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Grb7 is an adapter protein found to be overexpressed in several breast and other cancer cell types along with ErbB2. Grb7 is normally an interaction partner with focal adhesion kinase and in cancer cells also aberrantly interacts with ErbB2. It is thus implicated in the migratory and proliferative potential of cancer cells. Previous studies have shown that the phage display-derived cyclic nonphosphorylated inhibitor peptide, G7-18NATE, when linked to Penetratin, is able to interfere with the interaction of Grb7 with its upstream binding partners and to impact on both cell migration and proliferation. Here we report the synthesis of a biotinylated G7-18NATE covalently attached to just the last seven residues of Penetratin (G7-18NATE-P-Biotin). We demonstrate that this construct is taken up efficiently into MDA-MB-468 breast cancer cells and colocalizes with Grb7 in the cytoplasm. We also used isothermal titration calorimetry to determine the binding affinity of G7-18NATE-P-Biotin to the Grb7-SH2 domain, and showed that it binds with micromolar affinity (K(d) = 14.4 microM), similar to the affinity of G7-18NATE (K(d) = 35.4 microM). Together this shows that this shorter G7-18NATE-P-Biotin construct is suitable for further studies of the antiproliferative and antimigratory potential of this inhibitor.

The membrane interaction of two beta peptides was studied using a surface plasmon resonance biosensor. The two peptides are beta-17, a novel beta-amino acid based antimicrobial peptide and the corresponding scrambled-beta17--a non-antimicrobial beta-peptide analogue. Membrane interaction studies were performed with a series of phospholipid mixtures which mimic either mammalian cells (high in phosphatidylcholine and cholesterol) or microbial cells (high in phosphatidylethanolamine and phosphatidylglycerol). The results were compared with the membrane binding of the well-characterized antimicrobial peptide magainin 2. The secondary structure of these peptides were also determined in each lipid mixture by circular dichroism and correlated with the membrane-binding properties. Both beta-17 and the scrambled peptide have the same peptide length, charge and showed a similar secondary structure in both aqueous buffer and in the presence of liposomes. Both peptides also bound to a similar level on each membrane mixture, showing that the dramatic difference in biological activity is not based on the amount of peptide bound but rather differences in the degree of insertion and rate of membrane dissociation. Although beta-17 and the scrambled beta-17 peptide exhibited similar binding properties on all membrane mimics, both beta-peptides bound more to all membranes compared with magainin 2. Overall, the results further reveal the significant interplay between peptide amphipathicity and secondary structure induction on membrane binding., (Copyright 2009 Wiley Periodicals, Inc.)

The aggregation of the amyloid-beta-protein (Abeta) is an important step in the pathogenesis of Alzheimer's disease. As Abeta fibrils are not found in all brain regions, endogenous factors may influence Abeta fibril formation. In this study, atomic force microscopy was used to investigate the role of surface phenomena in directing amyloid aggregation. Abeta1-40 was applied to a surface of highly oriented pyrolytic graphite at a concentration of 0.5 microM. Steps formed by edge-plane surface defects on the graphite were found to act as a template to promote the assembly of Abeta into fibrils. Initially, after being deposited on the graphite surface, Abeta had a uniform beaded morphology. However, after incubating (aging) the Abeta on the surface for several hours, the Abeta assembled along step edges to form linear aggregates. After more prolonged incubation, the linear Abeta aggregates fused to form mature fibrils with a distinctive helical morphology. The results demonstrate that surface interactions can promote the aggregation of Abeta into amyloid fibrils and they suggest that similar interactions could promote amyloid aggregation in vivo., ((c) 2006 Wiley Periodicals, Inc.)