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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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Mapping of the peptide aptamer binding sites on RasGAP SH3 by AptaPrint.(A) RasGAP SH3 aminoacid substitutions. (B) AptaPrint assay. 11 LexA fusions with RasGAP SH3 mutants were expressed in EGY48α yeast containing a 8 lexAop-lacZ reporter gene. Peptide aptamer-B112 fusions were expressed in EGY42a yeast. A yeast two-hybrid mating assay was performed. (C) NMR structure of the RasGAP SH3 domain. Residues W317, L313 and D295/7 are located.
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pone-0002902-g003: Mapping of the peptide aptamer binding sites on RasGAP SH3 by AptaPrint.(A) RasGAP SH3 aminoacid substitutions. (B) AptaPrint assay. 11 LexA fusions with RasGAP SH3 mutants were expressed in EGY48α yeast containing a 8 lexAop-lacZ reporter gene. Peptide aptamer-B112 fusions were expressed in EGY42a yeast. A yeast two-hybrid mating assay was performed. (C) NMR structure of the RasGAP SH3 domain. Residues W317, L313 and D295/7 are located.

Mentions: We performed an AptaPrint assay to map the peptide aptamer binding sites on the RasGAP SH3 domain [11], [12]. Based on the NMR structure [13], we created 11 mutants to interrogate different molecular surfaces (Figure 3A). As shown in Figure 3B, our peptide aptamer collection showed different binding profiles against the RasGAP SH3 mutant panel, thus indicating that the aptamers bind to their target on different molecular surfaces. All aptamers (except RG28 and RG30) showed a decreased or abolished interaction phenotype against the W317K mutant, which suggests that this residue contributes to the integrity of most aptamer binding surfaces. Most aptamers (RG17, 18, 20, 21, 23, 26, 27, 29) failed to show an interaction phenotype when either L313 or D295/7 were mutated, which suggests that they bind to the pocket delineated by W317 and these three residues (Figure 3B and C). However, RG19 and RG25 seem to bind to a different surface as they retained their interaction phenotypes, either with the D295/7A and the L313A mutants (RG19), or with the D295/7A mutant (RG25). This AptaPrint did not provide information on the binding sites of RG28 and 30, since both aptamers retained their interaction phenotypes with all mutants.


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)

Mapping of the peptide aptamer binding sites on RasGAP SH3 by AptaPrint.(A) RasGAP SH3 aminoacid substitutions. (B) AptaPrint assay. 11 LexA fusions with RasGAP SH3 mutants were expressed in EGY48α yeast containing a 8 lexAop-lacZ reporter gene. Peptide aptamer-B112 fusions were expressed in EGY42a yeast. A yeast two-hybrid mating assay was performed. (C) NMR structure of the RasGAP SH3 domain. Residues W317, L313 and D295/7 are located.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0002902-g003: Mapping of the peptide aptamer binding sites on RasGAP SH3 by AptaPrint.(A) RasGAP SH3 aminoacid substitutions. (B) AptaPrint assay. 11 LexA fusions with RasGAP SH3 mutants were expressed in EGY48α yeast containing a 8 lexAop-lacZ reporter gene. Peptide aptamer-B112 fusions were expressed in EGY42a yeast. A yeast two-hybrid mating assay was performed. (C) NMR structure of the RasGAP SH3 domain. Residues W317, L313 and D295/7 are located.
Mentions: We performed an AptaPrint assay to map the peptide aptamer binding sites on the RasGAP SH3 domain [11], [12]. Based on the NMR structure [13], we created 11 mutants to interrogate different molecular surfaces (Figure 3A). As shown in Figure 3B, our peptide aptamer collection showed different binding profiles against the RasGAP SH3 mutant panel, thus indicating that the aptamers bind to their target on different molecular surfaces. All aptamers (except RG28 and RG30) showed a decreased or abolished interaction phenotype against the W317K mutant, which suggests that this residue contributes to the integrity of most aptamer binding surfaces. Most aptamers (RG17, 18, 20, 21, 23, 26, 27, 29) failed to show an interaction phenotype when either L313 or D295/7 were mutated, which suggests that they bind to the pocket delineated by W317 and these three residues (Figure 3B and C). However, RG19 and RG25 seem to bind to a different surface as they retained their interaction phenotypes, either with the D295/7A and the L313A mutants (RG19), or with the D295/7A mutant (RG25). This AptaPrint did not provide information on the binding sites of RG28 and 30, since both aptamers retained their interaction phenotypes with all mutants.

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