凝集特异性蛋白质互作组介导突变体p53促进癌症转移的机制研究

The mutation of tumor suppressor p53 is a key event in the process of tumor formation. The mutant p53 not only loses its tumor suppression function, but also gain new functions to promote tumorigenesis. The mechanism of p53 mutant gain of function (GOF) is pivotal for understanding the process of oncogenesis. In our previous study we found the aggregation of mutant p53 in the perinuclear region, which causes the sequestration of its family members p63 and p73 (Nat Chem Biol,2011). This study partially unvealed the mechanism of mutant p53 GOF. Since the whole human proteome bears numerous sequences that are homologous to the p53 aggregating sequence, the coaggregators of p53 mutant should not be limited within its family members. According to our preliminary data, we proposed a hypothesis of "aggregation-specific protein interactome of mutant p53". This research project will validate this hypothesis using multiple experimental procedures including in vitro peptide binding assay, protein coimmunoprecipitation, colocalization, mass spectrometry, fluorescence resonance energy transfer (FRET) and aggregation-specific fluorescence labeling, etc. By these efforts we aim to characterize the aggregation-mediated interactome of mutant p53 and study its effect on cellular proliferation, infiltration and other malignant phenotypes. This study will help to understand the molecular mechanism of tumorigenesis and provide foundamental basis for the development of new strategies for tumor grading and therapies.

抑癌基因p53的突变是癌症转移的重要相关因素。突变型p53不仅丧失了原有的抑癌功能，还获得了显著的促癌功能，其内在机制的研究对于深入理解癌症转移过程至关重要。我们发现错义突变导致p53蛋白的中心结构域错误折叠并形成凝集体，后者捕获了具有抑癌功能的家族蛋白p63和p73，从而部分地解释了其促癌功能的机制(Nat Chem Biol.2011)。由于人类蛋白质组中存在大量与p53相似的凝集序列，因此与p53共凝集的蛋白很可能不仅限于其家族成员的范围，据此我们提出"凝集特异性的蛋白质互作组"的假说。本研究将深入验证该假说，通过多肽体外结合筛选实验、荧光共振能量转移、免疫共沉淀联合质谱分析、凝集蛋白质特异性染色等方法识别突变型p53的凝集蛋白互作组，阐明靶蛋白的失活对细胞恶性表型的影响，并探讨其与肿瘤转移的关联。本研究将促进对细胞癌变过程的理解，有利于发展新的癌症分型和治疗思路。

本课题按照计划执完整地行了研究任务，探讨凝集特异性蛋白质互作组介导突变体p53促进癌症转移的机制，经过4年的研究，完成了预定的目标，筛选人类蛋白质组中能够与p53 251ILTIITL257 共凝集的多肽序列，明确序列特征与结合强度的关系；研究共凝集多肽相对应的蛋白质与p53 突变体共表达状态下的相互结合，分析内源表达的p53 突变体凝集特异性互作蛋白；阐明p53 突变体诱导的靶蛋白凝集对后者细胞内定位和功能的影响，明确共凝集对细胞增殖的调控作用；在肿瘤组织检测p53 突变体与靶蛋白的共凝集，分析与肿瘤恶性度及对放化疗敏感性的关联程度。阐明了p53基因突变体的多种促癌机制，包括调控GAPLINC长非编码RNA并促进CD44癌基因的功能、p53突变体通过激活多种miRNA影响胃肠癌的预后和转归、通过促进CYP3A4等药物代谢基因的表达而导致肿瘤产生对化疗药物的抵抗。此外，还发现了大肠癌中p53乙酰化的主要调控机制。这些工作对于揭示凝集特异性蛋白质互作组介导突变体p53促进癌症转移的作用具有启示意义。综上所述，原定研究计划已经执行完成。