Abstract
p53 is a tumor suppressor protein that prevents tumorigenesis through cell cycle arrest or apoptosis of cells in response to cellular stress such as DNA damage. Because the oncoprotein MDM2 interacts with p53 and inhibits its activity, MDM2-p53 interaction has been a major target for the development of anticancer drugs. While previous studies have used phage display to identify peptides (such as DI) that inhibit the MDM2-p53 interaction, these peptides were not sufficiently optimized because the size of the phage-displayed random peptide libraries did not cover all of the possible sequences. In this study, we performed selection of MDM2-binding peptides from large random peptide libraries in two stages using mRNA display. We identified an optimal peptide named MIP that inhibited the MDM2-p53 and MDMX-p53 interactions 29- and 13-fold more effectively than DI, respectively. Expression of MIP fused to the thioredoxin scaffold protein in living cells by adenovirus caused stabilization of p53 through its interaction with MDM2, resulting in activation of the p53 pathway. Furthermore, expression of MIP also inhibited tumor cell proliferation in a p53-dependent manner more potently than DI. These results show that two-stage, mRNA-displayed peptide selection is useful for the rapid identification of potent peptides that target oncoproteins.
Highlights
P53 is a tumor suppressor protein that prevents tumorigenesis through cell cycle arrest or apoptosis of cells in response to cellular stress such as DNA damage
P53 induces cell cycle arrest or apoptosis in cells that have genetic aberrations, and as such, inactivation of p53 leads to accumulation of the aberrations that may cause overexpression of several kinds of oncoproteins, resulting in tumorigenesis [5]. p53 retains its wildtype status in approximately 50% of human cancers
DI was not sufficiently optimized because it was selected by phage display from a 12-mer random library (4.161015 possible members) with a size of,108 that did not cover all of the possible sequences
Summary
P53 is a tumor suppressor protein that prevents tumorigenesis through cell cycle arrest or apoptosis of cells in response to cellular stress such as DNA damage. Expression of MIP inhibited tumor cell proliferation in a p53-dependent manner more potently than DI These results show that two-stage, mRNA-displayed peptide selection is useful for the rapid identification of potent peptides that target oncoproteins. We performed in vitro selection of MDM2-binding peptides from random peptide libraries using mRNA display [22], [23] This system based on cell-free translation is a potent method for screening large peptide libraries (,1013 unique members) that can cover all of the possible sequences in a 10-mer random library. We applied mRNA display to identify a highly optimized peptide that could disrupt the MDM2-p53 complex from a random library containing all of the possible sequences by dividing the selection process into two stages. We verified that a selected peptide could inhibit the MDM2-p53 interaction in living cells and block tumor cell growth
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