肿瘤抑制蛋白p53DNA结合结构域与靶基因相互作用的研究

【作者】 薛永来；

【导师】 冯喜增；

【作者基本信息】 南开大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 肿瘤抑制蛋白p53是一个广泛分布的磷蛋白,其具备维持基因组完整的功能。野生型的p53主要由三个功能结构域组成:C端四聚化结构域、N端转录激活结构域和一个中心DNA结合结构域（p53DBD）,其包含96-308氨基酸。p53被细胞压力激活以后,作为一个转录因子进一步激活下游基因,参与包括细胞周期调控、DNA修复、血管形成抑制、肿瘤转移抑制、细胞凋亡等生物学过程。所有这些已知的生物学功能都依赖于其DNA结合特性。野生型的p53通过序列特异性的DNA结合结构域（p53DBD）结合DNA。p53基因在50%的肿瘤当中发生突变,而这些突变主要发生在序列特异性的DNA结合结构域,突变的p53不能激活下游基因转录。因此,序列特异性的DNA结合和转录激活活性是p53最关键的生物学功能。p53DBD的晶体结构表明:p53核心结构域结构为:一个β-三明治结构形成脚手架,支撑两个大环和一个环-片层-螺旋模序。p53DBD上的三个半胱氨酸（Cys176、Cys238和Cys242）和一个组氨酸（His179）结合一个锌离子。锌离子的结合被认为是p53转录激活所必需的,去除锌离子将会使p53DBD降低DNA结合特异性。不过,镉离子、汞离子和铜离子结合p53蛋白,却导致p53构象和DNA结合活性的破坏。不同金属离子对p53DBD蛋白截然相反的作用可以预计:存在其他的金属离子或细胞因子能够结合并影响p53DBD。在本论文中,我们利用荧光滴定法研究了9种金属离子与p53DBD的结合反应,其结合能力依次为Fe³⁺＞Zn²⁺＞Cu²⁺＞Ca²⁺＞Mg²⁺＞Ba²⁺＞Mn²⁺＞Ni²⁺＞Co²⁺。圆二色谱研究结果表明,Ba²⁺、Ca²⁺、Co²⁺、Mn²⁺、Ni²⁺这些离子并未引起蛋白二级结构变化,Zn²⁺、Mg²⁺、Fe³⁺这三种离子诱导蛋白结构细微调整,而Cu²⁺离子结合导致蛋白螺旋结构大量丢失。ANS结合研究结果表明,Mg²⁺与Zn²⁺相似,诱导p53DBD蛋白表面疏水性增强,而Fe³⁺引起p53DBD蛋白表面疏水性降低。因此,Mg²⁺和Fe³⁺可能是潜在的影响或调节p53活性的因子之一。接着,我们研究了Mg²⁺对p53DBD与DNA结合的影响。竞争结合分析表明镁离子与锌离子竞争结合p53DBD。圆二色谱分析表明镁离子结合诱导蛋白结构发生细微的改变而不是激烈的变化。基于电泳迁移分析和荧光分析实验,我们推断:Mg²⁺以一种非序列特异性的方式刺激p53DBD蛋白结合DNA,这与Zn²⁺特异性的方式不同。基于细胞内镁离子浓度相对较高并且能够影响胞内酶的活性的事实,我们认为:镁离子是潜在的影响或调节p53转录激活活性的因子之一。金属离子对p53DBD结构稳定性影响研究表明:金属离子结合能增加p53DBD的结构稳定性和热稳定性。分析丙烯酰胺淬灭实验表明:金属离子结合p53DBD诱导蛋白结构发生变化,并且这种变化给蛋白提供一个保护作用,防止丙烯酰胺淬灭。综合起来,我们认为:p53DBD作为一个转录因子起作用的过程中,金属离子起到双重调节作用。金属离子不仅支持DNA结合活性,而且稳定蛋白结构。荧光共振能量转移（FRET）研究p53DBD与DNA的相互作用表明:p53DBD蛋白结合一个半位点p21启动子诱导DNA弯曲角度为23.6°。本论文关于肿瘤抑制蛋白p53DBD与靶基因相互作用的研究,为我们认识p53DBD蛋白与靶基因相互作用提供了更精确的单分子水平的信息,可以更深层次地认识肿瘤细胞的发生机制,为药物研究和设计提供帮助。更多还原

【Abstract】 The tumor suppressor protein p53 is a widely distributed phosphoprotein which functions to maintain the integrity of the genome. Wild-type p53 consists of three major functional domains, the C-terminal tetramerization domain, the N-terminal transactivation domain and the central DNA binding domain （p53DBD） encompassing amino acid residues from 96 to 308. It acts as a sequence-specific transcription factor to transactivate the downstream target genes which is activated in response to a variety of cell stress, and involves in many biological processes, such as cell cycle arrest, DNA repair, inhibition of angiogenesis, inhibition of metastasis and apoptosis. All of these presently known biological functions of p53 depend critically upon its DNA binding properties. Wild type p53 binds DNA through a sequence-specific DNA binding domain （p53DBD）. p53 is mutated in more than half of the human cancers. The overwhelming majority of these mutations occurs in the sequence-specific DNA binding domain and result in the loss of its DNA binding activity. Therefore, site-specific recognition and DNA-binding activity of p53 are crucial for its tumor suppressor function.The crystal structure of p53DBD reveals that the p53 core domain structure consists of a beta sandwich that serves as a scaffold for two large loops and a loop-sheet-helix motif. Zn²⁺ is coordinated to three Cys （C176, C238 and C242） and a His （H179） in p53DBD. Zinc coordination is thought to be necessary for transcriptional activation and removal of zinc reduces the DNA-binding specificity. Nevertheless, the binding of mercury, cadmium and copper to the protein results in disrupting the p53 conformation and the DNA-binding activity. The opposite effects of metal ions on p53 support the notion that additional metal ions or cellular factors can affect specific recognition.In this paper, we investigated the binding reaction between p53DBD and nine kinds of metal ions by fluorescence titration method, the binding affinity of metal ions to p53DBD is Fe³⁺>Zn²⁺>Cu²⁺>Ca²⁺>Mg²⁺>Ba²⁺>Mn²⁺>Ni²⁺>Co²⁺. Analysis of the far-UV CD data clearly suggested that the binding of Ba²⁺, Ca²⁺, Co²⁺, Mn and Ni did not induce changes in protein secondary structure. The binding of Zn²⁺, Mg²⁺ and Fe³⁺ induced a subtle conformational change, while the binding of Cu²⁺ resulted in a lot of loss in its helical content. Analysis of ANS binding data showed that the binding of Mg²⁺ enhanced hydrophobic exposure on protein surface like Zn²⁺, while Fe³⁺ decreased the hydrophobic exposure. Therefore, Mg²⁺ and Fe³⁺ may be one of potential factors to affect or regulate the transactivation of p53.Then, we investigated the influence of Mg²⁺ on the Binding of p53DBD to DNA. Analysis of competitive binding revealed that magnesium competed with zinc for binding to p53DBD. Analysis of the CD data clearly suggested that the binding of magnesium ion induced a subtle conformational change rather than a radical modification of the overall protein architecture. Based on the results of electrophoretic mobility shift assays and fluorescence experiments, we concluded that the binding of Mg²⁺ stimulated the binding of the protein to DNA in a sequence-independent manner, which differed from that of zinc ions in a sequence-specific manner. Based on the facts that Mg²⁺ exists at relatively high concentration in the cell and several cellular enzymes can be regulated by Mg²⁺, we propose that Mg²⁺ is one of potential factors to affect or regulate the transactivation of p53.Studies on the effect of metal ions on the structural stability of p53DBD showed that the binding of metal ions increased the structural and thermal stability. Analysis of acrylamide quenching experiments revealed that the binding of metal ions to p53DBD induced a structural modification of the protein and this change provided significant protection against acrylamide quenching. Overall, we propose that metal ions play a dual modulatory role in the process of p53DBD functioning as a transcription factor. The metal ions not only support the DNA-binding affinity of p53DBD, but also stabilize the structure of the protein.Studies on the interaction between p53DBD and DNA by FRET showed that the bend angle of a half-site of p21 promoter induced by the binding of p53DBD was 23.6°.Studies on the interaction between p53DBD and the target genes in this paper provide us more precise information to understand the interaction between p53 and DNA on the single molecule lever. It is helpful to understand the mechanism of carcinogenesis of tumor cells and provide a help to the study and design of medicine.更多还原