantification of Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Treponema denticola, and Tannerella forsythensis and total bacterial counts (TBCs). Statistical analysis included t test, kappa, and Spearman correlation. RESULTS: Higher TBC was harvested with curets than by paper points (P = 0.008). The plaque composition with regard to total target pathogens was similar for both sampling techniques. A strong positive correlation was found between curet and paper point samples for TBC and single target bacteria. CONCLUSIONS: Overall, there was a relatively good agreement for the results of paper point and curet sampling. Thus, both techniques seem to be suitable for microbiological diagnostics.

Ca2+-dependent binding and activation of dormant ezrin by dimeric S100P.

S100 proteins are EF hand type Ca2+ binding proteins thought to function in stimulus-response coupling by binding to and thereby regulating cellular targets in a Ca2+-dependent manner. To isolate such target(s) of the S100P protein we devised an affinity chromatography approach that selects for S100 protein ligands requiring the biologically active S100 dimer for interaction. Hereby we identify ezrin, a membrane/F-actin cross-linking protein, as a dimer-specific S100P ligand. S100P-ezrin complex formation is Ca2+ dependent and most likely occurs within cells because both proteins colocalize at the plasma membrane after growth factor or Ca2+ ionophore stimulation. The S100P binding site is located in the N-terminal domain of ezrin and is accessible for interaction in dormant ezrin, in which binding sites for F-actin and transmembrane proteins are masked through an association between the N- and C-terminal domains. Interestingly, S100P binding unmasks the F-actin binding site, thereby at least partially activating the ezrin molecule. This identifies S100P as a novel activator of ezrin and indicates that activation of ezrin's cross-linking function can occur directly in response to Ca2+ transients.