Short amino acid sequences that interact with the Ca2+ binding protein S-100b were identified by screening a bacteriophage random peptide display library. S-100b binding bacteriophages were selected by Ca(2+)-dependent affinity chromatography, and the sequence of the random peptide insert contained in 51 clones was determined. Alignment of the sequence of 44 unique S-100b binding peptides identified a common motif of eight amino acids. A subgroup of peptides that contained sequences with the highest degree of similarity had the consensus motif (K/R)(L/I)XWXXIL, in which predominantly P, S, and N were found in position 3, and S and D were found in position 5. Analysis of sequence databanks identified a similar sequence in the COOH-terminal region of the alpha-subunit of actin capping proteins. The peptide TRTKIDWNKILS (TRTK-12), corresponding to the region of greatest homology within this region of the subunit of actin capping proteins (e.g. amino acids 265-276 in CapZ alpha 1 and CapZ alpha 2), was synthesized and shown by fluorescence spectrophotometry to bind S-100b in a Ca(2+)-dependent manner. Gel overlay and cross-linking experiments demonstrated the interaction of S-100b with CapZ to be Ca2+ dependent. Moreover, this interaction was blocked by addition of TRTK-12 peptide. These results identify Ca(2+)-dependent S-100b target sequence epitopes and designate the carboxyl terminus of the alpha-subunit of actin capping proteins, like CapZ, to be a target of S-100b activity. The high level of conservation within this region of actin capping proteins and the apparent high affinity of this interaction strongly suggest that the interaction between S-100b and the alpha-subunit of actin capping proteins is biologically significant.
Using fluorescence histochemistry, 5-HT, histamine and heparin were colocalized in a large population of cells in the dura mater thereby identifying them as mast cells. In addition, because these cells were highly sensitive to compound 48/80 and were densely packed with granules of a consistent density, they were identified specifically as 'connective tissue' mast cells. Other types of mast cells, i.e. 'mucosal' or 'neurolipomastocytes', were not present in the rat dura mater. 5-HT immunohistochemistry was the best technique for demonstrating that there were populations of mast cells, one associated with each of the two layers of dura. Although shaped differently the type of mast cell in each layer was the same. It was observed that mast cell shape is dependent on the contiguity, density and orientation of its surrounding elements, not its type. In general, mast cells in the outer layer were aligned parallel to the middle meningeal artery and those in the inner layer were parallel to trigeminal nerve branches that coursed obliquely across the middle meningeal artery. Examination of cross-sections of dura revealed that most mast cells also were aligned at the interface between the two dural layers. The linear orientation of mast cells in two planes of each layer suggests a programmed lamellar seeding of these cells during development of the dura. This study also demonstrated that the majority of dural mast cells were more closely related to other connective tissue elements than to blood vessels and nerves. These results (1) are compatible with the suggestion that dural mast cells play a non-obligatory role in the neuroinflammatory response, (2) leave open to question the role of the dural mast cell in headache or the regulation of blood flow, and (3) support evidence that dural mast cells play an important role in connective tissue related functions, e.g. development, inflammatory response to injury and wound repair.
The calcium-binding protein S100B (an S100 dimer composed of two S100 monomers) is proposed to act as a calcium-sensory protein through interactions with a variety of proteins. While the nature of the exact targets for S100B has yet to be defined, random bacteriophage peptide mapping experiments have elucidated a calcium-sensitive "epitope" (TRTK-12) for S100B recognition. In this work, interactions of TRTK-12 with S100B have been shown to be calcium-sensitive. In addition, the interactions are enhanced by zinc binding to S100B, resulting in an approximate 5-fold decrease in the TRTK-12/S100B dissociation constant. Moreover, Zn 2؉ binding alone has little effect. TRTK-12 showed little evidence for binding to another S100 protein, S100A11 or to a peptide derived from the N terminus of S100B, indicating both a level of specificity for TRTK-12 recognition by S100B and that the N-terminal region of S100B is probably not involved in protein-protein interactions. NMR spectroscopy revealed residues most responsive to TRTK-12 binding that could be mapped to the surface of the three-dimensional structure of calcium-saturated S100B, revealing a common region indicative of a binding site.
The origin, density, and distribution of sympathetic nerve fibers in the supratentorial dura mater of the rat were examined in detail in the current study by using wheat germ agglutinin horseradish peroxidase (WGA-HRP) retrograde tracing procedures, glyoxylic acid-induced fluorescence, and dopamine beta-hydroxylase (DBH) immunocytochemical staining of dural whole mount preparations. Application of WGA-HRP to the superior sagittal sinus and adjacent areas of the supratentorial dura mater labeled numerous neurons in each of the left and right superior cervical ganglia. Glyoxylic acid and DBH immunocytochemical staining of fixed dural whole mount preparations revealed prominent plexuses of sympathetic nerves about the middle meningeal artery and its branches, about the superior sagittal and transverse sinuses, and "free" within the dura mater, i.e., apparently unassociated with any vasculature. Significantly, in all of these areas, the density of sympathetic innervation revealed in this study was considerably greater than that previously demonstrated by other workers. An impressive population of mast cells also was observed within the dura mater of the glyoxylic acid-treated preparations. The majority of these cells were perivascular; however, a significant number were also present within the dura unrelated to the vasculature, and occasional cells were seen in close apposition to fluorescent sympathetic nerve fibers. Taken together, the identification of a robust sympathetic plexus and prominent mast cell population associated with a dura mater that also receives significant sensory projections from the trigeminal system raises interest regarding the functional interactions of these elements. These observations warrant further consideration regarding their role in the pathogenesis of vascular headache and head pain.
Alignment of previously characterized S-100 (alpha and beta)-binding peptides (J. Biol. Chem. 270, 14651-14658) has enabled the identification of a putative S-100 target epitope within the head domain of glial fibrillary acidic protein (GFAP). The capacity of a known peptide inhibitor of S-100 protein (TRTK-12), homologous to this region, to perturb the interaction of S-100 (alpha and beta) and GFAP (J. Biol. Chem 268, 12669-12674) was investigated. Fluorescence spectrophotometry and chemical cross-linking analyses determined TRTK-12 to disrupt S-100:GFAP interaction in a dose- and Ca(2+_dependent manner. TRTK-12 also inhibited S-100's ability to block GFAP assembly and to mediate disassembly of preformed glial filaments. Each of these events was strictly dependent upon the presence of calcium and inhibitory peptide, maximal inhibition occurring at a concentration of TRTK-12 equivalent to the molar amount of S-100 monomer present. Together with our recent report demonstrating TRTK-12 also blocks the interaction of S-100 protein with the actin capping protein, CapZ, these results suggest TRTK-12 functions as a pleiotropic inhibitor of S-100 function. Availability of a functional inhibitor of S-100 will assist the further characterization of S-100 protein function in vitro and in vivo. Moreover, this report provides additional evidence supportive of a role for S-100 as a multi-faceted regulator of cytoskeletal integrity.
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