Zheleva D I, Zhelev N Z, Fischer P M, Duff S V, Warbrick E, Blake D G, Lane D P
Dundee Technopole, Cyclacel Ltd., Dundee,UK.
Biochemistry. 2000 Jun 27;39(25):7388-97. doi: 10.1021/bi992498r.
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 p21(Waf1/Cip1) [Cox, L. S., (1997) Trends Cell Biol. 7, 493-497]. This protein-protein interaction provides a particularly good model target for designing therapeutic agents to treat proliferative disorders such as cancer. In this study, the formation of complexes between PCNA and peptides derived from the C-terminus of p21 has been investigated at the molecular level and quantified using a competitive PCNA binding assay and isothermal titration calorimetry (ITC). The affinity constant for the interaction between p21 (141-160) peptide and PCNA has been determined to be 1.14 x 10(7) M(-)(1), corresponding to a K(d) 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 the minimum 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 significantly decreased, 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 measurements demonstrated 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 PCNA and interruption of the PCNA-p21 (141-160) complex. As a result of this screening, we have identified a 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 to inhibit in vitro replication of DNA originated from SV40. Such peptides could prove useful in assessing p21-mimetic strategies for cancer treatment.
增殖细胞核抗原（PCNA）在DNA复制、修复及细胞增殖控制中发挥着至关重要的作用，其活性可通过与p21（Waf1/Cip1）相互作用进行调节[考克斯，L.S.，（1997年）《细胞生物学趋势》7卷，493 - 497页]。这种蛋白质 - 蛋白质相互作用为设计治疗增殖性疾病（如癌症）的治疗药物提供了一个特别好的模型靶点。在本研究中，已在分子水平上研究了PCNA与源自p21 C末端的肽之间复合物的形成，并使用竞争性PCNA结合测定法和等温滴定量热法（ITC）进行了定量。已确定p21（141 - 160）肽与PCNA之间相互作用的亲和常数为1.14×10⁷ M⁻¹，对应解离常数Kd为87.7 nM。基于p21（141 - 160）序列的截短和取代类似物与PCNA相互作用的测量结果表明，上述肽的N末端部分（残基141 - 152）是PCNA结合所需的最小识别基序。然而，p21（153 - 160）C末端区域的截短显著抑制了这些肽与全长p21（141 - 160）竞争结合PCNA的能力。Met 147或Asp 149的丙氨酸突变分别完全消除或显著降低了PCNA结合水平以及对SV40 DNA复制的抑制作用。竞争性PCNA结合测定法和ITC测量所获得数据的比较证明了该测定法对于筛选可调节PCNA - p21相互作用的化合物的有用性。使用该测定法，我们合理筛选了与PCNA结合并中断PCNA - p21（141 - 160）复合物的设计肽。通过该筛选，我们鉴定出了一个具有以下序列的16个残基的肽（共有基序1肽）：SAVLQKKITDYFHPKK。共有基序1肽和p21（141 - 160）在结合PCNA方面具有相似的亲和力，并且在抑制源自SV40的DNA体外复制方面具有相似的能力。此类肽可能在评估用于癌症治疗的p21模拟策略中证明是有用的。
