Nihal U. Obeyesekere

Tight-binding inhibitory sequences against pp60(c-src) identified using a random 15-amino-acid peptide library

A bacteriophage peptide library containing a random 15‐amino‐acid insert was screened for identification of peptide sequence(s) that bind pp60 c−src . Sequencing the random insert from more than 100 virions indicated that more than 60% of the phage virions that bound to this enzyme contained a GXXG sequence motif in which X was frequently a hydrophobic residue. The GXXG sequence was often repeated as GXXGXXG. Two nonameric peptides were synthesized to determine whether or not the peptide inhibits pp60 c−src tyrosine kinase activity and the importance of the glycine residues within this sequence. The peptide containing glycine had a K i of μM, whereas replacing the glycines with proline increased the K i value to 3.1 mM.

p202 self-associates through a sequence conserved among the members of the 200-family proteins.

Murine p202 is an interferon‐inducible primarily nuclear phosphoprotein (52 kDa) whose expression in transfected cells inhibits colony formation. p202‐binding proteins include the pocket proteins (pRb, p107 and p130), a p53‐binding protein (sm53BP1), and transcription factors (e.g. NF‐κB (p50 and p65), AP‐1 (c‐Fos and c‐Jun), E2F‐1, E2F‐4, MyoD, and myogenin). p202 modulates the transcriptional activity of these factors in transfected cells. Here we demonstrate that p202 self‐associates directly and a sequence in p202, which is conserved among the members of the 200‐family proteins, was sufficient for self‐association in vitro. Our observations reported herein raise the possibility that self‐association of p202 may provide a mechanism for the regulation of its activity.

Use of synthetic peptides and copolymers to study the substrate specificity and inhibition of the protein tyrosine kinase pp60c-src

The ability of synthetic peptides and polypeptides to act as substrates and/or inhibitors of pp60c-src was examined. The random copolymer, poly(K4Y) had a threefold lower specificity than poly(E4Y). Peptides containing lysine vs. glutamate were also found to have a lower substrate specificity (Vmax:Km ratio). In order to assess the substrate specificity of acidic peptides, an assay protocol using DEAE-membranes was developed. Peptides containing a (YXE)5YXD motif (X = G, A, V, P, or norvaline) were tested as inhibitors and substrates of pp60c-src. The glycine-containing peptide was the best substrate having a specificity 16,000-fold higher than 5Val-angiotensin II, the most commonly used peptide substrate. Most of the peptides, except for the proline containing peptide, had Ki values of 20-100 microM. In a series of (XGE)5XGD peptides, where X = Y or F, tyrosine at position 10 was found to be the preferred site for accepting a phosphate. Analogs in which the glycine was replaced with alanine indicated that loss of flexibility around position 10 was detrimental to substrate specificity. Results suggest that conformational requirements of the peptides tested was important and substrate specificity was a more sensitive parameter than binding as measured by Ki values.

Enhanced phagocytosis activity of cyclic analogs of tuftsin

Cyclic analogs of the physiological immunostimulating peptide tuftsin (Thr-Lys-Pro-Arg), cyclo(Thr-Lys-Pro-Arg-Gly) (ctuf-G) and cyclo(Thr-Lys-Pro-Arg-Asp) (ctuf-D), were synthesized based on molecular modeling studies, and assayed for the ability to stimulate phagocytosis by human polymorphonuclear leukocytes. As predicted, the synthesis of ctuf-D resulted in two isomers with the correct molecular mass and amino acid composition. In phagocytosis assays, tuftsin, ctuf-G and two isomers of ctuf-D showed the usual bell-shaped activity profiles. The optimum concentration of ctuf-G was 50-fold less than that of tuftsin, whereas the degree of stimulation was similar. One isomer of ctuf-D was almost inactive, and the other ctuf-D exhibited the same degree of phagocytosis as tuftsin but its optimum concentration was 5-fold lower. The enhanced potency of ctuf-G and one isomer of ctuf-D may be due to conformational effects and/or to the possibility that these cyclic peptides are resistant to proteolytic degradation.

Stereospecific synthesis of 4-carboxyphenylalanine and derivatives for use in Fmoc-based solid-phase peptide synthesis

Abstract Starting from N α -benzyloxycarbonyl-L-tyrosine( O -triflate)benzyl ester, we have prepared enantiomerically pure 4-carboxyphenylalanine and 4-methoxycarbonylphenylalanine derivatives using palladium (0) catalyzed carbonylation reactions. These unnatural amino acids were suitably protected and were used in solid-phase peptide synthesis using the Fmoc/ t -butyl approach.