Wang-Qing Liu

A Sos-derived peptidimer blocks the Ras signaling pathway by binding both Grb2 SH3 domains and displays antiproliferative activity

With the aim of interrupting the growth factor‐stimulated Ras signaling pathway at the level of the Grb2–Sos interaction, a peptidimer, made of two identical proline‐rich sequences from Sos linked by a lysine spacer, was designed using structural data from Grb2 and a proline‐rich peptide complexed with its SH3 domains. The peptidimer affinity for Grb2 is 40 nM whereas that of the monomer is 16 μM, supporting the dual recognition of both Grb2 SH3 domains by the dimer. At 50 nM, the peptidimer blocks selectively Grb2–Sos complexation in ER 22 (CCL 39 fibroblasts overexpressing epidermal growth factor receptor) cellular extracts. The peptidimer specifically recognizes Grb2 and does not interact with PI3K or Nck, two SH3 domam‐containing adaptors. The peptidimer was modified to enter cells by coupling to a fragment of Antennapedia homeo‐domain. At 10 μM, the conjugate inhibits the Grb2–Sos interaction (100%) and MAP kinase (ERK1 and ERK2) phosphorylation (60%) without modifying cellular growth of ER 22 cells. At the same concentration, the conjugate also inhibits both MAP kinase activation induced by nerve growth factor or epidermal growth factor in PC12 cells, and differentiation triggered by nerve growth factor. Finally, when tested for its antiproliferative activity, the conjugate was an efficient inhibitor of the colony formation of transformed NIH3T3/HER2 cells grown in soft agar, with an IC50 of around 1 μM. Thus, the designed peptidimers appear to be interesting leads to investigate signaling and intracellular processes and for designing selective inhibitors of tumorigenic Ras‐dependent processes.—Cussac, D., Vidal, M., LePrince, C., Liu, W.‐Q., Cornille, F., Tiraboschi, G., Roques, B. P., Garbay, C. A Sos‐derived peptidimer blocks the Ras signaling pathway by binding both Grb2 SH3 domains and displays antiproliferative activity. FASEB J. 13, 31–38 (1999)

The synaptic acetylcholinesterase tetramer assembles around a polyproline II helix

Functional localization of acetylcholinesterase (AChE) in vertebrate muscle and brain depends on interaction of the tryptophan amphiphilic tetramerization (WAT) sequence, at the C‐terminus of its major splice variant (T), with a proline‐rich attachment domain (PRAD), of the anchoring proteins, collagenous (ColQ) and proline‐rich membrane anchor. The crystal structure of the WAT/PRAD complex reveals a novel supercoil structure in which four parallel WAT chains form a left‐handed superhelix around an antiparallel left‐handed PRAD helix resembling polyproline II. The WAT coiled coils possess a WWW motif making repetitive hydrophobic stacking and hydrogen‐bond interactions with the PRAD. The WAT chains are related by an ∼4‐fold screw axis around the PRAD. Each WAT makes similar but unique interactions, consistent with an asymmetric pattern of disulfide linkages between the AChE tetramer subunits and ColQ. The P59Q mutation in ColQ, which causes congenital endplate AChE deficiency, and is located within the PRAD, disrupts crucial WAT–WAT and WAT–PRAD interactions. A model is proposed for the synaptic AChET tetramer.

STAT3-Stathmin Interactions Control Microtubule Dynamics in Migrating T-cells

T-cell migration is a complex highly coordinated process that involves cell adhesion to the high endothelial venules or to the extracellular matrix by surface receptor/ligand interactions, cytoskeletal rearrangements, and phosphorylation-dependent signaling cascades. The mechanism(s) that regulates T-cell migration is of considerable relevance for understanding the pathogenesis of various diseases, such as chronic inflammatory diseases and cancer metastasis. This study was designed to identify potential involvement of STAT3, a latent transcription factor, in mediating integrin-induced T-cell migration. Using our previously characterized in vitro model for lymphocyte migration, we demonstrate that STAT3 is activated and translocated to the nucleus during the process of active motility of Hut78 T-lymphoma cells triggered via LFA-1. Blocking STAT3 signaling by multiple approaches inhibited LFA-1-induced T-cell locomotion via destabilization of microtubules and post-translational modification of tubulin. Here, we show that STAT3 physically interacts with stathmin to regulate microtubule dynamics in migrating T-cells. These observations strongly indicate that STAT3 is critically important for T-cell migration and associated signaling events.

Assembly of Acetylcholinesterase Tetramers by Peptidic Motifs from the Proline-rich Membrane Anchor, PRiMA COMPETITION BETWEEN DEGRADATION AND SECRETION PATHWAYS OF HETEROMERIC COMPLEXES

The membrane-bound form of acetylcholinesterase (AChE) constitutes the major component of this enzyme in the mammalian brain. These molecules are hetero-oligomers, composed of four AChE catalytic subunits of type T (AChET), associated with a transmembrane protein of type 1, called PRiMA (proline-rich membrane anchor). PRiMA consists of a signal peptide, an extracellular domain that contains a proline-rich motif (14 prolines with an intervening leucine, P4LP10), a transmembrane domain, and a cytoplasmic domain. Expression of AChET subunits in transfected COS cells with a truncated PRiMA, without its transmembrane and cytoplasmic domains (Pstp54 mutant), produced secreted heteromeric complexes (T4-Pstp54), instead of membrane-bound tetramers. In this study, we used a series of deletions and point mutations to analyze the interaction between the extracellular domain of PRiMA and AChET subunits. We confirmed the importance of the polyproline stretches and defined a peptidic motif (RP4LP10RL), which induces the assembly and secretion of a heteromeric complex with four AChET subunits, nearly as efficiently as the entire extracellular domain of PRiMA. It is noteworthy that deletion of the N-terminal segment preceding the prolines had little effect. Interestingly, short PRiMA mutants, truncated within the proline-rich motif, reduced both cellular and secreted AChE activity, suggesting that their interaction with AChET subunits induces their intracellular degradation.

Grb2‐SH3 ligand inhibits the growth of HER2+ cancer cells and has antitumor effects in human cancer xenografts alone and in combination with docetaxel

HER2 represents an important signaling pathway in breast and other cancers. Herceptin has demonstrated clinical activity, but resistance is common. Thus, new therapeutic approaches to the HER2 pathway are needed. Grb2 is an adaptor protein involved in Ras‐dependent signaling induced by HER2 receptors. A specific Grb2‐SH3 ligand, designed and synthesized in our laboratory, called peptidimer‐c, inhibited colony formation in HER2 overexpressing SKBr3 cancer cells. Combined treatment of peptidimer‐c with docetaxel further inhibited both colony formation and tumor cell survival compared to docetaxel treatment alone. Efficacy of this combined treatment was correlated with a reduction in the phosphorylation of MAPK and AKT. Finally, peptidimer‐c reduced the growth of a HER2+ human breast cancer (BK111) xenograft in nude mice and potentiated the antitumor effect of docetaxel in a HER2+ hormone‐independent human prostate adenocarcinoma (PAC120 HID28) xenograft. These results validate Grb2 as a new target for the HER2 pathway.

Synthesis of constrained 4-(phosphonomethyl)phenylalanine derivatives as hydrolytically stable analogs of O-phosphotyrosine

Abstract In order to elucidate the role of protein tyrosine phosphorylation involved in various intracellular signaling pathways, peptides containing O -phosphotyrosine have been developed. However, in order to improve the stability of the phosphorylated amino acid, we have designed some years ago a hydrolytically stable analogue, the 4-(phosphonomethyl)phenylalanine (Pmp). Introduced in peptide sequences, this residue, which is resistant to phosphatase action, was shown also able to inhibit substrate recognition by protein targets. With the aim to design peptidomimetics endowed with improved affinity and selectivity, we report in this study the synthesis of five new sterically hindered amino acids derived from Pmp. These modifications include α-methyl, β-methyl, β,β-dimethyl substitutions, α,β-cyclization of Pmp and methyl substitution on the phosphomomethyl group of Pmp.

Inhibition of the ras-dependent mitogenic pathway by phosphopeptide prodrugs with antiproliferative properties.

Phosphopeptide prodrugs bearing two S-acyl-2-thioethyl (SATE) biolabile phosphate protections were developed. They are capable to inhibit the Shc/Grb2 interaction and MAP kinases (ERK1 and ERK2) phosphorylation in cellular assay. The S-acetyl-2-thioethyl (MeSATE) analogue showed an IC50 of 1 microM in the inhibition of the colony formation of tumor cell line NIH3T3/HER2.

ENANTIOSELECTIVE SYNTHESIS OF N-FMOC PROTECTED DI-TERT-BUTYL 4-PHOSPHONOMETHYL-L-PHENYLALANINE: A HYDROLYTICALLY STABLE ANALOGUE OF O-PHOSPHOTYROSINE

Abstract Fmoc-L-Pmp(tBu) 2 -OH was prepared with high enantiomeric purity by an asymmetric synthetic pathway, using a camphor sultam as chiral auxiliary.

Synthesis of L-2,3,5,6-tetrafluoro-4-(phosphonomethyl) phenylalanine, a novel non-hydrolyzable phosphotyrosine mimetic and L-4-(phosphonodifluoromethyl)phenylalanine

A new non-hydrolyzable phosphotyrosine analogue, L-F4Pmp and its N-Fmoc protected derivative were prepared by using an enantioselective synthetic pathway with camphor sultam as chiral auxiliary. The side chain pKa2 (6.9) of L-F4Pmp was determined. A new synthesis of L-F2Pmp was also described.

Enantioselective synthesis of (2S)-2-(4-phosphonophenylmethyl)-3-aminopropanoic acid suitably protected for peptide synthesis

Abstract Protected d -β-2-(4-phosphono) phenylalanine was prepared by a multistep synthesis, involving the diastereoselective alkylation of a chiral enolate. This new compound can be further incorporated into peptidic backbone by means of solid-phase peptide synthesis in order to synthesize short peptides adopting β-turn or α-helix conformations.