Figure 1.
Schematic representation of in vitro selection of MDM2-binding peptides using mRNA display.
(1) A DNA library encoding randomized peptides is transcribed. (2) The resulting RNA library is ligated with a PEG-Puro spacer and (3) in vitro translated to form a peptide-mRNA conjugate library. (4) The mRNA-displayed peptide library is incubated with MDM2 immobilized on beads through an affinity selection tag containing a ZZ domain and a TEV protease cleavage site [25], and unbound molecules are washed away. (5) The bound molecules are eluted by cleavage with the TEV protease, and (6) their mRNA portion is amplified by RT-PCR. The resulting DNA can be used for the next rounds of selection or analyzed by cloning and sequencing.
Figure 2.
A multiple-sequence alignment of the selected peptides.
Peptide sequences selected from randomized mRNA-displayed peptide libraries were aligned using the ClustalW program. The peptide sequences are shown using the single-letter code. (A) 16-mer peptides selected after four rounds of selection. Three amino acid residues, Phe, Trp and Leu, which were conserved in nearly every peptide, are shown in bold. (B) 12-mer peptides after five rounds of selection. Fixed amino acid residues are shown in bold. Values below the displayed p5317–28 sequence indicate the position of amino acids in the p53 protein. (C) Sequence logos representations were created with webLogo version 2.8.2 (http://weblogo.berkeley.edu/) based on the 83 peptide sequences obtained using the mRNA display system to select peptides that bound to MDM2 from the 12-mer partially randomized peptide library. The height of each column reflects the bias of particular residues. Polar amino acids containing an amide group and the amino acids that do not contain an amide group are shown in purple and green, respectively. Acidic and basic charged residues are shown in red and blue, respectively, while the hydrophobic residues are shown in black.
Figure 3.
Inhibition of MDM2-p53 and MDMX-p53 interactions by synthetic peptides.
(A) GFP-tagged MIP was generated by a transcription/translation reaction and used for in vitro binding assays as described in the “Materials and Methods” section. I, input; F, flow-through; B, beads. (B) MDM2 or (C) MDMX was generated by an in vitro transcription reaction, bound to His6-p53 immobilized on copper-coated plates in the presence of various concentrations of synthetic MIP (circle), DI (triangle), 3A (diamond) or p53 (square) peptides and quantified by ELISA. The IC50 values are shown in Table 1.
Table 1.
IC50 value for MDM2 and MDMX-p53 interaction in ELISA.
Figure 4.
Functional analyses of GFP-MIP in living cells.
HCT116-p53+/+ cells or SW480 cells (p53 mt) were transfected with plasmids encoding GFP-fused MIP, GFP-fused FLAG or GFP alone. (A) Immunoprecipitation assays with anti-GFP were performed followed by western blot with an anti-MDM2 antibody. I, input; F, flow-through; B, beads. (B) The whole cell lysates were analyzed by western blot with antibodies against p53, MDM2, p21 and β-actin. (C) mRNA levels of p53, MDM2, p21 and GAPDH were determined by quantitative reverse transcription-PCR using total RNA extracted from the cells. GAPDH was used for normalization.
Figure 5.
Activation of the p53 pathway by inhibiting the MDM2-p53 interaction.
(A) HCT116-p53+/+ cells were infected with 400 MOI of Ad-3A, DI or MIP and 8 MOI of Ad-Cre. After 48 h, immunoprecipitation assay with anti-FLAG antibody was performed followed by western blot with anti-MDM2 or anti-FLAG antibody. (B) HCT116-p53+/+ or HCT116-p53−/− cells were infected with 50 MOI of Ad-3A, DI or MIP and 1 MOI of Ad-Cre. After 24 h, the whole cell lysates were analyzed by western blot with antibodies against p53, p21 and β-actin. (C) HCT116-p53+/+ or HCT116-p53−/− cells were infected with 50 MOI of Ad-3A, DI or MIP and 1 MOI of Ad-Cre. After 24 h, mRNA levels of p53, MDM2, p21 and GAPDH were determined by quantitative reverse transcription-PCR using total RNA extracted from the cells. GAPDH was used for normalization.
Figure 6.
Inhibition of cell growth by Ad-MIP.
HCT116 cells p53+/+ or p53−/− were infected with the indicated MOI of Ad-3A, DI or MIP and 1/50 MOI of Ad-Cre. After 72 h, cell viability was analyzed by the WST-1 assay.