Recovering Protein-Protein and Domain-Domain Interactions from Aggregation of IP-MS Proteomics of Coregulator Complexes

Coregulator proteins (CoRegs) are part of multi-protein complexes that transiently assemble with transcription factors and chromatin modifiers to regulate gene expression. In this study we analyzed data from 3,290 immuno-precipitations (IP) followed by mass spectrometry (MS) applied to human cell lines aimed at identifying CoRegs complexes. Using the semi-quantitative spectral counts, we scored binary protein-protein and domain-domain associations with several equations. Unlike previous applications, our methods scored prey-prey protein-protein interactions regardless of the baits used. We also predicted domain-domain interactions underlying predicted protein-protein interactions. The quality of predicted protein-protein and domain-domain interactions was evaluated using known binary interactions from the literature, whereas one protein-protein interaction, between STRN and CTTNBP2NL, was validated experimentally; and one domain-domain interaction, between the HEAT domain of PPP2R1A and the Pkinase domain of STK25, was validated using molecular docking simulations. The scoring schemes presented here recovered known, and predicted many new, complexes, protein-protein, and domain-domain interactions. The networks that resulted from the predictions are provided as a web-based interactive application at http://maayanlab.net/HT-IP-MS-2-PPI-DDI/

A Potential Peptide Therapeutic Derived from the Juxtamembrane Domain of the Epidermal Growth Factor Receptor

<div><p>The epidermal growth factor receptor (EGFR) is involved in many cancers and EGFR has been heavily pursued as a drug target. Drugs targeting EGFR have shown promising clinical results for several cancer types. However, resistance to EGFR inhibitors often occurs, such as with KRAS mutant cancers, therefore new methods of targeting EGFR are needed. The juxtamembrane (JXM) domain of EGFR is critical for receptor activation and targeting this region could potentially be a new method of inhibiting EGFR. We hypothesized that the structural role of the JXM region could be mimicked by peptides encoding a JXM amino acid sequence, which could interfere with EGFR signaling and consequently could have anti-cancer activity. A peptide encoding EGFR 645–662 conjugated to the Tat sequence (TE-64562) displayed anti-cancer activity in multiple human cancer cell types with diminished activity in non-EGFR expressing cells and non-cancerous cells. In nude mice, TE-64562 delayed MDA-MB-231 tumor growth and prolonged survival, without inducing toxicity. TE-64562 induced non-apoptotic cell death after several hours and caspase-3-mediated apoptotic cell death with longer treatment. Mechanistically, TE-64562 bound to EGFR, inhibited its dimerization and caused its down-regulation. TE-64562 reduced phosphorylated and total EGFR levels but did not inhibit kinase activity and instead prolonged it. Our analysis of patient data from The Cancer Genome Atlas supported the hypothesis that down-regulation of EGFR is a potential therapeutic strategy, since phospho- and total-EGFR levels were strongly correlated in a large majority of patient tumor samples, indicating that lower EGFR levels are associated with lower phospho-EGFR levels and presumably less proliferative signals in breast cancer. Akt and Erk were inhibited by TE-64562 and this inhibition was observed <em>in vivo</em> in tumor tissue upon treatment with TE-64562. These results are the first to indicate that the JXM domain of EGFR is a viable drug target for several cancer types.</p> </div

Effect of Tat-645-662 on cell viability and colony growth of various human cancer and normal cell lines.

<p>(<b>A–B</b>) The indicated cell line was plated overnight, then serum starved overnight and treated with TE-64562 for 24 hours. For the HMEC and MDA-MB-231 cells were treated in HMEC media. Representative dose response curves are shown from one experiment run in triplicate with error bars representing the standard error of the mean. In the legend, the mean EC₅₀ values (± standard deviation from two to three independent experiments) derived from the nonlinear fit of dose response curves are shown (generated and fitted in Prism 5.0 GraphPad Software, Inc., USA). Also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049702#pone.0049702.s002" target="_blank">Figure S2</a>. (<b>C</b>) MDA-MB-231 (breast), A-549 (non-small cell lung), DLD-1 (colo-rectal) MIA-PaCa-2 (pancreatic) and SK-N-MC (neuroepithelioma, EGFR-null) cells were grown in soft agar containing 5% serum alone or treated with Tat peptide (20 µM) or Tat-645-62 peptide (10 or 20 µM) and allowed to grow for 2 weeks with addition of medium or medium containing peptide every 2 days. Means of the counts of four or more plates from at least two independent experiments are plotted. The significantly difference between the mean counts (*P<0.04) for TE-64562 treatment was assessed by comparison to untreated control.</p

TE-64562 treatment reduces Akt and Erk phosphorylation in MDA-MB-231 xenograft tumor tissue.

<p>(<b>A–B</b>) Nude mice bearing subcutaneous, MDA-MB-231 xenographic tumors were injected with the TE-64562 peptide (40 mg/kg; 7 µmol/kg), Tat-peptide (20 mg/kg; 7 µmol/kg) or vehicle (saline), intraperitoneally for four days, once per day. On the last day, the mice were injected 30 minutes prior to extracting the tumor. Frozen tumor sections were stained for (<b>A</b>) phospho-Akt (S473) or (<b>B</b>) phospho-Erk and counterstained with DAPI. Representative stained tumor sections are shown with the area in the box enlarged in the images below each section. Large scale bars = 500 µm and small scale bars = 50 µm. (<b>C</b>) A ∼1–2 mm cross-sectional slice of the tumor was lysed in RIPA buffer by sonication and the resulting lysates were analyzed by Western blot. Each lane represents a tumor from a different mouse. (<b>D</b>) Western blot data is quantified and plotted. Each treatment group was compared statistically (*P≤0.0364). Error bars represent the standard error of the mean. Also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049702#pone.0049702.s006" target="_blank">Figure S6</a>.</p

Fluorescent confocal microscopy and activity of EGFR juxtamembrane peptides and control peptides in MDA-MB-231 cells.

<p>(<b>A</b>) The Tat-conjugated EGFR JMA peptide (TE-64562) was added to the MDA-MB-231 cells, in 2.5% serum (gray triangles) or serum free media (black squares). The non-Tat-conjugated EGFR JMA peptide (E-645-662, diamonds), the Tat peptide (Tat, black circles) or Tat-conjugated EGFR JMB peptide (TE-66482, black inverted triangles) were added to the MDA-MB-231 cells in serum free media. The cell viability, measured after overnight treatment, is plotted. The plot represents one of three independent experiments, run in triplicate. Error bars represent standard error of the mean of triplicate values from one experiment. (<b>B–D</b>) Peptides were N-terminally labeled with 5-carboxyfluorescein (FAM). (<b>B</b>) MDA-MB-231 cells were treated with FAM-TE-64562 (1.25 µM) for the indicated amounts of time. Scale bar = 15 µm. (<b>C</b>) Cells were treated overnight with FAM-TE-64562 (5.0 µM), FAM-645-662 (5.0 µM), FAM-Tat (2.5 µM) or FAM-664-682 (5.0 µM) and imaged the following day. Scale bars are 40 µm. Also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049702#pone.0049702.s001" target="_blank">Figure S1</a>.</p

TE-64562 causes EGFR down-regulation and prolongs EGFR phosphorylation.

<p>(<b>A</b>) Serum starved MDA-MB-231 cells were treated with EGF for 2 minutes followed by TE-64562 (10 µM) treatment for the indicated amounts of time. The intensity of the EGFR bands was quantified with respect to the intensity of the respective α-tubulin bands. Mean intensity values are plotted from two independent experiments and error bars represent the standard error of the mean. Significance was assessed by a two-tailed, unpaired t test (*P<0.026). (<b>B</b>) Serum starved MDA-MB-231 cells were treated with TE-64562 (2.5, 5.0, 10.0 and 20.0 µM), Tat (20 µM), an EGFR specific tyrosine kinase inhibitor (TKI, 2.0 µM) or vehicle for 30 minutes, followed by EGF treatment (10 ng/mL) for 10 minutes. Results are representative of three independent experiments. The intensity of the EGFR (N = 3 to 7, depending on concentration) or phospho-EGFR (N = 2) bands were quantified with respect to the intensity of the respective α-tubulin band. Mean intensity values are plotted from two (phospho) or three or more (total) independent experiments and error bars represent the standard error of the mean. Significant differences (Mann-Whitney test) were assessed between each treatment condition and untreated control (*P<0.03). (<b>C</b>) Serum starved MDA-MB-231 cells were treated with TE-64562 (5.0, 10.0 and 20.0 µM) or T-Poly-Ala control peptide (5.0, 10.0 and 20.0 µM) or vehicle for 30 minutes, followed by EGF treatment (10 ng/mL) for 10 minutes. Results represent one of three independent experiments. (<b>D</b>) Correlation plot of breast cancer data from The Cancer Genome Atlas. The normalized EGFR protein expression data from antibody array data was plotted for each individual in the study. Shown in red are the 320 individuals who showed a positive correlation between EGFR expression and phospho-EGFR Y1173 levels. Shown in blue are the 32 individuals who did not show a correlation. Plotting and linear regression were performed in Prism 5.0 (GraphPad Software, Inc., USA). (<b>E</b>) Serum starved MDA-MB-231 cells were treated with TE-64562 (10 µM) or vehicle for 30 minutes followed by EGF (10 ng/mL) for 5, 10, 30, 60 120 or 240 minutes. Cell lysates were collected and analyzed by Western blot for phospho-EGFR (Y1173) and EGFR. Blots were stripped and re-probed for α-tubulin. Also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049702#pone.0049702.s005" target="_blank">Figure S5</a>.</p

TE-64562 treatment causes early non-apoptotic cell death and induces apoptosis after longer treatment.

<p>Serum starved (<b>A</b>) MDA-MB-231 cells or (<b>B</b>) SK-N-MC cells were treated overnight with 5, 10 or 20 µM of TE-64562 for 0.5, 1 or 3 hours and assayed for cell viability. Data were averaged from three independent experiments and plotted with the error bars representing the standard deviation from the mean. Significant differences were assessed between each treatment condition and untreated control (*P<0.05 from a two-tailed unpaired t test). (<b>C</b>) MDA-MB-231 breast cancer cells were serum starved overnight then treated with 0 (control), 6 or 12 µM TE-64562 for 18 hours. Cells were stained with Annexin-V and propidium iodide (PI) and the mean results from four independent experiments are plotted. Unstained cells represent viable cells; total Annexin V staining (Anx-V) is the sum of the Anx-V-stained and dually stained cells which are undergoing apoptosis or are fully apoptotic or necrotic; and Anx-V only stained cells are undergoing apoptosis. Significant differences were assessed between each treatment condition and untreated control (*P = 0.028; **P = 0.008; ***P = 0.004). (<b>D</b>) Serum starved MDA-MB-231 cells were treated with 0 (control), 3, 6 or 12 µM TE-64562 for 18 hours. Cells were lysed, analyzed by Western blot and probed for the presence of cleaved caspase-3. Numbers below the blot images represent the quantifications of each band normalized to the respective control. The blot is representative of two independent experiments. All error bars represent standard error of the mean. Also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049702#pone.0049702.s003" target="_blank">Figure S3</a>.</p

Activity of EGFR juxtamembrane peptides in viability assay of MDA-MB-231 cells.

a<p>Values are shown with standard deviation values (S.D.) from at least three independent experiments.</p

TE-64562 interacts with EGFR and inhibits dimerization.

<p>(<b>A</b>) SK-N-MC cells were transfected with the intracellular domain (ICD) of EGFR (645–1186) or the ICD of EGFR lacking the entire JXM region (ΔJM) or the JMA region (ΔJMA). Biotinylated peptides at a concentration of 0.1 µM (+) or 0.5 µM (++) were incubated with SK-N-MC cells for 2 hours and precipitated from cellular lysates with streptavidin-coated beads. The resulting bead-precipitates were analyzed by Western blot for the presence of the EGFR constructs. Results are representative of three independent experiments. (<b>B</b>) Streptavidin beads were pre-bound with biotinylated peptides and incubated with transfected SK-N-MC lysates. The non-biotinylated version of TE-64562 was added to compete for binding in lanes 3 and 4. The resulting bead-precipitates and lysates were analyzed by Western blot for the presence of the EGFR constructs. Results are representative of two independent experiments. (<b>C</b>) Serum starved MDA-MB-231 cells were treated with TE-64562 (12.5 and 25.0 µM), an EGFR specific tyrosine kinase inhibitor (TKI, 1.0 µM), Tat (25.0 µM) or vehicle for 30 minutes, followed by EGF treatment (25 ng/mL) for 10 minutes. Cellular proteins were cross-linked using bis(sulfosuccinimidyl) suberate (BS3), cells were lysed and lysates analyzed by Western blot for EGFR. The quantification of the dimer band is shown. The EGFR dimer band 25.0 µM TE-64562 was not detectable (N.D.). Results are representative of three independent experiments.</p

TE-64562 effects downstream EGFR signaling.

<p>(<b>A</b>) Serum starved MDA-MB-231 cells were treated with TE-64562 (0.6, 1.2, 2.5 5.0 10 and 20 µM), an EGFR specific tyrosine kinase inhibitor (2.0 µM, TKI) or vehicle for 30 minutes, followed by EGF treatment (10 ng/mL) for 10 minutes. Cell lysates were collected and analyzed by Western blot for (<b>A</b>) phospho-Akt (S273), total Akt, phospho-Erk (p44/p42, T202/Y204) and total Erk. Blots were stripped and re-probed for α-tubulin. The Erk (N = 3) and Akt (N = 4) data from separate experiments, all of which are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049702#pone.0049702.s006" target="_blank">Figure S6</a>, were analyzed. The normalized (ratio of phospho to total intensity) data were plotted as mean values from with the error bars representing the standard error of the mean. The significant differences (*P<0.04) were assessed between each treatment condition and the EGF-treated control (lane 2). (<b>B</b>) Serum starved MDA-MB-231 cells were treated with TE-64562 (0.6, 1.2, 2.5 5.0 10 and 20 µM), an EGFR specific tyrosine kinase inhibitor (2.0 µM, TKI), Tat (20 µM) or vehicle for 30 minutes, followed by EGF treatment (10 ng/mL) for 10 minutes. Results represent one of three independent experiments. Also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049702#pone.0049702.s006" target="_blank">Fig. S6</a>. (<b>C</b>) Serum starved MDA-MB-231 cells were treated with TE-64562 (5.0, 10.0 and 20.0 µM) or T-Poly-Ala control peptide (5.0, 10.0 and 20.0 µM) or vehicle for 30 minutes, followed by EGF treatment (10 ng/mL) for 10 minutes. Results are representative of three independent experiments.</p