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David J. Weber - One of the best experts on this subject based on the ideXlab platform.
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Solution structure of S100A1 bound to the CapZ peptide (TRTK12).
Journal of molecular biology, 2009Co-Authors: Nathan T. Wright, Paul T. Wilder, Kristen M. Varney, Brian R. Cannon, Michael T. Morgan, Danna B. Zimmer, David J. WeberAbstract:Abstract As is typical for S100–target Protein interactions, a Ca2+-dependent conformational change in S100A1 is required to bind to a 12-residue peptide (TRTK12) derived from the actin-capping Protein CapZ. In addition, the Ca2+-binding affinity of S100A1 is found to be tightened (greater than threefold) when TRTK12 is bound. To examine the biophysical basis for these observations, we determined the solution NMR structure of TRTK12 in a complex with Ca2+-loaded S100A1. When bound to S100A1, TRTK12 forms an amphipathic helix (residues N6 to S12) with several favorable hydrophobic interactions observed between W7, I10, and L11 of the peptide and a well-defined hydrophobic binding pocket in S100A1 that is only present in the Ca2+-bound state. Next, the structure of S100A1–TRTK12 was compared to that of another S100A1–target complex (i.e., S100A1–RyRP12), which illustrated how the binding pocket in Ca2+-S100A1 can accommodate peptide targets with varying amino acid sequences. Similarities and differences were observed when the structures of S100A1–TRTK12 and S100B–TRTK12 were compared, providing insights regarding how more than one S100 Protein can interact with the same peptide target. Such comparisons, including those with other S100–target and S100–drug complexes, provide the basis for designing novel small-molecule inhibitors that could be specific for blocking one or more S100–target Protein interactions.
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The Three-dimensional Solution Structure of Ca2+-bound S100A1 as Determined by NMR Spectroscopy
Journal of molecular biology, 2005Co-Authors: Nathan T. Wright, Kristen M. Varney, Danna B. Zimmer, Karen C. Ellis, Joseph Markowitz, Rossitza K. Gitti, David J. WeberAbstract:S100A1 is an EF-hand-containing Ca 2+ -binding Protein that undergoes a conformational change upon binding calcium as is necessary to interact with Protein targets and initiate a biological response. To better understand how calcium influences the structure and function of S100A1, the three-dimensional structure of calcium-bound S100A1 was determined by multidimensional NMR spectroscopy and compared to the previously determined structure of apo. In total, 3354 nuclear Overhauser effect-derived distance constraints, 240 dihedral constraints, 160 hydrogen bond constraints, and 362 residual dipolar coupling restraints derived from a series of two-dimensional, three-dimensional, and four-dimensional NMR experiments were used in its structure determination (>21 constraints per residue). As with other dimeric S100 Proteins, S100A1 is a symmetric homodimer with helices 1, 1′, 4, and 4′ associating into an X-type four-helix bundle at the dimer interface. Within each subunit there are four α-helices and a short antiparallel β-sheet typical of two helix-loop-helix EF-hand calcium-binding domains. The addition of calcium did not change the interhelical angle of helices 1 and 2 in the pseudo EF-hand significantly; however, there was a large reorientation of helix 3 in the typical EF-hand. The large conformational change exposes a hydrophobic cleft, defined by residues in the hinge region, the C terminus, and regions of helix 3, which are important for the interaction between S100A1 and a peptide (TRTK-12) derived from the actin-capping Protein CapZ.
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Solution NMR structure of S100B bound to the high-affinity target peptide TRTK-12
Journal of molecular biology, 2002Co-Authors: Keith G. Inman, Richard R. Rustandi, Ruiqing Yang, Kristine E. Miller, Donna M. Baldisseri, David J. WeberAbstract:Abstract The solution NMR structure is reported for Ca2+-loaded S100B bound to a 12-residue peptide, TRTK-12, from the actin capping Protein CapZ (α1 or α2 subunit, residues 265–276: TRTKIDWNKILS). This peptide was discovered by Dimlich and co-workers by screening a bacteriophage random peptide display library, and it matches exactly the consensus S100B binding sequence ((K/R)(L/I)XWXXIL). As with other S100B target Proteins, a calcium-dependent conformational change in S100B is required for TRTK-12 binding. The TRTK-12 peptide is an amphipathic helix (residues W7 to S12) in the S100B–TRTK complex, and helix 4 of S100B is extended by three or four residues upon peptide binding. However, helical TRTK-12 in the S100B–peptide complex is uniquely oriented when compared to the three-dimensional structures of other S100–peptide complexes. The three-dimensional structure of the S100B–TRTK peptide complex illustrates that residues in the S100B binding consensus sequence (K4, I5, W7, I10, L11) are all involved in the S100B–peptide interface, which can explain its orientation in the S100B binding pocket and its relatively high binding affinity. A comparison of the S100B–TRTK peptide structure to the structures of apo- and Ca2+-bound S100B illustrates that the binding site of TRTK-12 is buried in apo-S100B, but is exposed in Ca2+-bound S100B as necessary to bind the TRTK-12 peptide.
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Role of the C-terminal extension in the interaction of S100A1 with GFAP, tubulin, the S100A1- and S100B-inhibitory peptide, TRTK-12, and a peptide derived from p53, and the S100A1 inhibitory effect on GFAP polymerization.
Biochemical and biophysical research communications, 1999Co-Authors: Marisa Garbuglia, Marco Verzini, Richard R. Rustandi, Dirk Osterloh, David J. Weber, Volker Gerke, Rosario DonatoAbstract:Whereas native and recombinant S100A1 inhibited GFAP assembly, a truncated S100A1 lacking the last six C-terminal residues (Phe88-Ser93) (S100A1Delta88-93) proved unable to do so. The inhibitory effects of native and recombinant S100A1 on GFAP assembly were blocked by both TRTK-12, a synthetic peptide derived from the alpha-subunit of the actin capping Protein, CapZ, and a synthetic peptide derived from the tumor-suppressor Protein, p53, in a dose-dependent manner. By fluorescent spectroscopy, TRTK-12 and the p53 peptide, like GFAP and tubulin, caused a dose- and Ca2+-dependent blue-shift of the fluorescence maximum of acrylodan-S100A1. In contrast, GFAP, tubulin, TRTK-12, or the p53 peptide caused no significant changes in the fluorescence spectrum of acrylodan-S100A1Delta88-93. By chemical crosslinking, both TRTK-12 and the p53 peptide strongly reduced or blocked the formation of GFAP-S100A1 or tubulin-S100A1 complexes, respectively, and S100A1Delta88-93 was unable to complex with tubulin, whereas a remarkably reduced complexation of GFAP with the truncated Protein was observed. All the above observations show that the C-terminal extension of S100A1 is an essential part of the S100A1 site implicated in the recognition of GFAP, tubulin, p53, and the alpha-subunit of CapZ.
Vasily V. Ivanenkov - One of the best experts on this subject based on the ideXlab platform.
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Hydrophobic residues in the C-terminal region of S100A1 are essential for target Protein binding butnot for dimerization
Cell calcium, 1998Co-Authors: Dirk Osterloh, Vasily V. Ivanenkov, Volker GerkeAbstract:Abstract S100 Proteins are a family of small dimeric Proteins characterized by two EF hand type Ca 2+ binding motifs which are flanked by unique N- and C-terminal regions. Although shown unequivocally in only a few cases S100 Proteins are thought to function by binding to, and thereby regulating, cellular target Proteins in a Ca 2+ dependent manner. To describe for one member of the family, S100A1, structural requirements underlying target Protein binding, we generated specifically mutated S1 00A1 derivatives and characterized their interaction with the a subunit of the actin capping Protein CapZ shown here to represent a direct binding partner for S100A1. Chemical cross-linking, ligand blotting and fluorescence emission spectroscopy reveal that removal of, or mutations within, the sequence encompassing residues 88–90 in the unique C-terminal region of S1 00A1 interfere with binding to CapZa and to TRTK12, a synthetic CapZa peptide. The S1 00A1 sequence identified contains a cluster of three hydrophobic residues (Phe-88, Phe-89 and Trp-90) at least one of which - as revealed by qualitative phenyl Sepharose binding and hydrophobic fluorescent probe spectroscopy - is exposed on the Protein surface of Ca 2+ bound S100A1. As homologous hydrophobic residues in the closely related S100B Protein were shown by NMR spectroscopy of Ca 2+ -free S100B dimers to provide intersubunit contacts [Kilby P.M., van Eldik L.J., Roberts G.C.K. The solution structure of the bovine S100B dimer in the calcium-free state. Structure 1996; 4 : 1041–1052; Drohat A.C., Amburgey J.C., Abildgaard F., Starich M.R., Baldisseri D., Weber D.J. Solution structure of rat apo-S100B (beta beta) as determined by NMR spectroscopy. Biochemistry 1996; 35 : 11577–11588], we characterized the physical state of the various S100Al derivatives. Analytical gel filtration and chemical cross-linking show that dimer formation is not compromised in S100A1 mutants lacking residues 88–90 or containing specific amino acid substitutions in this sequence. Thus a cluster of hydrophobic residues in the C-terminal region of S100Al is essential for target Protein binding but dispensable for dimerization, a situation possibly met in other S100 Proteins as well.
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S-100 (α and β) binding peptide (TRTK-12) blocks S-100/GFAP interaction: identification of a putative S-100 target epitope within the head domain of GFAP
Biochimica et biophysica acta, 1996Co-Authors: Roberta Bianchi, Vasily V. Ivanenkov, Ruth V.w. Dimlich, Gordon A. Jamieson, Marisa Garbuglia, Marco Verzini, Ileana Giambanco, Rosario DonatoAbstract:Abstract Alignment of previously characterized S-100 (α and β)-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 (α and β) 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 Ca2+-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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Interaction of S100a0Protein with the Actin Capping Protein, CapZ: Characterization of a Putative S100a0Binding Site in CapZα-Subunit
Biochemical and biophysical research communications, 1996Co-Authors: Vasily V. Ivanenkov, Ruth V.w. Dimlich, Gordon A. JamiesonAbstract:Abstract S100a 0 , a Ca 2+ -binding Protein expressed predominantly in cardiac and skeletal muscle tissues, was demonstrated by chemical cross-linking to interact in a Ca 2+ -dependent manner with the actin capping Protein CapZ. TRTK-12, a peptide contained within the COOH-terminal region of CapZα, inhibited S100a 0 : CapZ interaction in a dose-dependent manner. TRTK-12 was shown by cross-linking to bind S100a 0 in the presence of Ca 2+ , and by fluorescence spectrophotometry to interact in a saturable manner with the anionic phospholipid and a regulator of CapZ activity, phosphatidylinositol 4-monophosphate; but not with the neutral phospholipid, phosphatidylcholine. These data suggest S100a 0 and polyphosphoinositides bind to the same COOH-terminal region of CapZα, thus potentially modulating CapZ activity.
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Characterization of S-100b Binding Epitopes. IDENTIFICATION OF A NOVEL TARGET, THE ACTIN CAPPING Protein, CapZ
The Journal of biological chemistry, 1995Co-Authors: Vasily V. Ivanenkov, Gordon A. Jamieson, Eric Gruenstein, Ruth V.w. DimlichAbstract:Abstract Short amino acid sequences that interact with the Ca binding Protein S-100b were identified by screening a bacteriophage random peptide display library. S-100b binding bacteriophages were selected by Ca-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 α-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 CapZα1 and CapZα2), was synthesized and shown by fluorescence spectrophotometry to bind S-100b in a Ca-dependent manner. Gel overlay and cross-linking experiments demonstrated the interaction of S-100b with CapZ to be Ca dependent. Moreover, this interaction was blocked by addition of TRTK-12 peptide. These results identify Ca-dependent S-100b target sequence epitopes and designate the carboxyl terminus of the α-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 α-subunit of actin capping Proteins is biologically significant.
Brenda Russell - One of the best experts on this subject based on the ideXlab platform.
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PKC epsilon signaling effect on actin assembly is diminished in cardiomyocytes when challenged to additional work in a stiff microenvironment.
Cytoskeleton (Hoboken N.J.), 2018Co-Authors: Michael A. Mkrtschjan, Christopher Solís, Admasu Y. Wondmagegn, Janki Majithia, Brenda RussellAbstract:The stiffness of the microenvironment surrounding a cell can result in cytoskeletal remodeling, leading to altered cell function and tissue macrostructure. In this study, we tuned the stiffness of the underlying substratum on which neonatal rat cardiomyocytes were grown in culture to mimic normal (10 kPa), pathological stiffness of fibrotic myocardium (100 kPa), and a nonphysiological extreme (glass). Cardiomyocytes were then challenged by beta adrenergic stimulation through isoproterenol treatment to investigate the response to acute work demand for cells grown on surfaces of varying stiffness. In particular, the PKCɛ signaling pathway and its role in actin assembly dynamics were examined. Significant changes in contractile metrics were seen on cardiomyocytes grown on different surfaces, but all cells responded to isoproterenol treatment, eventually reaching similar time to peak tension. In contrast, the assembly rate of actin was significantly higher on stiff surfaces, so that only cells grown on soft surfaces were able to respond to acute isoproterenol treatment. Forster Resonance Energy Transfer of immunofluorescence on the cytoskeletal fraction of cardiomyocytes confirmed that the molecular interaction of PKCɛ with the actin capping Protein, CapZ, was very low on soft substrata but significantly increased with isoproterenol treatment, or on stiff substrata. Therefore, the stiffness of the culture surface chosen for in vitro experiments might mask the normal signaling and affect the ability to translate basic science more effectively into human therapy.
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myofibril growth during cardiac hypertrophy is regulated through dual phosphorylation and acetylation of the actin capping Protein CapZ
Cellular Signalling, 2016Co-Authors: Chad M Warren, Jieli Li, Timothy A Mckinsey, Brenda RussellAbstract:The mechanotransduction signaling pathways initiated in heart muscle by increased mechanical loading are known to lead to long-term transcriptional changes and hypertrophy, but the rapid events for adaptation at the sarcomeric level are not fully understood. The goal of this study was to test the hypothesis that actin filament assembly during cardiomyocyte growth is regulated by post-translational modifications (PTMs) of CapZβ1. In rapidly hypertrophying neonatal rat ventricular myocytes (NRVMs) stimulated by phenylephrine (PE), two-dimensional gel electrophoresis (2DGE) of CapZβ1 revealed a shift toward more negative charge. Consistent with this, mass spectrometry identified CapZβ1 phosphorylation on serine-204 and acetylation on lysine-199, two residues which are near the actin binding surface of CapZβ1. Ectopic expression of dominant negative PKCɛ (dnPKCɛ) in NRVMs blunted the PE-induced increase in CapZ dynamics, as evidenced by the kinetic constant (Kfrap) of fluorescence recovery after photobleaching (FRAP), and concomitantly reduced phosphorylation and acetylation of CapZβ1. Furthermore, inhibition of class I histone deacetylases (HDACs) increased lysine-199 acetylation on CapZβ1, which increased Kfrap of CapZ and stimulated actin dynamics. Finally, we show that PE treatment of NRVMs results in decreased binding of HDAC3 to myofibrils, suggesting a signal-dependent mechanism for the regulation of sarcomere-associated CapZβ1 acetylation. Taken together, this dual regulation through phosphorylation and acetylation of CapZβ1 provides a novel model for the regulation of myofibril growth during cardiac hypertrophy.
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Cyclic mechanical strain of myocytes modifies CapZβ1 post translationally via PKCε
Journal of Muscle Research and Cell Motility, 2015Co-Authors: Ying-hsi Lin, Michael A. Mkrtschjan, Erik R. Swanson, Brenda RussellAbstract:The heart is exquisitely sensitive to mechanical stimuli and adapts to increased demands for work by enlarging the cardiomyocytes. In order to determine links between mechano-transduction mechanisms and hypertrophy, neonatal rat ventricular myocytes (NRVM) were subjected to physiologic strain for analysis of the dynamics of the actin capping Protein, CapZ, and its post-translational modifications (PTM). CapZ binding rates were assessed after strain by fluorescence recovery after photobleaching (FRAP) of green fluorescent Protein (GFP) expressed by a GFP-CapZβ1 adenovirus. To assess the role of the Protein kinase C epsilon isoform (PKCε), rest or cyclic strain were combined with specific PKCε activation by constitutively active PKCε, or by inhibition with dominant negative PKCε (dnPKCε) expression. Significant increases of CapZ FRAP kinetics with strain were blunted by dnPKCε, suggesting that PKCε is involved in mechano-transduction signaling. Similar combinations of strain and PKC regulation in NRVMs were studied by PTM profiles of CapZβ1 using quantitative two-dimensional gel electrophoresis. The significantly increased charge on CapZ seen with mechanical strain was reversed by the addition of dnPKCε. Potential clinical relevance was confirmed in vivo by PTMs of CapZ in the failing heart of one-year old transgenic mice over-expressing PKCε. Furthermore, with strain there was significant PKCε translocation to the Z-disc and co-localization with CapZβ1 or α-actinin, which was quantified on confocal images. A hypothetical model is presented proposing that one destination of the mechanotransduction signaling pathways might be for PTMs of CapZ thereby regulating actin capping and filament assembly.
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Cyclic mechanical strain of myocytes modifies CapZβ1 post translationally via PKCε.
Journal of Muscle Research and Cell Motility, 2015Co-Authors: Ying-hsi Lin, Michael A. Mkrtschjan, Erik R. Swanson, Brenda RussellAbstract:The heart is exquisitely sensitive to mechanical stimuli and adapts to increased demands for work by enlarging the cardiomyocytes. In order to determine links between mechano-transduction mechanisms and hypertrophy, neonatal rat ventricular myocytes (NRVM) were subjected to physiologic strain for analysis of the dynamics of the actin capping Protein, CapZ, and its post-translational modifications (PTM). CapZ binding rates were assessed after strain by fluorescence recovery after photobleaching (FRAP) of green fluorescent Protein (GFP) expressed by a GFP-CapZβ1 adenovirus. To assess the role of the Protein kinase C epsilon isoform (PKCe), rest or cyclic strain were combined with specific PKCe activation by constitutively active PKCe, or by inhibition with dominant negative PKCe (dnPKCe) expression. Significant increases of CapZ FRAP kinetics with strain were blunted by dnPKCe, suggesting that PKCe is involved in mechano-transduction signaling. Similar combinations of strain and PKC regulation in NRVMs were studied by PTM profiles of CapZβ1 using quantitative two-dimensional gel electrophoresis. The significantly increased charge on CapZ seen with mechanical strain was reversed by the addition of dnPKCe. Potential clinical relevance was confirmed in vivo by PTMs of CapZ in the failing heart of one-year old transgenic mice over-expressing PKCe. Furthermore, with strain there was significant PKCe translocation to the Z-disc and co-localization with CapZβ1 or α-actinin, which was quantified on confocal images. A hypothetical model is presented proposing that one destination of the mechanotransduction signaling pathways might be for PTMs of CapZ thereby regulating actin capping and filament assembly.
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Acetylation and Phosphorylation Post-Translational Modifications of the CapZ β1 Subunit Regulate FRAP Dynamics Leading to Myocyte Hypertrophy
Biophysical Journal, 2014Co-Authors: Ying-hsi Lin, Chad M Warren, Brenda RussellAbstract:The mechanism by which more thin filaments are built during cardiac hypertrophy is not fully understood. Very rapid increases the dynamics both actin and the actin capping Protein (CapZ) following mechanical flexing suggest that a post-translational regulation is the underlying mechanism. Neonatal rat ventricular myocytes in culture were stimulated to hypertrophy by a neurohormone (10μM phenylephrine, PE, for 24 hr). CapZ dynamics were analyzed by fluorescence recovery after photobleaching (FRAP) using CapZβ1-GFP. After PE treatment, CapZ dynamics increased above resting controls by ∼3.17 fold (p=0.0004). Post-translational modifications of CapZ were analyzed by 2D gel electrophoresis. After PE treatment, 2D spots of CapZβ1-GFP have an increased negative shift, suggesting that post-translational modification of CapZ is up regulated. To identify the types of post-translational modifications, 2D western blotting and mass spectrometry (MS) was applied. Increased post-translational spots included the acetylation of K199 and phosphorylation of S204, which are both close to the actin-binding region of CapZ. To test whether CapZ acetylation was mediated by HDAC3, located at the Z-disc of myocytes, the class I HDAC inhibitor (5μM trichostatin A / 5hr) and HDAC3 activator (10μM theophylline / 24hr) were applied. CapZ dynamics with trichostatin A increased by four-fold (p=0.01), and the effect of PE on CapZ dynamics was blunted by theophylline (p=0.09). Thus, the increased sarcomere remodeling during cardiac hypertrophy may be induced via altered acetylation of CapZ, which is mediated by HDAC3. In addition to MS identification of phosphorylation sites, post-translational modifications were reduced by dominant negative PKCɛ, suggesting a regulatory role. Together the acetyl and phospho posttranslational modifications of CapZ reduce the capping property and may increase thin filament assembly. NIH HL62426 (BR) and AHA 12PRE12050371 (Y-H L).
Rosario Donato - One of the best experts on this subject based on the ideXlab platform.
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Role of the C-terminal extension in the interaction of S100A1 with GFAP, tubulin, the S100A1- and S100B-inhibitory peptide, TRTK-12, and a peptide derived from p53, and the S100A1 inhibitory effect on GFAP polymerization.
Biochemical and biophysical research communications, 1999Co-Authors: Marisa Garbuglia, Marco Verzini, Richard R. Rustandi, Dirk Osterloh, David J. Weber, Volker Gerke, Rosario DonatoAbstract:Whereas native and recombinant S100A1 inhibited GFAP assembly, a truncated S100A1 lacking the last six C-terminal residues (Phe88-Ser93) (S100A1Delta88-93) proved unable to do so. The inhibitory effects of native and recombinant S100A1 on GFAP assembly were blocked by both TRTK-12, a synthetic peptide derived from the alpha-subunit of the actin capping Protein, CapZ, and a synthetic peptide derived from the tumor-suppressor Protein, p53, in a dose-dependent manner. By fluorescent spectroscopy, TRTK-12 and the p53 peptide, like GFAP and tubulin, caused a dose- and Ca2+-dependent blue-shift of the fluorescence maximum of acrylodan-S100A1. In contrast, GFAP, tubulin, TRTK-12, or the p53 peptide caused no significant changes in the fluorescence spectrum of acrylodan-S100A1Delta88-93. By chemical crosslinking, both TRTK-12 and the p53 peptide strongly reduced or blocked the formation of GFAP-S100A1 or tubulin-S100A1 complexes, respectively, and S100A1Delta88-93 was unable to complex with tubulin, whereas a remarkably reduced complexation of GFAP with the truncated Protein was observed. All the above observations show that the C-terminal extension of S100A1 is an essential part of the S100A1 site implicated in the recognition of GFAP, tubulin, p53, and the alpha-subunit of CapZ.
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S-100 (α and β) binding peptide (TRTK-12) blocks S-100/GFAP interaction: identification of a putative S-100 target epitope within the head domain of GFAP
Biochimica et biophysica acta, 1996Co-Authors: Roberta Bianchi, Vasily V. Ivanenkov, Ruth V.w. Dimlich, Gordon A. Jamieson, Marisa Garbuglia, Marco Verzini, Ileana Giambanco, Rosario DonatoAbstract:Abstract Alignment of previously characterized S-100 (α and β)-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 (α and β) 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 Ca2+-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.
Gordon A. Jamieson - One of the best experts on this subject based on the ideXlab platform.
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S-100 (α and β) binding peptide (TRTK-12) blocks S-100/GFAP interaction: identification of a putative S-100 target epitope within the head domain of GFAP
Biochimica et biophysica acta, 1996Co-Authors: Roberta Bianchi, Vasily V. Ivanenkov, Ruth V.w. Dimlich, Gordon A. Jamieson, Marisa Garbuglia, Marco Verzini, Ileana Giambanco, Rosario DonatoAbstract:Abstract Alignment of previously characterized S-100 (α and β)-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 (α and β) 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 Ca2+-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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Interaction of S100a0Protein with the Actin Capping Protein, CapZ: Characterization of a Putative S100a0Binding Site in CapZα-Subunit
Biochemical and biophysical research communications, 1996Co-Authors: Vasily V. Ivanenkov, Ruth V.w. Dimlich, Gordon A. JamiesonAbstract:Abstract S100a 0 , a Ca 2+ -binding Protein expressed predominantly in cardiac and skeletal muscle tissues, was demonstrated by chemical cross-linking to interact in a Ca 2+ -dependent manner with the actin capping Protein CapZ. TRTK-12, a peptide contained within the COOH-terminal region of CapZα, inhibited S100a 0 : CapZ interaction in a dose-dependent manner. TRTK-12 was shown by cross-linking to bind S100a 0 in the presence of Ca 2+ , and by fluorescence spectrophotometry to interact in a saturable manner with the anionic phospholipid and a regulator of CapZ activity, phosphatidylinositol 4-monophosphate; but not with the neutral phospholipid, phosphatidylcholine. These data suggest S100a 0 and polyphosphoinositides bind to the same COOH-terminal region of CapZα, thus potentially modulating CapZ activity.
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Characterization of S-100b Binding Epitopes. IDENTIFICATION OF A NOVEL TARGET, THE ACTIN CAPPING Protein, CapZ
The Journal of biological chemistry, 1995Co-Authors: Vasily V. Ivanenkov, Gordon A. Jamieson, Eric Gruenstein, Ruth V.w. DimlichAbstract:Abstract Short amino acid sequences that interact with the Ca binding Protein S-100b were identified by screening a bacteriophage random peptide display library. S-100b binding bacteriophages were selected by Ca-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 α-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 CapZα1 and CapZα2), was synthesized and shown by fluorescence spectrophotometry to bind S-100b in a Ca-dependent manner. Gel overlay and cross-linking experiments demonstrated the interaction of S-100b with CapZ to be Ca dependent. Moreover, this interaction was blocked by addition of TRTK-12 peptide. These results identify Ca-dependent S-100b target sequence epitopes and designate the carboxyl terminus of the α-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 α-subunit of actin capping Proteins is biologically significant.
