Marc Duchesne

A Ras-GTPase-activating protein SH3-domain-binding protein

We report the purification of a Ras-GTPase-activating protein (GAP)-binding protein, G3BP, a ubiquitously expressed cytosolic 68-kDa protein that coimmunoprecipitates with GAP. G3BP physically associates with the SH3 domain of GAP, which previously had been shown to be essential for Ras signaling. The G3BP cDNA revealed that G3BP is a novel 466-amino-acid protein that shares several features with heterogeneous nuclear RNA-binding proteins, including ribonucleoprotein (RNP) motifs RNP1 and RNP2, an RG-rich domain, and acidic sequences. Recombinant G3BP binds effectively to the GAP SH3 domain G3BP coimmunoprecipitates with GAP only when cells are in a proliferating state, suggesting a recruitment of a GAP-G3BP complex when Ras is in its activated conformation.

A Role for Sam68 in Cell Cycle Progression Antagonized by a Spliced Variant within the KH Domain

Sam68 is the main tyrosine-phosphorylated and Src-associated protein in mitotic cells. Sam68 exhibits a conserved functional KH (hnRNPK homology) RNA binding domain and binds single strand nucleic acids. Tyrosine phosphorylation mediates the interaction of Sam68 with many SH3- and SH2-containing proteins and negatively regulates its nucleic acid binding properties. But the function and the impact of Sam68 on cell signaling and cell proliferation remains elusive. We report here the identification of a natural isoform of Sam68 with a deletion within the KH domain. This isoform, called Sam68ΔKH, is specifically expressed at growth arrest upon confluency in normal cells. In cells that do not enter quiescence at confluency such as Src-transformed cells, no recruitment of Sam68ΔKH is observed. Transfected Sam68ΔKH inhibits serum-induced DNA synthesis and cyclin D1 expression. Sam68 overcomes these effects, suggesting that isoforms of Sam68 are involved, through KH domain signaling, in cell proliferation, and more precisely in G1/S transition.

Constrained analogs of KCVFM with improved inhibitory properties against farnesyl transferase

Abstract Constrained analogs of KCVFM, reported thus far as one of the most active peptidic inhibitors of farnesyl transferase, have been synthesized. Replacement of Val-Phe with Val-Tic and (N-Me)Val-Tic led to dramatically more active analogs possessing favored extended conformations. Based on molecular modelling studies the design and synthesis of various conformational probes to be substituted for Val and Phe led to a good correlation between the ratio of extended conformers and biological activity.

Intracellular expression and functional properties of an anti-p21Ras scFv derived from a rat hybridoma containing specific λ and irrelevant κ light chains

Abstract A rat single-chain Fv (Y238 scFv) was derived from the Y13–238 monoclonal antibody, a non-neutralizing anti-Ras antibody. The Y13–238 hybridoma expresses two functional light chains. N-terminus microsequencing of these chains showed the presence of the Y3 Ag1.2.3 Vk chain derived from the rat fusion partner and of a rat Vl chain. Primers designed for rat Vl amplification allowed the cloning of a functional scFv that could bind p21Ras. The kinetics of interaction of purified Y238 scFv with the p21Ras protein was evaluated by BIAcore® with a NTA sensor chip and gave an apparent affinity constant in the nanomolar range (KD=4.58±0.63 nM). Immunoprecipitation experiments of Y238 scFv expressed in Xenopus laevis oocytes confirmed the specificity of the scFv for the Ras protein. Y238 scFv could be intracellularly expressed in oocytes and in mammaliam cells without adverse effect on the Ras signalling cascade. This scFv was therefore used as control in experiments where another anti-Ras scFv (Y259 scFv, derived from the neutralizing anti-Ras mAb Y13–259) blocked the Ras pathway in vitro and led to tumor regression in a nude mouse model [Cochet, O., Kenigsberg, M., Delumeau, I., Virone-Oddos, A., Multon, M.C., Fridman, W.H., Schweighoffer, F., Teillaud, J.L., Tocque, B., 1998. Intracellular expression of an antibody fragment-neutralizing p21 ras promotes tumor regression. Cancer Res. 58, 1170–1176.]. Finally, BIAcore® analyses indicated that the epitopes recognized by Y238 and Y259 scFvs are not overlapping and allowed a more precise definition of the Y13–238 epitope.

Local constrained shifty pseudopeptides inhibitors of rasfarnesyl transferase

Abstract Pseudopeptide analogues related to the C-terminal tetrapeptide of ras-protein (Cys-Val-X-Met) were synthesized and evaluated for inhibition of ras farnesyl transferase (FTase). We demonstrate that the introduction of a shifty amino acid related to Cys instead of Cys-Val and a tetrahydroisoquinoline carboxylic acid (TIC) instead of Phe lead to potent inhibitors of FTase on isolated enzyme or on cell based tests. One of the pseudopeptides, conceived as a prodrug, suppressed specifically the ability of ras transformed cells to form colonies in soft agar.

Synthesis and conformational analysis of peptide inhibitors of farnesyltransferase

Farnesylation of the ras oncogene product by Farnesyl Transferase (FTase) is known to be a critical step in cell transformation leading to uncontrolled proliferation. The peptide CysValTicMet is a potent FTase inhibitor, but its degradation by amino-peptidases and its only weak internalization into cells make it a bad candidate for a future cancer drug. We have prepared improved CysValTicMet analogues using several approaches: (i) amino terminal modifications or introduction of pseudopeptides or non-natural amino acids to increase proteolytic stability, (ii) introduction of hydrophobic aliphatic chains to increase cell internalization and metabolic stability and (iii) transformation into prodrugs. Additionally, we have carried out comparative conformational analysis studies by molecular dynamics of some of the here presented peptides and of our recently described peptidomimetic inhibitors of FTase.

Monoclonal antibodies against synovial collagenase: use for immunopurification and characterization of the latent and active enzyme.

Monoclonal antibodies have been produced against porcine synovial collagenase which recognize both the active enzyme and its inactive precursor. These antibodies inhibited the collagenolytic activity of collagenase, but not its activity with a synthetic peptide substrate. The antibodies were also able to recognize human synovial, human skin fibroblast and human chondrocyte collagenase but not the enzyme from human granulocytes. One of the monoclonal antibodies was successfully used for the immunopurification of the porcine enzyme and these experiments led to the demonstration of an endogenous activator of procollagenase in the synovial cell culture medium.

From Random Screening of Chemical Libraries to the Optimization of FPP-Competitive Inhibitors of Farnesyltransferase

Together with gene alterations of the p53 tumor suppressor gene, mutations of the ras genes represent the most frequent gene modification umancancers. Ras mutations are found in at least 90% of pancreas, 50% of colon, and 30% of both lung and thyroid cancers (1,2).