Molecular analysis of methylated p53

描述(申请人提供)：肿瘤抑制基因p53在大约一半的人类恶性肿瘤中发生突变。在大多数剩余的癌症中，由于调节P53活性的信号通路的改变，P53的功能受到了损害。尽管最近取得了进展，但癌症患者的总体5年生存率仍然很低，迫切需要开发更有效的抗癌疗法。P53是一个有吸引力的治疗靶点，了解其调控的分子机制对于设计新的治疗策略至关重要。我们的初步数据显示，P53的活性可以被识别P53上甲基化赖氨酸标记的蛋白质效应物53BP(P53结合蛋白1)和L3MBT(致命的3种恶性脑瘤)所调节。P53这些新的相互作用背后的分子机制尚不清楚，将在拟议的研究中阐明。对这些机制的详细了解对于理解P53的生理和致瘤活性是非常重要的。我们推测，L3MBT的MBT结构域与单甲基化的P53(P53K382me1)结合可抑制P53的反式激活，而53BP的Tudor结构域与二甲基化的P53(P53K382me2)结合可促进P53在DNA损伤部位的积聚，促进DNA修复。此外，我们认为附近的P53翻译后修饰(PTM)改变了这些效应物的结合特性，并微调了P53的功能。我们试图确定53BP和L3MBT与甲基化的p53(P53me)相互作用的结构基础和功能后果。本项目的具体目的是：(1)确定53bp识别P53的分子基础；(2)阐明L3MBT靶向P53的分子机制。53BP Tudor和L3MBT MBT与单链和双链修饰的P53多肽形成的复合体的三维结构将通过X射线结晶学或核磁共振来确定。P53的特异性、结合亲和力和PTM之间的串扰将被检测。突变的结合部位残基将被突变，突变的53BP和L3MBT蛋白将在体外通过荧光光谱、等温滴定量热法和核磁共振进行检测，并在体内通过免疫沉淀(IP)、染色质IP、PCR和DNA损伤检测。深入研究p53me效应子的生化、结构和功能特征将有助于全面了解p53的生理和致癌活性的机制，并可能有助于开发新的抗癌治疗方法。

DESCRIPTION (provided by applicant): The tumor suppressor p53 is mutated in about half of all human malignancies. In most of the remaining cancers, the function of p53 is compromised due to alterations in signaling pathways that regulate p53 activity. Despite recent advances, the overall 5-year survival rate for cancer patients remains very low and there is an urgent need for the development of more efficient anti-cancer therapies. p53 is an attractive therapeutic target and understanding the molecular mechanisms of its regulation is essential for the design of new treatment strategies. Our preliminary data reveal that p53 activity can be modulated by protein effectors 53BP (p53-binding protein 1) and L3MBT (lethal 3 malignant brain tumor) that recognize methylated lysine marks on p53. The molecular mechanisms underlying these novel interactions of p53 are unknown and will be elucidated in the proposed studies. Detailed knowledge of these mechanisms is of fundamental importance for understanding the physiological and tumorigenic activities of p53. We hypothesize that binding of the MBT domain of L3MBT to monomethylated p53 (p53K382me1) represses p53 transactivation, whereas binding of the 53BP Tudor domain to dimethylated p53 (p53K382me2) facilitates p53 accumulation at DNA damage sites and promotes DNA repair. Furthermore, we propose that nearby posttranslational modifications (PTMs) of p53 alter the binding properties of these effectors and fine-tune p53 functions. We seek to define the structural basis and functional consequences of the interactions of 53BP and L3MBT with methylated p53 (p53me). The specific aims of this project are: (1) to determine the molecular basis of p53 recognition by 53BP and (2) to elucidate the molecular mechanism of p53 targeting by L3MBT. The three-dimensional structures of 53BP Tudor and L3MBT MBT in complex with singly and doubly modified p53 peptides will be determined by X-ray crystallography or NMR. The specificities, binding affinities and the crosstalk between PTMs of p53 will be examined. The binding site residues will be mutated and the mutant 53BP and L3MBT proteins will be tested in vitro by fluorescence spectroscopy, isothermal titration calorimetry and NMR and in vivo by immunoprecipitation (IP), chromatin IP, PCR and DNA damage assays. In-depth biochemical, structural and functional characterization of the p53me effectors will provide a comprehensive understanding of the mechanistic principles underlying physiological and oncogenic activities of p53 and may facilitate the development of novel anti-cancer therapies.