Jones R E, Heimbrook D C, Huber H E, Wegrzyn R J, Rotberg N S, Stauffer K J, Lumma P K, Garsky V M, Oliff A
Department of Cancer Research, Merck Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486.
J Biol Chem. 1992 Jan 15;267(2):908-12.
Complex formation between the human papilloma virus type 16 E7 protein (HPV-16 E7) and the retinoblastoma growth suppressor protein (RB) is believed to contribute to the process of cellular transformation that leads to cervical carcinoma. Genetic analysis of the HPV-16 E7 protein has shown that the segment of E7 homologous to the conserved region 2 of adenovirus 5 E1A protein is involved in both RB binding and E7-mediated cell transformation. We have previously shown that a peptide colinear with HPV-16 E7 residues 21-29 was able to block immobilized species of E7 from binding to RB protein. The current study reports the effects of different chemical modifications of this peptide. One type of modification, methylation of the alpha-amino nitrogens contributed by Leu22, Tyr25, and Leu28, resulted in a 45-fold increase in E7/RB binding antagonist activity. This increased antagonist activity is sequence-specific since methylation of the amino groups contributed by Tyr23, Cys24, or Glu26 resulted in a profound loss of binding antagonist activity. Using a newly developed binding assay we determined that the apparent dissociation constant for recombinant HPV-16 E7 protein binding to recombinant human RB protein is 1.3 nM. The peptide Ac[N-MeLeu22,N-Me-Tyr25,N-MeLeu28]-(21-29)-E7 amide was determined to be a competitive inhibitor of HPV-16 E7 binding to RB with a Ki value of 32 nM.
人乳头瘤病毒16型E7蛋白（HPV-16 E7）与视网膜母细胞瘤生长抑制蛋白（RB）之间形成的复合物被认为与导致宫颈癌的细胞转化过程有关。对HPV-16 E7蛋白的基因分析表明，E7中与腺病毒5 E1A蛋白保守区域2同源的片段参与了RB结合和E7介导的细胞转化。我们之前已经表明，与HPV-16 E7第21-29位残基共线性的肽能够阻止固定化的E7与RB蛋白结合。当前研究报道了该肽不同化学修饰的作用。一种修饰类型，即Leu22、Tyr25和Leu28贡献的α-氨基氮甲基化，导致E7/RB结合拮抗剂活性增加了45倍。这种增加的拮抗剂活性具有序列特异性，因为Tyr23、Cys24或Glu26贡献的氨基甲基化导致结合拮抗剂活性大幅丧失。使用新开发的结合试验，我们确定重组HPV-16 E7蛋白与重组人RB蛋白结合的表观解离常数为1.3 nM。肽Ac[N-MeLeu22,N-Me-Tyr25,N-MeLeu28]-(21-29)-E7酰胺被确定为HPV-16 E7与RB结合的竞争性抑制剂，Ki值为32 nM。
