Design and development of tumor suppressor protein p53 variant that cannot form heterooligomers with the wild-type protein

肿瘤抑制蛋白p53是一种含有393个氨基酸的磷酸化蛋白，是四聚体形式的转录增强子，在基因毒性应激反应中抑制细胞周期进程。p53的四聚结构域对于有效的位点特异性DNA结合至关重要，并有助于p53激活天然启动子转录的能力。p53四聚结构域位于326-356残基。超过50%的人类癌症与p53基因突变有关，而p53基因突变多位于序列特异性DNA结合域。错义突变体p53通过异寡聚化以显性负性方式失活野生型p53。在本研究中，我们设计并合成了p53四聚域40个氨基酸的两个肽文库，以筛选不能与野生型p53形成异聚物的肽。在第一个文库p53lib-4中，位于两个二聚体界面α-螺旋上的4个疏水残基与Tip、Tyr、Phe、Met、Lew、Ire和Val等7个疏水氨基酸随机结合。在第二个文库p53lib-9中，形成p53四聚体疏水核心的9个残基以同样的方式随机结合。筛选了两个具有野生型序列的非异聚肽库。筛选后的片段CD谱几乎相同，表明片段中的肽可以形成野生型四聚体结构。第二个文库是根据第一次筛选的肽的特性设计的。图书馆接受了第二次筛选。结果表明，通过这种筛选分离的肽片段可能含有自己形成同源四聚体的肽，而不会形成野生型的异聚物。结果表明，该方法可用于筛选不形成异聚寡聚物的肽。

The tumor suppressor protein p53 is a 393 amino acid phosphoprotein that is a transcriptional enhancer in tetrameric form and suppresses cell cycle progression in response to genotoxic stress. The tetramerization domain of p53 is essential for efficient site-specific DNA binding and contributes to p53's ability to activate transcription from natural promoters. The p53 tetramerization domain is located at residues 326-356. More than 50% of human cancer is associated with p53 gene mutation, which is mostly located at sequence specific DNA binding domain. The missense mutant p53 inactivates a wild-type p53 in a dominant-negative manner via hetero-oligomerization.In this study, we designed and synthesized two peptide libraries of the 40 amino acid p53 tetramerization domain to screen peptides that cannot form hetero-oligomer with the wild-type p53. For the first library, termed p53lib-4, four hydrophobic residues, which are located on the α-helix at the interface of two dimers, were randomized with seven hydrophobic amino acids including Tip, Tyr, Phe, Met, Lew, Ire, and Val. In the second library, p53lib-9, all nine residues that form the hydrophobic core of p53 tetramer were randomized in the same way. Two peptide libraries were screened for non-heterooligomerizing peptides with the wild-type sequence. The screened fraction showed the almost identical CD spectrum, indicating that peptides in the fraction could form a wild-type tetrameric structure. Second library was designed by characterization of the screened peptides on the first screening. The library was subjected for second screening. The results indicated that a peptide fraction isolated by this screening could contain peptides that form homo-tetramers by themselves and do not form hetero-oligomers with wild type. The results demonstrated that the strategy used in this study could be applied to screen peptide that does not form hetero-oligomers.