A nanotechnological, molecular-modeling, and immunological approach to study the interaction of the anti-tumorigenic peptide p28 with the p53 family of proteins

Abstract

p28 is an anionic, amphipathic, cell-penetrating peptide derived from the cupredoxin azurin that binds to the DNA-binding domain (DBD) of the tumor suppressor protein, p53, and induces a post-translational increase in the level of wild type and mutated p53 in a wide variety of human cancer cells. As p63 and p73, additional members of the p53 superfamily of proteins, also appear to be involved in the cellular response to cancer therapy and are reportedly required for p53-induced apoptosis, we asked whether p28 also binds to p63 and p73. Atomic force spectroscopy demonstrates that p28 forms a stable, high-affinity complex with full-length p63, the DBD of p63, and full-length p73. Exposure to p28 decreased the level of TAp63α and ΔNp63α, the truncated form of p63, in p53 wild type and mutated human breast cancer cells, respectively. p28 increased the level of TAp73α, but not ΔNp73α, in the same breast cancer cell lines. In contrast, p28 increased the level of the TA and ΔN isoforms of p63 in p53 wild type, but not in p53 mutated melanoma cells, while decreasing TA p73α in p53 wild type and mutated human melanoma cells. All changes were mirrored by an associated change in the expression of the HECT E3 ligases Itch/AIP4, AIP5, and the RING E3 ligase Pirh2, but not in the receptor for activated C kinase or the RING E3 ligases Mdm2 and Cop1. Collectively, the data suggest that molecules such as p28 bind with high affinity to the DBD of p63 and p73 and alter their expression independent of the Mdm2 and Cop1 pathways.

Keywords: anticancer peptide; molecular interaction; p53 superfamily of proteins.

Figure 1

Docking model of p28 and p63 DBD. Notes: (A) The structure for the p28-p63 DBD complex was obtained from the best docking model. Blue: overall ribbon diagram of the p63 DBD, green: S4, S6, S7, S9, and S10 loops. (B) Ribbon diagram of the p63 DBD (yellow) superimposed on the electrostatic potential plot. p28 (green) binds to the hydrophobic region. (C) Relationship of p28 (green) and S4, S6, S7, S9, and S10 (orange) of the p63 DBD. (D) Amino acid sequence alignment of the DBD of p53 and p63, data from Chen et al. p28 binding residues are indicated in red on the aa sequence of p53 DBD (top) and p63 DBD (bottom). The Pirh2 binding motif on p53 is indicated by the blue bar. S, sheet structure; orange box, DNA-binding sites; green box, zinc finger sites. Abbreviation: DBD, DNA-binding domain.

Figure 2

Tapping mode AFM images of protein monolayer. Notes: Tapping mode AFM images of: (A) p63 DBD monolayer immobilized on an amino-silane and glutaraldehyde functionalized glass slide; (B) p63 DBD monolayer after p28 deposition. All images were acquired in ambient air. Abbreviations: AFM, atomic force microscopy; DBD, DNA-binding domain.

Figure 3

Schematic representation of AFS analysis. Notes: Left panel shows illustration of AFS analysis showing the p28 bound to the tip via a flexible linker and p63, p73, or p63 DBD immobilized on glass substrates. Right panel shows schematic representation of a force–piezo displacement cycle, showing a typical unbinding event. Abbreviations: AFS, atomic force spectroscopy; DBD, DNA-binding domain.

Figure 4

AFS analysis of p28 binding. Notes: (A) Histogram of the unbinding force for the p63 DBD/p28 complex recorded at a loading rate of 7 nN/s before (red bars) and after (green bars) blocking the p63 DBD monolayer. (B) Plot of the most probable unbinding forces versus the natural logarithm of the different loading rates of the p63 DBD/p28 interaction. The line represents the fit of the experimental data by the Bell–Evans model (Equation 1); the goodness of the fit being assessed by both the linear coefficient correlation (ρ=0.96) and the reduced chi-square (χ²=1.4). The kinetic parameters obtained are reported in the inset with the relative uncertainties. (C) Histogram of the unbinding force distribution for the full-length p63/p28 complex acquired at the loading rate 7 nN/s before (red bars) and after (green bars) blocking the p63 monolayer. (D) Plot of the most probable unbinding forces versus the natural logarithm of the different loading rates of the full-length p63/p28 interaction. The line represents the fit of the experimental data by the Bell–Evans model (Equation 1); the goodness of the fit being assessed by both the linear coefficient correlation (ρ=0.97) and the reduced chi-square (χ²=1.3). Kinetic values appear in the inset with their relative uncertainties. (E) Histogram of the unbinding force distribution for the full-length p73/p28 complex acquired at the loading rate 7 nN/s before (red bars) and after (green bars) blocking the p73 monolayer. (F) Plot of the most probable unbinding forces versus the natural logarithm of the different loading rates of the full-length p73/p28 interaction. The line represents the ft of the experimental data by the Bell–Evans model (Equation 1); the goodness of the fit being assessed by both the linear coefficient correlation (ρ=0.98) and the reduced chi-square (χ²=1.2). The kinetic parameters obtained are reported in the inset with the relative uncertainties. Abbreviations: DBD, DNA-binding domain; AFS, atomic force spectroscopy.

Figure 5

Interaction of p28 with p53, p63, and p73. Notes: GST pull-down assay showing complex formation between GST-p28 and p53, p63, and p73. p53, p63, or p73 were detected with antibodies to anti-p53, p63, or p73. Whole-cell lysates (Lysate) from each cell line served as a positive control. The numbers indicated below each band are expressed relative to each control (Lysate) expressed as 100%. Abbreviations: GST, glutathione S-transferase; wt, wild-type; mut, mutant.

Figure 6

Competitive immunoprecipitation assay for Cop1 and Pirh2. Notes: GST-p63 DBD and GST alone were immobilized on glutathione-Sepharose 4B beads and incubated in absence (−) or presence of p28 (+: 10, ++: 100 mole excess), followed by addition of MCF-7 lysates containing Cop1 and Pirh2. Samples were separated by SDS-PAGE and immunoblotted with either anti-Cop1 or anti-Pirh2 antibodies. Lysate: whole-cell lysates of MCF-7 used in assay stably expressed Cop1 and Pirh2. Numbers below Pirh2 bands are the relative percentage to the level of Pirh2 bound to p63 DBD in the absence of p28. Abbreviations: DBD, DNA-binding domain; GST, glutathione S-transferase; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; IB, immunoblotting.

Figure 7

Effect of p28 on p63, p73, and E3 ligases on p53^wt,mut breast cancer cells. Notes: MCF-7, MDD2, MDA-MB-231, and MCF-10A cells were treated with 50 μM p28 for 24–72 hours, and protein levels determined by immunoblotting (A). The expression of each gene was determined by RT-PCR (B). The numbers indicated below each band represent the level of expression relative to the control (control expressed as 100%). Abbreviations: RT-PCR, reverse transcription polymerase chain reaction; wt, wild-type; mut, mutant; dom, dominant; neg, negative.

Figure 8

Effect of p28 on p63, p73, and E3 ligases on p53^wt,mut melanoma cells. Notes: (A) Mel-29, Mel-23, and Mel-6 cells were treated with 50 μM p28 for 24–72 hours, and protein levels determined by immunoblotting. (B) The expression of each gene was determined by RT-PCR. The numbers below each band represent the level of expression relative to the control (control expressed as 100%). Abbreviations: RT-PCR, reverse transcription polymerase chain reaction; wt, wild-type; mut, mutant.