Figure 1.
RasGAP SH3 peptide aptamer binding specificity.
(A) Protein domain mapping of the RasGAP protein. PPP: Proline-rich region; PH: Pleckstrin homology domain; C2: pKC-conserved region 2. (B) Sequence alignment of RasGAP, Nck, and Grb2 SH3 domains. Residues that are conserved between at least two proteins are highlighted in green. (C) Yeast two-hybrid interaction mating assay. LexA fusion proteins (“baits”) were expressed in EGY191α yeast containing a 8 lexAop-lacZ reporter gene. Control and selected peptide aptamers (fused to the B112 activation domain) were expressed in EGY42a yeast. Strains were mated and diploids were grown on X-gal-containing solid medium. 10M is a Cdk2 peptide aptamer and Control is a peptide aptamer randomly picked from the library.
Figure 2.
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.
Figure 3.
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.
Figure 4.
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.
Figure 5.
Cell growth inhibitory activity of RG27 peptide aptamer.
(A) Colony formation assays. A control peptide aptamer or RG27 were stably expressed for two weeks in HeLa, HCT116 or Panc 10.05 cells. Cells were stained with crystal violet. (B) Quantification of results shown in (a) and in two other experiments **: p value <0.01.
Figure 6.
Cytotoxic activity of RG27 peptide aptamer.
(A) Trypan blue exclusion assay. Control peptide aptamer (C6) and RG27 were transiently expressed in untransformed fibroblasts or in HeLa, HCT116 or Panc 10.05 tumor cell lines. The percentage of cells that excluded trypan blue was determined at different times after transfection. (B) Propidium iodide/Annexin V labeling assay. The percentage of cells that incorporated propidium iodide and that were positively labeled with Annexin V was determined by flow cytometry 36 h post-transfection for HeLa cells and 48 h post-transfection for HCT116 cells, Panc 10.05 cells and untransformed fibroblasts. Three independent experiments were performed in all assays. **: p value <0.01. Transfection rates were 75% for HeLa cells, 55% for HCT116, 40% for Panc 10.05 cells, and 70% for untransformed fibroblasts.
Figure 7.
Nuclear fragmentation of RG27-expressing cells.
The nuclei of HeLa cells untransfected or transfected with control aptamer or RG27 expression plasmids were labeled by DAPI 36 h post-transfection and observed using an epifluorescence microscope.
Figure 8.
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.
Figure 9.
Aurora kinase and Survivin involvement in RG27-induced cell death.
(A) Silencing of Aurora A and B expression. HeLa cells were transfected with scrambled (Scr) siRNAs or with siRNAs directed against Aurora A or B (Cont: untransfected cells). Aurora A and B expression was revealed by Western blot experiments using specific antibodies, and γ-tubulin expression was used as a control (B) HeLa cells were co-transfected with a plasmid directing the expression of either control aptamer C6 or RG27, and with siRNAs directed against Aurora A, Aurora B, or Survivin (“Untreated”: no siRNA; “Scr”: scrambled siRNA). Cells were labeled and analyzed as in Figure 6B.
Figure 10.
RG27-induced inhibition of RasGAP-Aurora B interaction and of Aurora B kinase activity.
(A,B,C) RasGAP/Aurora B co-capture assay. Upper panels: A RasGAP pulldown assay was performed from HeLa cells (A), HCT116 cells (B) and Panc 10.05 cells (C) expressing control aptamer C6 or RG27, using either a RasGAP-SH2-interacting peptide (DOK) coupled to CNBr-sepharose beads or uncoupled beads (Cont.). Captured RasGAP and Aurora B proteins were revealed by Western blotting. Lower panels: Western blot experiments were performed from a fraction of the total HeLa, HCT116, and Panc 10.05 cell lysates used in the pulldown assays, and from lysates of non-transfected cells (NT), using RasGAP and Aurora B specific antibodies. (D) Aurora B kinase activity. The content of phosphorylated Histone H3 was determined by Western blot from soluble protein extracts of HeLa cells expressing either C6 or RG27 peptide aptamers. A Western blot using a γ-tubulin antibody was performed to obtain a loading control. Histogram shows the quantification of three independent experiments.
Table 1.
RasGAP SH3 peptide aptamer sequences