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

本研究的目的是(i)开发用于胃肠道癌症诊断的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；（ii）开发用于胃肠道癌症治疗的p53相关分子装置，包括用于p53介导的基因治疗的改进的p53 cDNA和用于恢复失活突变p53功能的小分子化合物。我们开发了几个重要的p53相关分子器件。首先，利用全面的定点诱变技术和基于酵母的功能测定，构建、表达和评估了2314个p53突变体，这些突变体代表了整个蛋白质中由点突变引起的所有可能的氨基酸替换（每个残基5.9个替换），以及与结构和肿瘤衍生突变相关的p53功能。其次，为了确定控制蛋白质功能的关键温度敏感（ts）结构元件，我们从一个包含2,314个p53错义突变的综合突变文库中筛选了p53突变体…More s，并通过酵母的p53结合序列对其序列特异性进行了交易。我们分离了142个ts p53突变体，包括131个未报道的ts突变体，发现p53核心dna结合域的分子内β -sheet是控制该蛋白功能的关键结构元件，为寻找能够挽救p53突变体功能的分子机制提供了线索。第三，利用p53突变文库，我们确定了常见p53突变的第二位点抑制（sss）突变。鉴定的sss突变通过分子内和分子间的方式恢复了两个常见突变。结果表明，特异p53突变体的失活功能可能通过外分子恢复。第四，通过对p53突变文库的深入筛选，我们发现至少有几个突变型p53比野生型p53具有更强的诱导凋亡活性。这些p53突变体可能成为癌症基因治疗的有用cDNA资源。最后，我们通过分子对接模拟方法在计算机上筛选可能恢复突变体p53失活功能的小分子化合物，并找到了几个候选分子。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