| Abstract
| - Proliferating cell nuclear antigen (PCNA) plays an essential role in DNA replication, repair,and control of cell proliferation, and its activity can be modulated by interaction with p21Waf1/Cip1 [Cox,L. S., (1997) Trends Cell Biol. 7, 493−497]. This protein−protein interaction provides a particularlygood model target for designing therapeutic agents to treat proliferative disorders such as cancer. In thisstudy, the formation of complexes between PCNA and peptides derived from the C-terminus of p21 hasbeen investigated at the molecular level and quantified using a competitive PCNA binding assay andisothermal titration calorimetry (ITC). The affinity constant for the interaction between p21 (141−160)peptide and PCNA has been determined to be 1.14 × 107 M-1, corresponding to a Kd of 87.7 nM.Measurement of the interaction of truncation and substitution analogues based on the p21 (141−160)sequence with PCNA revealed that the N-terminal part (residues 141−152) of the above peptide is theminimum recognition motif, required for PCNA binding. Truncation of the C-terminal region p21 (153−160), though, inhibited significantly the ability of the peptides to compete with the full-length p21 (141−160) for binding to PCNA. Alanine mutation of Met 147 or Asp 149 completely abolished or significantlydecreased, respectively, the level of the PCNA binding and the inhibition of SV40 DNA replication.Comparison of the data obtained by the competitive PCNA binding assay and the ITC measurementsdemonstrated the usefulness of this assay for screening for compounds that could modulate the PCNA−p21 interaction. Using this assay, we have screened rationally designed peptides for binding to PCNAand interruption of the PCNA−p21 (141−160) complex. As a result of this screening, we have identifieda 16-residue peptide (consensus motif 1 peptide) with the following sequence: SAVLQKKITDYFHPKK.Consensus motif 1 peptide and p21 (141−160) have similar affinities for binding PCNA and abilities toinhibit in vitro replication of DNA originated from SV40. Such peptides could prove useful in assessingp21-mimetic strategies for cancer treatment.
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