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This paper introduces a flexible assay for characterizing the activities of the histone deacetylase enzymes. The approach combines mass spectrometry with self-assembled monolayers that present acetylated peptides and enables a label-free and one-Step assay of this biochemical activity. The assay was used to characterize the activity of HDAC8 toward peptides taken from the N-terminal tail of the H4 histone and reveals that a distal region of the peptide substrate interacts with the deacetylase at an exosite and contributes to the activity of the substrate. Specifically, a peptide corresponding to residues 8-19 of H4 and having lysine 12 acetylated is an active substrate, but removal of the KRHR (residues 16-19) sequence abolishes activity. Mutation of glycine 11 to arginine in the peptide lacking the KRHR sequence restores activity, demonstrating that both local and distal sequences act synergistically to regulate the activity of the HDAC. Assays with peptides bearing multiply acetylated residues, but in which each acetyl group is isotopically labeled, permit studies of the processive deacetylation of peptides. Peptide substrates having an extended sequence that includes K20 were used to demonstrate that methylation of this residue directly affects HDAC8 activity at K12. This work provides a mechanistic basis for the regulation of HDAC activities by distal sequences and may contribute to studies aimed at evaluating the role of the histone code in regulating gene expression. [ABSTRACT FROM AUTHOR]

This work reports on the role of the synergy peptide PHSRN in mediating the adhesion of cells. The attachment of baby hamster kidney cells and 3T3 Swiss fibroblasts to model substrates presenting either GRGDS or PHSRN was evaluated using self-assembled monolayers of alkanethiolates on gold presenting the peptide ligands mixed with tri(ethylene glycol) groups. These substrates permit rigorous control over the structures and densities of peptide ligands and at the same time prevent nonspecific interactions with adherent cells. Both cell types attached efficiently to monolayers presenting either the RGD or the PHSRN peptide but not to monolayers presenting scrambled peptide GRDOS or HRPSN. Cell attachment was comparable on substrates presenting either peptide ligand but less efficient than on substrates presenting the protein fibronectin. The degree of cell spreading, however, was substantially higher on substrates presenting RGD relative to PHSRN. Staining of 3T3 fibroblasts with anti-vinculin and phalloidin revealed clear cytoskeletal filaments and focal adhesions for cells attached by way of either RGD or PHSRN. Inhibition experiments showed that the attachment of 3T3 fibroblasts to monolayers presenting RGD could be inhibited completely by a soluble RGD peptide and partially by a soluble PHSRN peptide. IMR 90 fibroblast attachment to monolayers presenting PHSRN could be inhibited with anti-integrin α5 or anti-integrin β antibody. This work demonstrates unambiguously that PHSRN alone can support the attachment of cells and that the ROD and PHSRN bind competitively to the integrin receptors. [ABSTRACT FROM AUTHOR]

The adhesion of mammalian cells is mediated by the binding of cell-surface integrin receptors to peptide ligands from the extracellular matrix and the clustering of these receptors into focal adhesion complexes. This paper examines the effect of one mechanistic variable, ligand affinity, on the assembly of focal adhesions (FAs) in order to gain mechanistic insight into this process. This study uses self- assembled monolayers of alkanethiolates on gold as a substrate to present either a linear or cyclic Arg- Gly-Asp peptide at identical densities. Inhibition assays showed that the immobilized cyclic RGD is a higher affinity ligand than linear RGD. 3T3 Swiss fibroblasts attached to substrates presenting the cyclic peptide at twice the rate they attached to substrates presenting the linear peptide. Quantitation of focal adhesions revealed that cells on cyclic RGD had twice the number of FAs as did cells on linear RGD and that these focal adhesions were on average smaller. These findings show that affinity affects the assembly of integrins into focal adhesions and support a model based on competing rates of nucleation and growth of FAs to explain the change in distribution of FAs with ligand affinity. This study is important because it provides a model system that is well-suited for biophysical studies of integrin-mediated cell adhesion and reveals insight into one mechanism utilized by cells to perceive environmental changes. [ABSTRACT FROM AUTHOR]

Studies the sequence specificity of the natural product distamycin. Synthesis of pyridine-2-carboxamidonetropsin and 1-methylimidazole-2-carboxamidonetropsin; Equilibrium association constants for three peptides at four different binding sites; Use of a high-resolution footprinting reagent for accommodating closely-spaced binding sites; Methods for the alteration of relative specificity.