Molecular analysis of methylated p53

甲基化 p53 的分子分析

基本信息

批准号：
8447202
负责人：
TATIANA G KUTATELADZE
金额：
$ 36.35万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-08-01 至 2017-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8447202
关键词：
Active Sites Affinity Binding Binding Sites Biochemical Biological Biological Assay Biological Process Calorimetry Cancer Patient Cells Chromatin Chromatin Modeling Complex DNA Damage DNA Repair Data Development Fluorescence Spectroscopy Gene Expression Regulation Gene Targeting Human Immunoprecipitation Knowledge Light Lysine Malignant Neoplasms Malignant neoplasm of brain Measures Mediating Mediator of activation protein Methyltransferase Modeling Molecular Molecular Analysis Mutagenesis Mutate Normal Cell Oncogenic Peptides Physiological Post-Translational Protein Processing Property Protein p53 Proteins Regulation Resolution Role Signal Pathway Signal Transduction Site Solutions Specificity Structure Survival Rate Testing Therapeutic Intervention Titrations Transactivation Tumor Suppressor Proteins X-Ray Crystallography base cancer therapy chromatin immunoprecipitation design gene repression human 53BP1 protein in vitro testing in vivo insight loss of function mutant novel promoter public health relevance research study response therapeutic target three dimensional structure treatment strategy tumorigenesis tumorigenic

项目摘要

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.

描述（由申请人提供）：大约一半的人类恶性肿瘤中肿瘤抑制因子 p53 发生突变。在大多数其余癌症中，由于调节 p53 活性的信号通路发生改变，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，了解其调节的分子机制对于设计新的治疗策略至关重要。我们的初步数据表明，p53 活性可以通过识别 p53 上甲基化赖氨酸标记的蛋白效应器 53BP（p53 结合蛋白 1）和 L3MBT（致命 3 恶性脑肿瘤）来调节。 p53 这些新颖相互作用的分子机制尚不清楚，将在拟议的研究中得到阐明。详细了解这些机制对于理解 p53 的生理和致瘤活性至关重要。我们假设 L3MBT 的 MBT 结构域与单甲基化 p53 (p53K382me1) 的结合抑制 p53 反式激活，而 53BP Tudor 结构域与二甲基化 p53 (p53K382me2) 的结合促进 p53 在 DNA 损伤位点积累并促进 DNA 修复。此外，我们建议 p53 附近的翻译后修饰 (PTM) 会改变这些效应子的结合特性并微调 p53 功能。我们试图定义 53BP 和 L3MBT 与甲基化 p53 (p53me) 相互作用的结构基础和功能后果。该项目的具体目标是：（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 与单修饰和双修饰 p53 肽复合物的三维结构将通过 X 射线晶体学或 NMR 确定。将检查 p53 PTM 之间的特异性、结合亲和力和串扰。结合位点残基将发生突变，突变的 53BP 和 L3MBT 蛋白将通过荧光光谱、等温滴定量热法和 NMR 进行体外测试，并通过免疫沉淀 (IP)、染色质 IP、PCR 和 DNA 损伤测定进行体内测试。 p53me效应子的深入生化、结构和功能表征将提供对p53生理和致癌活性的机制原理的全面理解，并可能促进新型抗癌疗法的开发。