Limits...
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.

Show MeSH

Related in: MedlinePlus

Peptide aptamer interactions in a mammalian cell context.(A) Mammalian cell two-hybrid assay. HeLa cells were co-transfected with a plasmid directing the expression of a GAL4-RasGAP SH3 bait protein, a plasmid containing a luciferase two-hybrid reporter gene, and plasmids directing the expression of VP16-peptide aptamer fusions. Luminescence was measured 48 h after transfection. Two independent experiments were performed. SH3-pACT: RasGAP bait with empty prey plasmid; pBIND-pACT: empty bait and prey plasmids; Control-pACT: empty prey plasmid only. (B) Pull-down assay from total protein extract. Left: A mouse liver total protein extract was added to glutathion sepharose beads, uncoated (−) or coated with GST-RG27 or GST-Control fusion proteins. Captured native RasGAP protein was revealed with a monoclonal antibody directed against the SH3 domain. Right: Coomassie staining of a SDS-PAGE revealing comparable amounts of GST-Control and GST-RG27 fusion proteins immobilized on glutathion sepharose beads.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2483412&req=5

pone-0002902-g004: Peptide aptamer interactions in a mammalian cell context.(A) Mammalian cell two-hybrid assay. HeLa cells were co-transfected with a plasmid directing the expression of a GAL4-RasGAP SH3 bait protein, a plasmid containing a luciferase two-hybrid reporter gene, and plasmids directing the expression of VP16-peptide aptamer fusions. Luminescence was measured 48 h after transfection. Two independent experiments were performed. SH3-pACT: RasGAP bait with empty prey plasmid; pBIND-pACT: empty bait and prey plasmids; Control-pACT: empty prey plasmid only. (B) Pull-down assay from total protein extract. Left: A mouse liver total protein extract was added to glutathion sepharose beads, uncoated (−) or coated with GST-RG27 or GST-Control fusion proteins. Captured native RasGAP protein was revealed with a monoclonal antibody directed against the SH3 domain. Right: Coomassie staining of a SDS-PAGE revealing comparable amounts of GST-Control and GST-RG27 fusion proteins immobilized on glutathion sepharose beads.

Mentions: We set out to determine the ability of the peptide aptamers to bind to the full length RasGAP protein in mammalian cells. To this end, we performed two-hybrid assays in HeLa cells that expressed a full-length RasGAP bait protein and all the selected aptamers, as preys. We failed to detect any significant interaction signal (data not shown). We performed the same experiments using a RasGAP SH3 bait protein. RG27 was the only peptide aptamer that strongly interacted with RasGAP SH3 in this assay (Figure 4A). We then performed a pull-down experiment using a GST-RG27 fusion protein and a mouse liver total protein extract. We successfully captured the native endogenous RasGAP protein from the extract using the GST-RG27 fusion but not a GST-control aptamer fusion (Figure 4B). From all these observations, we conclude that aptamer RG27, at least, can interact with the native RasGAP protein in a mammalian cell context and that the lack of two-hybrid interaction phenotypes between the peptide aptamers and the full length RasGAP bait proteins is probably caused by an improper folding of the RasGAP moiety or, more probably, by a masking of the SH3 domain when the entire protein is expressed in yeast or human cell nuclei (see Discussion).


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)

Peptide aptamer interactions in a mammalian cell context.(A) Mammalian cell two-hybrid assay. HeLa cells were co-transfected with a plasmid directing the expression of a GAL4-RasGAP SH3 bait protein, a plasmid containing a luciferase two-hybrid reporter gene, and plasmids directing the expression of VP16-peptide aptamer fusions. Luminescence was measured 48 h after transfection. Two independent experiments were performed. SH3-pACT: RasGAP bait with empty prey plasmid; pBIND-pACT: empty bait and prey plasmids; Control-pACT: empty prey plasmid only. (B) Pull-down assay from total protein extract. Left: A mouse liver total protein extract was added to glutathion sepharose beads, uncoated (−) or coated with GST-RG27 or GST-Control fusion proteins. Captured native RasGAP protein was revealed with a monoclonal antibody directed against the SH3 domain. Right: Coomassie staining of a SDS-PAGE revealing comparable amounts of GST-Control and GST-RG27 fusion proteins immobilized on glutathion sepharose beads.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0002902-g004: Peptide aptamer interactions in a mammalian cell context.(A) Mammalian cell two-hybrid assay. HeLa cells were co-transfected with a plasmid directing the expression of a GAL4-RasGAP SH3 bait protein, a plasmid containing a luciferase two-hybrid reporter gene, and plasmids directing the expression of VP16-peptide aptamer fusions. Luminescence was measured 48 h after transfection. Two independent experiments were performed. SH3-pACT: RasGAP bait with empty prey plasmid; pBIND-pACT: empty bait and prey plasmids; Control-pACT: empty prey plasmid only. (B) Pull-down assay from total protein extract. Left: A mouse liver total protein extract was added to glutathion sepharose beads, uncoated (−) or coated with GST-RG27 or GST-Control fusion proteins. Captured native RasGAP protein was revealed with a monoclonal antibody directed against the SH3 domain. Right: Coomassie staining of a SDS-PAGE revealing comparable amounts of GST-Control and GST-RG27 fusion proteins immobilized on glutathion sepharose beads.
Mentions: We set out to determine the ability of the peptide aptamers to bind to the full length RasGAP protein in mammalian cells. To this end, we performed two-hybrid assays in HeLa cells that expressed a full-length RasGAP bait protein and all the selected aptamers, as preys. We failed to detect any significant interaction signal (data not shown). We performed the same experiments using a RasGAP SH3 bait protein. RG27 was the only peptide aptamer that strongly interacted with RasGAP SH3 in this assay (Figure 4A). We then performed a pull-down experiment using a GST-RG27 fusion protein and a mouse liver total protein extract. We successfully captured the native endogenous RasGAP protein from the extract using the GST-RG27 fusion but not a GST-control aptamer fusion (Figure 4B). From all these observations, we conclude that aptamer RG27, at least, can interact with the native RasGAP protein in a mammalian cell context and that the lack of two-hybrid interaction phenotypes between the peptide aptamers and the full length RasGAP bait proteins is probably caused by an improper folding of the RasGAP moiety or, more probably, by a masking of the SH3 domain when the entire protein is expressed in yeast or human cell nuclei (see Discussion).

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