Molecular analysis of methylated p53

描述（由申请人提供）：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年生存率仍然非常低，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活性可以通过识别p53上的甲基化赖氨酸标记的蛋白效应物53 BP（p53结合蛋白1）和L3 MBT（致死性3恶性脑肿瘤）来调节。p53的这些新的相互作用的分子机制是未知的，将在拟议的研究中阐明。这些机制的详细知识是了解p53的生理和致瘤活性的根本重要性。 我们推测，L3 MBT的MBT结构域与单甲基化p53（p53 K382 me 1）的结合抑制了p53的反式激活，而53 BP Tudor结构域与二甲基化p53（p53 K382 me 2）的结合促进了p53在DNA损伤位点的积累并促进DNA修复。此外，我们建议附近的翻译后修饰（PTM）的p53改变这些效应器的结合特性和微调p53功能。我们试图定义的结构基础和功能的53 BP和L3 MBT与甲基化p53（p53 ME）的相互作用的后果。 本课题的具体目标是：（1）确定53 BP识别p53的分子基础;（2）阐明L3 MBT靶向p53的分子机制。 53 BP Tudor和L3 MBT MBT与单修饰和双修饰的p53肽复合的三维结构将通过X射线晶体学或NMR确定。将检查p53的特异性、结合亲和力和PTM之间的串扰。将突变结合位点残基，并通过荧光光谱法、等温滴定量热法和NMR在体外以及通过免疫沉淀（IP）、染色质IP、PCR和DNA损伤试验在体内检测突变53 BP和L3 MBT蛋白。 深入的生化，结构和功能特性的p53效应器将提供一个全面的了解的机制原则的生理和致癌活动的p53，并可能促进新的抗癌疗法的发展。

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.