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

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

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

Modeled interactions between apoptin and Bcr-Abl.

<p>(<b>A</b>) Shows the interaction between apoptin and the SH3-domain of Bcr-Abl (solid ribbon view, showing the two terminals of two proteins) obtained by performing virtual docking experiment between apoptin model and the X-ray structure of Bcr-Abl-SH3 domain (PDB: 2ABL). (<b>B</b>) Shows the space filling docking view of the interactions between apoptin (pink) and the SH3-domain of Bcr-Abl (blue), the 13 residues (red) of Bcr-Abl and 13 residues (light blue) of apoptin that are within 2.5 Å to each other; some of the proline-rich (PxxP) SH3-binding residues (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028395#pone-0028395-t002" target="_blank">Table 2</a>) are present and at least five direct hydrogen bonding are possible in between them (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028395#pone-0028395-t003" target="_blank">Table 3</a>). Additional information on apoptin interaction with BcrAbl could be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028395#pone.0028395.s004" target="_blank">Coordinates S4</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028395#pone.0028395.s005" target="_blank">S5</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028395#pone.0028395.s006" target="_blank">S6</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

Sequence alignment and interactions between apoptin and adopter proteins CrkL, Akt1 and STAT5.

<p>Space filling views and similarity in sequences between apoptin and the SH2-domain of the adopter protein CrkL (<b>A</b>), apoptin's SH3-binding domain residues (<b>B</b>), 81 to 86 (PKPPSK), are within the SH2-domain of CrkL (<b>C</b>). In addition, the similarity in sequences between apoptin and the adopter proteins Akt1 (<b>D</b>) and STAT5 (<b>E</b>) suggesting that apoptin might directly interact in the CrkL, Akt1 and STAT5 interacting sites in addition to the SH3-binding domain of Bcr-Abl and could block further propagation of survival and proliferation signaling.</p

rEtpE-C-coated beads enter macrophages by a pathway similar to one that mediates <i>E. chaffeensis</i> entry.

<p>(A) Latex beads (red) coated with rEtpE-C by anti-EtpE-C labeling (green) under fluorescence microscopy. Scale bar, 1 µm. (B) Fluorescence and phase contrast merged images of rEtpE-C-coated beads incubated with mouse BMDMs. Cells were pretreated with DMSO (solvent control), MDC, genistein, or PI-PLC for 45 min followed by trypsin treatment to remove beads that were not internalized. Scale bar, 10 µm. (C and D) Numbers of internalized rEtpE-C-coated (C) and non-coated (D) beads/cell incubated with mouse BMDMs pretreated with MDC, genistein, or PI-PLC, relative to DMSO treatment (solvent control) set as 100. Data represent the mean and standard deviation of triplicate samples and are representative of three independent experiments. *Significantly different (<i>P</i><0.05).</p