Development of p53-associated molecular devices for diagnosis and treatment of gastrointestinal cancer

本研究的目的是（i）开发用于诊断胃肠癌的p53相关分子装置，包括检测p53基因突变和突变p53蛋白的新方法，和（ii）开发用于治疗胃肠癌的p53相关分子装置，包括用于p53介导的基因治疗的改进的p53cDNA和用于恢复失活突变型p53功能的小分子化合物。我们开发了几个重要的p53相关的分子装置。首先，通过使用全面的定点突变技术和基于酵母的功能测定来构建、表达和评估2，314个p53突变体，其代表了整个蛋白质中由点突变引起的所有可能的氨基酸取代（每个残基5.9个取代），以及与结构和肿瘤衍生突变相关的p53功能。其次，为了鉴定控制蛋白质功能的关键温度敏感（ts）结构元件，我们筛选了ts p53突变体， ...更多信息 从一个综合性突变库中筛选出2，314个p53错义突变，它们通过p53结合序列在酿酒酵母中具有序列特异性的反式作用。我们分离了142个ts p53突变体，其中包括131个未报道的ts突变体，并表明p53核心DNA结合结构域中的分子内β折叠是控制蛋白功能的关键结构元件，并为寻找能够拯救突变p53功能的分子机制提供了线索。第三，通过使用p53突变库，我们确定了常见的p53突变的第二位点抑制（sss）突变。所鉴定的sss突变通过分子内和分子间的方式恢复了两种常见的突变。这些结果表明，特异性p53突变体的失活功能可以通过外源分子的作用而恢复。第四，作为p53突变文库的密集筛选的结果，我们鉴定了至少几种突变型p53，其具有比野生型p53更强的诱导细胞凋亡的活性。这些突变型p53可能是肿瘤基因治疗的有用cDNA资源。最后，我们正在通过分子对接模拟方法筛选可能恢复突变型p53失活功能的小分子化合物，并发现了几个候选分子。少

The objectives of this study were (i) development of p53-associated molecular devices for diagnosis of gastrointestinal cancer, including novel method of detecting p53 gene mutations and mutant p53 proteins, and (ii) development of p53-associated molecular devices for treatment of gastrointestinal cancer, including improved p53 cDNA for p53-mediated gene therapy and small molecule compounds for restoration of inactivated mutant p53 function. We developed several important p53-related molecular devices. First, by using a comprehensive site-directed mutagenesis technique and a yeast-based functional assay to construct, express, and evalute 2,314 p53 mutants representing all possible amino acid substitutions caused by a point mutation throughout the protein (5.9 substitutions per residue), and cerrelated p53 function with structure-and tumor-derived mutations. Second, to identify key temperature-sensitive (ts) structural elements controlling the protein function, we screened ts p53 mutant … More s from a comprehensive mutation library consisting of 2,314 p53 missense mutations for their sequence-specific transactivity through p53-binding sequences in Saccharomyces cerevisiae. We isolated 142 ts p53 mutants, including 131 unreported ts mutants and showed that the intramolecular beta-sheet in the core DNA-binding domain of p53 was a key structural element controlling the rotein function and provided a clue for finding a molecular mechanism that enables the rescue of the mutant p53 function. Third, by using the p53 mutation library, we identified second-site suppressor (sss) mutations for common p53 mutations. The identified sss mutations restored two of the common mutations by both intramolecular and intermolecular manner. The results suggested that inactivated function of specific p53 mutants might be recovered by extramolecules. Fourth, as a result of intensive screening of the p53 mutation library, we identified at least several mutant p53 that had the stronger activity to induce apoptosis than wild-type p53. These mutant p53 might be useful cDNA resources for cancer gene therapy. Finally, we are screening in silico of small molecular compounds that might restore the inactivated function of mutant p53 through a molecular docking simulation method, and found several candidate molecules. Less