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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

Caspase involvement in RG27-induced cell death.(A) Propidium iodide/Annexin V labeling assay in presence of caspase inibitor. The percentage of HeLa cells (transfected with control aptamer or RG27 expression plasmids) that incorporated propidium iodide and that were positively labeled with Annexin V was determined by flow cytometry 36 h post-transfection in presence or in absence of 50 µM pan-caspase inhibitor Z-VAD-fmk. (B) Same experiment as in (A), on HeLa cells treated with 0.1 µM Staurosporine for 36 h. (C) Caspase activity assays. Caspase 3, 8, 9 activities were measured by flow-cytometry in HeLa cells 36 h post-transfection or after 36 h of treatment with 0.1 µM Staurosporine. Values are expressed as “fold induction”, as compared to basal caspase activity values measured in non-transfected, untreated cells (D) Detection of active caspases. Western blots were performed from HeLa cell extracts obtained 36 h after transfection or treatment with 0.1 µM Staurosporine, using antibodies directed against active caspases. NT: non-transfected; Cont: control peptide aptamer.
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pone-0002902-g008: Caspase involvement in RG27-induced cell death.(A) Propidium iodide/Annexin V labeling assay in presence of caspase inibitor. The percentage of HeLa cells (transfected with control aptamer or RG27 expression plasmids) that incorporated propidium iodide and that were positively labeled with Annexin V was determined by flow cytometry 36 h post-transfection in presence or in absence of 50 µM pan-caspase inhibitor Z-VAD-fmk. (B) Same experiment as in (A), on HeLa cells treated with 0.1 µM Staurosporine for 36 h. (C) Caspase activity assays. Caspase 3, 8, 9 activities were measured by flow-cytometry in HeLa cells 36 h post-transfection or after 36 h of treatment with 0.1 µM Staurosporine. Values are expressed as “fold induction”, as compared to basal caspase activity values measured in non-transfected, untreated cells (D) Detection of active caspases. Western blots were performed from HeLa cell extracts obtained 36 h after transfection or treatment with 0.1 µM Staurosporine, using antibodies directed against active caspases. NT: non-transfected; Cont: control peptide aptamer.

Mentions: To determine whether RG27-induced cell death involved caspases, we performed an Annexin V/propidium iodide double-labeling on transfected HeLa cells, in presence and in absence of the pan-caspase inhibitor Z-VAD-fmk [14]. As shown in Figure 8A, cell death induced by RG27 was not affected by Z-VAD-fmk, which inhibited Staurosporine-induced cell death, as expected (Figure 8B). In accordance with this result, we observed that RG27-expressing cells did not exhibit significant caspase 3, 8 and 9 activities, which could be detected upon Staurosporine treatment (Figure 8C). We also failed to detect active caspases in RG27-expressing cells (Figure 8D). Altogether, these observations establish that RG27 induces caspase-independent tumor cell death.


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)

Caspase involvement in RG27-induced cell death.(A) Propidium iodide/Annexin V labeling assay in presence of caspase inibitor. The percentage of HeLa cells (transfected with control aptamer or RG27 expression plasmids) that incorporated propidium iodide and that were positively labeled with Annexin V was determined by flow cytometry 36 h post-transfection in presence or in absence of 50 µM pan-caspase inhibitor Z-VAD-fmk. (B) Same experiment as in (A), on HeLa cells treated with 0.1 µM Staurosporine for 36 h. (C) Caspase activity assays. Caspase 3, 8, 9 activities were measured by flow-cytometry in HeLa cells 36 h post-transfection or after 36 h of treatment with 0.1 µM Staurosporine. Values are expressed as “fold induction”, as compared to basal caspase activity values measured in non-transfected, untreated cells (D) Detection of active caspases. Western blots were performed from HeLa cell extracts obtained 36 h after transfection or treatment with 0.1 µM Staurosporine, using antibodies directed against active caspases. NT: non-transfected; Cont: control peptide aptamer.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0002902-g008: Caspase involvement in RG27-induced cell death.(A) Propidium iodide/Annexin V labeling assay in presence of caspase inibitor. The percentage of HeLa cells (transfected with control aptamer or RG27 expression plasmids) that incorporated propidium iodide and that were positively labeled with Annexin V was determined by flow cytometry 36 h post-transfection in presence or in absence of 50 µM pan-caspase inhibitor Z-VAD-fmk. (B) Same experiment as in (A), on HeLa cells treated with 0.1 µM Staurosporine for 36 h. (C) Caspase activity assays. Caspase 3, 8, 9 activities were measured by flow-cytometry in HeLa cells 36 h post-transfection or after 36 h of treatment with 0.1 µM Staurosporine. Values are expressed as “fold induction”, as compared to basal caspase activity values measured in non-transfected, untreated cells (D) Detection of active caspases. Western blots were performed from HeLa cell extracts obtained 36 h after transfection or treatment with 0.1 µM Staurosporine, using antibodies directed against active caspases. NT: non-transfected; Cont: control peptide aptamer.
Mentions: To determine whether RG27-induced cell death involved caspases, we performed an Annexin V/propidium iodide double-labeling on transfected HeLa cells, in presence and in absence of the pan-caspase inhibitor Z-VAD-fmk [14]. As shown in Figure 8A, cell death induced by RG27 was not affected by Z-VAD-fmk, which inhibited Staurosporine-induced cell death, as expected (Figure 8B). In accordance with this result, we observed that RG27-expressing cells did not exhibit significant caspase 3, 8 and 9 activities, which could be detected upon Staurosporine treatment (Figure 8C). We also failed to detect active caspases in RG27-expressing cells (Figure 8D). Altogether, these observations establish that RG27 induces caspase-independent tumor cell death.

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