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A RasGAP SH3 peptide aptamer inhibits RasGAP-Aurora interaction and induces caspase-independent tumor cell death.

Pamonsinlapatham P, Hadj-Slimane R, Raynaud F, Bickle M, Corneloup C, Barthelaix A, Lepelletier Y, Mercier P, Schapira M, Samson J, Mathieu AL, Hugo N, Moncorgé O, Mikaelian I, Dufour S, Garbay C, Colas P - PLoS ONE (2008)

Bottom Line: However, RasGAP also acts as a positive effector of Ras and exerts an anti-apoptotic activity that is independent of its GAP function and that involves its SH3 (Src homology) domain.It disrupts the interaction between RasGAP and Aurora B kinase.This work identifies the above-mentioned pocket as an interesting therapeutic target to pursue and points its cognate peptide aptamer as a promising guide to discover RasGAP small-molecule drug candidates.

View Article: PubMed Central - PubMed

Affiliation: UFR Biomédicale, Laboratoire de Pharmacochimie Moléculaire et Cellulaire, INSERM U648, Université Paris Descartes, Paris, France.

ABSTRACT
The Ras GTPase-activating protein RasGAP catalyzes the conversion of active GTP-bound Ras into inactive GDP-bound Ras. However, RasGAP also acts as a positive effector of Ras and exerts an anti-apoptotic activity that is independent of its GAP function and that involves its SH3 (Src homology) domain. We used a combinatorial peptide aptamer approach to select a collection of RasGAP SH3 specific ligands. We mapped the peptide aptamer binding sites by performing yeast two-hybrid mating assays against a panel of RasGAP SH3 mutants. We examined the biological activity of a peptide aptamer targeting a pocket delineated by residues D295/7, L313 and W317. This aptamer shows a caspase-independent cytotoxic activity on tumor cell lines. It disrupts the interaction between RasGAP and Aurora B kinase. This work identifies the above-mentioned pocket as an interesting therapeutic target to pursue and points its cognate peptide aptamer as a promising guide to discover RasGAP small-molecule drug candidates.

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Related in: MedlinePlus

in vitro binding of peptide aptamers to RasGAP SH3.Pull-down assay with recombinant purified RasGAP SH3. Recombinant purified 6xHis-RasGAP SH3 was added to GST-peptide aptamer fusion proteins, coupled to glutathion-sepharose beads. “Control” is a peptide aptamer that was selected against another target protein. Captured protein was revealed with an anti-His antibody. GST-aptamer fusions coupled to beads were stained with Coomassie blue.
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pone-0002902-g002: in vitro binding of peptide aptamers to RasGAP SH3.Pull-down assay with recombinant purified RasGAP SH3. Recombinant purified 6xHis-RasGAP SH3 was added to GST-peptide aptamer fusion proteins, coupled to glutathion-sepharose beads. “Control” is a peptide aptamer that was selected against another target protein. Captured protein was revealed with an anti-His antibody. GST-aptamer fusions coupled to beads were stained with Coomassie blue.

Mentions: To confirm some of the yeast two-hybrid phenotypes, we performed pull-down experiments using 6 GST-RasGAP aptamer fusion proteins and recombinant purified 6xHis-RasGAP SH3. We confirmed the binding between RasGAP SH3 and all 6 peptide aptamers tested, with highly variable apparent binding affinities that were not determined by the amounts of GST-aptamer fusion proteins coupled to glutathione-sepharose beads and that did not match the intensities of the yeast two-hybrid phenotypes observed in the yeast two-hybrid mating assay (Figure 2).


A RasGAP SH3 peptide aptamer inhibits RasGAP-Aurora interaction and induces caspase-independent tumor cell death.

Pamonsinlapatham P, Hadj-Slimane R, Raynaud F, Bickle M, Corneloup C, Barthelaix A, Lepelletier Y, Mercier P, Schapira M, Samson J, Mathieu AL, Hugo N, Moncorgé O, Mikaelian I, Dufour S, Garbay C, Colas P - PLoS ONE (2008)

in vitro binding of peptide aptamers to RasGAP SH3.Pull-down assay with recombinant purified RasGAP SH3. Recombinant purified 6xHis-RasGAP SH3 was added to GST-peptide aptamer fusion proteins, coupled to glutathion-sepharose beads. “Control” is a peptide aptamer that was selected against another target protein. Captured protein was revealed with an anti-His antibody. GST-aptamer fusions coupled to beads were stained with Coomassie blue.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2483412&req=5

pone-0002902-g002: in vitro binding of peptide aptamers to RasGAP SH3.Pull-down assay with recombinant purified RasGAP SH3. Recombinant purified 6xHis-RasGAP SH3 was added to GST-peptide aptamer fusion proteins, coupled to glutathion-sepharose beads. “Control” is a peptide aptamer that was selected against another target protein. Captured protein was revealed with an anti-His antibody. GST-aptamer fusions coupled to beads were stained with Coomassie blue.
Mentions: To confirm some of the yeast two-hybrid phenotypes, we performed pull-down experiments using 6 GST-RasGAP aptamer fusion proteins and recombinant purified 6xHis-RasGAP SH3. We confirmed the binding between RasGAP SH3 and all 6 peptide aptamers tested, with highly variable apparent binding affinities that were not determined by the amounts of GST-aptamer fusion proteins coupled to glutathione-sepharose beads and that did not match the intensities of the yeast two-hybrid phenotypes observed in the yeast two-hybrid mating assay (Figure 2).

Bottom Line: However, RasGAP also acts as a positive effector of Ras and exerts an anti-apoptotic activity that is independent of its GAP function and that involves its SH3 (Src homology) domain.It disrupts the interaction between RasGAP and Aurora B kinase.This work identifies the above-mentioned pocket as an interesting therapeutic target to pursue and points its cognate peptide aptamer as a promising guide to discover RasGAP small-molecule drug candidates.

View Article: PubMed Central - PubMed

Affiliation: UFR Biomédicale, Laboratoire de Pharmacochimie Moléculaire et Cellulaire, INSERM U648, Université Paris Descartes, Paris, France.

ABSTRACT
The Ras GTPase-activating protein RasGAP catalyzes the conversion of active GTP-bound Ras into inactive GDP-bound Ras. However, RasGAP also acts as a positive effector of Ras and exerts an anti-apoptotic activity that is independent of its GAP function and that involves its SH3 (Src homology) domain. We used a combinatorial peptide aptamer approach to select a collection of RasGAP SH3 specific ligands. We mapped the peptide aptamer binding sites by performing yeast two-hybrid mating assays against a panel of RasGAP SH3 mutants. We examined the biological activity of a peptide aptamer targeting a pocket delineated by residues D295/7, L313 and W317. This aptamer shows a caspase-independent cytotoxic activity on tumor cell lines. It disrupts the interaction between RasGAP and Aurora B kinase. This work identifies the above-mentioned pocket as an interesting therapeutic target to pursue and points its cognate peptide aptamer as a promising guide to discover RasGAP small-molecule drug candidates.

Show MeSH
Related in: MedlinePlus