Interacting amino acid residues of apoptin and Bcr-Abl.

<p>*The SH3 interacting amino acids in the proline rich PxxP region of apoptin are marked as bold.</p

Amino acid residues forming hydrogen bond between apoptin and Bcr-Abl.

<p>*Hydrogen bonding forming residues between the Bcr-Abl and the proline rich PxxP region of apoptin are shown in <b>bold</b>.</p

MMP-12 cleavage abolishes agrins capability to bind to laminin.

<p>(A) Dose-dependency of binding of miniagrin to laminin-coated plates indicates a specific interaction between full-length miniagrin with immobilized laminin. This interaction is abolished by MMP-12 processing of miniagrin. (B) The dose-dependent binding of the isolated NtA-FS domain to laminin is abolished by MMP-12 cleavage. Incubation with Prinomastat confirms that the MMP-12 does not process the immobilized laminin to reduce NtA interaction. Detection of the polyhistidine tag on the miniagrin and NtA-Fs was carried out with a mouse anti-his antibody, anti-mouse secondary antibody conjugated to alkaline phosphatase and alkaline phosphatase substrate. Colour development was detected at 405 nm.</p

Agrin does not inhibit MMPs.

<p>Comparison of inhibitory action of miniagrin, NtA-Fs and TIMP-1 for: (A) MMP-2 (1 nM), MMP-7 (5.6 nM) and MMP-14 (4 nM) and MMP-13 (10 nM) with 50 nM TIMP-1, 100 nM Nta-FS and 1 µM miniagrin; (B) MMP-12 (1 nM) with a range of NtA-FS concentrations, 1 µM miniagrin and 50 nM TIMP-1; Values are expressed as a percentage of the uninhibited MMP activity +/− standard deviation. (C) Superimposition of NtA (Cα-backbone in pink) and TIMP-1 (Cα-backbone in steelblue) projected into the active site cleft of MMP-3 (pdb-code 1uea). Essential elements of MMP are highlighted in different color schemes (sIII-v, hB and both histidine fingers). The key disulfide bridges of TIMP-1 (Cys¹–Cys⁷⁰ and Cys³–Cys⁹⁹) and NtA (Cys²–⁷⁴) are labeled accordingly. Structural (Zn^s) and catalytic zinc (Zn^c) ions are shown as red spheres. The N-terminal sequences for NtA (in pink) and TIMP-1 (in black) reveal the missing Cys in position 1.</p

Interaction of apoptin with Abl and Bcr-Abl^p210.

<p>(<b>A</b>) Indirect immunofluorescence showing the nuclear localization of Bcr-Abl. 32D^P210 and 32D^DSMZ cells were transiently transfected with GFP-apoptin (green) and subjected to (immuno)fluorescence staining and detection. Apoptin localization was by GFP and Bcr-Abl was detected by staining with Bcr-Abl^p210 with Cy3 tagged (red) secondary antibody. Nuclei were co-stained with DAPI (4, 6-diamidino-2-phenylindole: blue). Column 1 shows DAPI stained nuclei; columns 2 and 3 show the nuclear localization of Bcr-Abl and apoptin. Column 4 shows the merged image of nuclear co-localized of Bcr-Abl^p210 and GFP-apoptin as small clusters (yellow). Abbreviations: Tx = cells transfected with GFP-apoptin, No Tx = no transfection with GFP-apoptin. (<b>B</b>) To demonstrate apoptin and Bcr-Abl interactions, 5–10 µg of GST-apoptin was used in the ‘pull-down assay’. The interaction was tested either on 500 µg of total cell lysates from Bcr-Abl expressing 32D^p210 cells, or on Bcr-Abl non-expressing 32D^DMSZ cells. Lanes from the left: (1) the pull-down products of Bcr-Abl in 32D^DMSZ extracts (negative control), (2) 32D ^p210 extract (positive control), (3) 32D ^p210 extract treated with glutathione-sepharose beads (beads control), and (4) 32D^p210 extract incubated with GST-Apoptin captured with glutathione-sepharose beads. (<b>C</b>) In order to detect apoptin and Bcr-Abl interaction by co-immunoprecipitation assay, 32D^p210 cells were transiently transfected with GFP-apoptin (3 µg of pEGFP-apoptin plasmid for 2×10⁶ cells per transfection using lipofectamine transfection reagent) and cell lysates were incubated with anti-Bcr-Abl antibody followed by immunoprecipitation by protein G-sepharose beads; washed IP products were tested for the presence of apoptin (GFP-apoptin: 40 kDa) by immunoblot using anti-apoptin antibody. Lanes from the left: 1 - GST-Apoptin (positive control), 2 - GFP-apoptin Co-IP from transfected 32D^p210 cells by anti-Bcr-Abl antibody, 3 - Co-IP supernatant/immunodepleted fraction from transfected 32D^p210 cell lysates, 4 - 32D^p210 transfected with GFP (Co-IP, negative control), and 5 - Co-IP from 32D^p210 cells without transfection (Co-IP, negative control). (<b>D</b>) The Abl SH3 domain in Bcr-Abl^p210 facilitates Bcr-Abl interaction with apoptin. 32D^DSMZ cells were transfected with various Bcr-Abl mutant constructs by lipofectamine using 3–4 µg purified plasmid DNA per 2×10⁶ cells. Specific mutant clones of transfected cells were selected by G418. Pull-down assays were performed 7–10 days following the selection and expressed proteins were detected by immunoblotting. The upper representative immunoblot shows various mutants of Bcr-Abl expressed in various 32D^DSMZ clones. The lower immunoblot shows results of GST-apoptin pull-down assay. The Src-homology domain mutant of Bcr-Abl^p210 and GST-apoptin were used in this ‘pull-down’ (><) experiments using lysates from various Bcr-Abl mutant protein expressing 32D^DSMZ clones. The protein-protein complexes were analyzed for apoptin interaction by immunoblotting with rabbit anti-Bcr antibody. As seen (lane 6–9), the presence of an intact SH3 domain in the Bcr-Abl molecule is essential for its interaction with apoptin. Some degree of Bcr-Abl></p

Processing of miniagrin and NtA-Fs by matrix metalloproteases.

<p>SDS-PAGE analysis of (A) Miniagrin and (B) NtA-Fs after incubation for 18 h at 37°C alone, or with a 10∶1 molar ratio with MMP-1, 2, 7, 8, 9, 12, 13 or 14 (+). Each MMP was also incubated alone (−). Products were analysed by (A) 9% Tris-glycine (upper panel) and 15% Tris-tricine (lower panel) and (B) 15% Tris-tricine SDS-PAGE with silver staining. Following transfer to PVDF, excised products were subjected to Edman sequencing to determine cleavage sites. (C) Topology scheme of miniagrin with MMP target sites highlighted. Miniagrin is a mosaic protein composed of the following domains: NtA, N-terminal Agrin; Fs, follistatin-like; EGF, EGF–like domains 1–4 and G, globular domains 1–3. Sites A to E correspond to sites detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043669#pone-0043669-t001" target="_blank">Table 1</a>.</p

List of template proteins used to build apoptin model.

<p>*Data source: Protein Data Bank (RCSB-PDB).</p

Schematic representation of the primary structure and functional domains of apoptin, its cytotoxic potency and inhibition of Bcr-Abl phosphorylation.

<p>(<b>A</b>) The SH3 binding domain is merged within NLS1 (amino acids, aa: 82–88). A pictorial representation of apoptin sequences (UniProtKB/Swiss-Prot entry P54094), LRS = Lecine-Rich Sequence, NLS = Nuclear-Localization Signal, NES = Nuclear Export Signal. (<b>B</b>) Cytotoxic activity of apoptin on Bcr-Abl positive 32D^p210 cells: 32D^p210 were grown in 96-well plates (10⁴ cells per well). Cells (in triplicates for each treatment) were treated with 1 µM Tat-apoptin, and Tat-GFP (negative control), or Imatinib for 0, 4, 8, 12, 18 and 24 h periods respectively. The percentage of viable cells, as assessed by MTT assay indicates that apoptin and Imatinib are both toxic to 32D^p210 cells, and that apoptin's cytotoxic effect favorably compares to that of imatinib. Results are expressed as a percent of cell survival (mean ± SD). (<b>C</b>) Apoptin inhibits Bcr-Abl phosphorylation: K562 and 32D^p210 cells were treated with 1 µM Tat-apoptin, Tat-GFP (negative control) or 1 µM imatinib (positive control). Cells were then harvested after 16 hrs and cell lysates were prepared. Representative Western blots show the expression levels of total and phosphorylated Bcr-Abl; equal loading was checked by the loading control, eIF4E. The upper panel of bands shows the expression of K562 cells and the lower panel shows the expression of 32D^p210 cells. Lanes from the left: (1) no-treatment control cell, (2) Tat-GFP treated cell, (3) imatinib treated cell, and (4) Tat-apoptin treated cell respectively in both cell lines. (<b>D</b>) For quantitation, band intensities from immunoblots were scanned by Image Quant software (version 5.2, Molecular Dynamics®). During quantitation, the imatinib expression data was omitted in order to enable visualization of the apoptin effect with greater clarity. Bcr-Abl phosphorylation was significantly inhibited by apoptin. The quantitation data were normalized to the loading control (eIF4E) and expressed as a ratio of phosphorylated to the total Bcr-Abl and presented as mean ± SEM of three independent experiments.</p

Visualization of pathways, interacting network, and validation of selected downstream regulators.

<p>(<b>A</b>) Bcr-Abl and its downstream effectors are shown by BioCarta pathways. Global gene expression data of K562 leukemia cells was taken from public database, analyzed, and visualized (GenMAPP). The expression values of signal log base e ratio (SLR) are shown outside the colored boxes. The software generated color codes denote up-regulation (dark-red) and down-regulation (blue-green). (<b>B</b>) Direct and indirect interacting network associated with Bcr-Abl was built utilizing global gene expression data and visualized by IPA. The up-regulated genes are shown in red and down-regulated genes are shown as green. The gene expression values (SLR) are also shown. (<b>C, E, G</b>) Apoptin induced inhibition of Bcr-Abl phosphorylation leads to the down-regulation of downstream regulators, STAT5, Akt, and CrkL respectively. K562 and 32D^p210 cells were treated with 1 µM Tat-apoptin, Tat-GFP (negative control) and 1 µM imatinib (positive control) and cell lysates were prepared by harvesting cells after 16 h. Representative Western blots (divided into upper and lower panels representing the K562 cells and the second panel represents the 32D^p210 cells) show the ratio of the expression levels of phosphorylated and total STAT5 (<b>D</b>), Akt (<b>F</b>), and CrkL (<b>H</b>) respectively. In all the immunoblots, lane 1 from no-treatment control cells, lane 2 is from Tat-GFP treated cells, lane 3 is from Tat-apoptin treated cells respectively for both cell lines. STAT5 phosphorylation was significantly inhibited by apoptin indicating that apoptin induced inhibition of Bcr-Abl phosphorylation decreases the activation of STAT5 through phosphorylation. On the other hand, Akt phosphorylation was higher, indicating that apoptin induced Akt activation, as previously published. For CrkL, apoptin induced inhibition of Bcr-Abl phosphorylation lead to the down-regulation of CrkL resulting in lower phosphorytion indicating that apoptin decreases the activation of CrkL, a down-stream substrate of Bcr-Abl. For quantitation, band intensities were scanned by Image Quant software (version 5.2, Molecular Dynamics®). During quantitation, imatinib expression data was omitted in order to enable greater visualization of the apoptin effect. The quantitation data were normalized to the loading control (eIF4E/β-tubulin) and expressed as a ratio of phosphorylated to the total protein and presented as mean ± SEM of three independent experiments.</p

Sequence alignment and 3D model of Apoptin (aa: 1–121).

<p>(<b>A</b>) A representative sequence alignment between apoptin residues and the residues of one of the templates from a group of templates (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028395#pone-0028395-t001" target="_blank">Table1</a>) is shown. (<b>B</b>) Solid ribbon view of full-length (aa: 1–121) of 3D model for apoptin and its amino and carboxyl terminals are shown. (<b>C</b>) Space filling view of apoptin model, showing the potential hydrophobic proline rich interacting area (PKPPSK, aa: 81–86, pink colored region, top right) is shown. (<b>D</b>) Ramachandran plot showing the N-Cα and Cα-C bonds in the apoptin polypeptide chain represented by the torsion angles phi (φ) and psi (ψ); quality of the model was examined by this plot (all atoms are within the allowed regions) and by the G-factors values (the overall value for G-factors is −0.35). (<b>E</b>) Solvent accessible surface area shows the regions of hydrophobic (large cream colored region at the surface) where protein-protein interactions could occur and the hydrophilic regions that are involved in hydrogen bonding, hydrogen bond acceptors (red color) and hydrogen bond donors (blue color). Additional information on apoptin structure could be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028395#pone.0028395.s001" target="_blank">Coordinates S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028395#pone.0028395.s002" target="_blank">S2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028395#pone.0028395.s003" target="_blank">S3</a>.</p