Intrinsic disorder controls the function of p53 and other cancer associated IDPs

内在紊乱控制 p53 和其他癌症相关 IDP 的功能

基本信息

批准号：
9279187
负责人：
Jiande Chen
金额：
$ 31.18万
依托单位：
UNIVERSITY OF SOUTH FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-06-01 至 2020-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9279187
关键词：
Affect Affinity Alanine Binding Binding Sites Biological Assay Biology Calorimetry Cell Cycle Cell Cycle Arrest Cell physiology Cells Charge Chemicals Complex Coupled DNA Damage Disease Entropy Fluorescence Microscopy Gene Expression Gene Targeting Goals In Vitro Kinetics Malignant Neoplasms Mediating Molecular Conformation Monitor Mutate Mutation NMR Spectroscopy Nature Phosphorylation Phosphotransferases Post-Translational Protein Processing Post-Translational Regulation Predisposition Proline Property Protein p53 Proteins Publications Reaction Reporter Residual state Signal Transduction Structure TP53 gene Tertiary Protein Structure Testing Titrations Transcriptional Activation Domain Tumor Suppressor Proteins design disorder control improved inter-alpha-inhibitor mutant neoplastic cell prevent protein protein interaction protein structure public health relevance transcription factor ubiquitin-protein ligase

项目摘要

DESCRIPTION (provided by applicant): Intrinsic disorder controls the function of p53 and other cancer-associated IDPs PI's Daughdrill/Chen Project Summary/Abstract -- p53 is a tumor suppressor and cell cycle regulator that is activated by protein-protein interactions and posttranslational modifications (PTMs). Deletion or mutation of p53 can dramatically increase susceptibility to cancer. p53 is also an intrinsically disordered protein (IDP). IDPs are highly dynamic, do not form stable tertiary structures, and contain variable amounts of transient secondary structure. IDP domains are hotspots for PTMs and they frequently mediate protein-protein interactions through coupled folding and binding. IDP domains that interact with other proteins can contain defined levels of transient secondary structure that resemble their complex-bound structure. These levels of residual structure can modulate binding affinities with other proteins by tuning the change in conformational entropy that occurs during the coupled folding and binding reaction. Our recent publication in Nature Chemical Biology showed that levels of residual helicity in the disordered p53 transcriptional activation domain (p53TAD) controlled the binding affinity to the E3 ubiquitin ligase Mdm2, both in vitro and inside living cells. The levelsof residual helicity in free p53TAD were controlled by conserved prolines flanking the Mdm2 binding site. Mutating these prolines to alanine resulted in higher p53TAD helicity and stronger Mdm2 binding. This stronger Mdm2 binding abrogates the effects of PTMs leading to more rapid degradation of p53 following DNA damage. Lower levels of p53 reduce target gene expression and prevent cell cycle arrest. Our results suggest that precise levels of intrinsic disorder and residual helicity are necessary for regulating the p53-signaling network and changing the levels of disorder can modify the effects of phosphorylation and other PTMs. Studies from other groups have shown that PTMs can change intrinsic levels of disorder. Together levels of intrinsic disorder and PTM status allow IDP domains to dynamically respond to signaling changes in cellular networks. We propose to change the levels of intrinsic disorder in p53 and determine the effects on activation dynamics and target gene expression. We will also determine how intrinsic disorder combines with PTMs to control protein-protein interactions. Finally, we will investigate how the levels of intrinsic disorder in other cancer-associated IDPs control structure and function. The following specific aims are designed to accomplish these goals: Aim 1) Determine how intrinsic disorder controls the function of p53, Aim 2) Determine how intrinsic disorder combines with PTMs to control protein-protein interactions, and Aim 3) Determine how intrinsic disorder controls binding affinity and binding kinetics. To test these aims we will monitor the activation dynamics and target gene expression of p53 mutants using single-cell fluorescence microscopy, qPCR arrays, and reporter assays. To investigate how intrinsic disorder combines with PTMs to control protein-protein interactions and how intrinsic disorder controls binding affinity and binding kinetics we will primarily use NMR spectroscopy, isothermal titration calorimetry, and stopped-flow kinetics.

描述（由适用提供）：内在障碍控制p53和其他与癌症相关的IDPS PI的Daughdrill/Chen Project Project摘要/摘要-P53是肿瘤抑制剂和细胞周期调节剂，该肿瘤抑制剂和细胞周期调节剂被蛋白质 - 蛋白质蛋白相互作用和发射后修饰（PTMS）激活。 p53的缺失或突变可以显着增加对癌症的敏感性。 p53也是一种本质上无序的蛋白质（IDP）。 IDP是高度动态的，不会形成稳定的三级结构，并且包含可变量的瞬态二级结构。 IDP结构域是PTM的热点，它们经常通过折叠和结合来介质蛋白质 - 蛋白质相互作用。与其他蛋白质相互作用的IDP结构域可以包含类似于其复杂结构结构的定义的瞬态二级结构。这些残留结构水平可以通过调整在耦合折叠和结合反应期间发生的会议熵的变化来调节与其他蛋白质的结合亲和力。我们最近在自然界的化学生物学中发表的表明，无序的p53转录激活结构域（p53tad）的残留螺旋水平控制着与E3泛素连接酶MDM2的结合亲和力，无论是体外和活细胞内的。自由p53tad中残留的螺旋度的水平由构成MDM2结合位点的脯氨酸控制。将这些脯氨酸突变为丙氨酸会导致较高的p53tad螺旋性和更强的MDM2结合。这种更强的MDM2结合使PTM的影响导致DNA损伤后p53的更快降解。较低的p53降低靶基因表达并防止细胞周期停滞。我们的结果表明，对于调节p53信号网络和改变疾病的水平，需要精确的内在障碍和残留螺旋性水平可以改变磷酸化和其他PTM的影响。来自其他小组的研究表明，PTM可以改变内在疾病水平。内在障碍和PTM状态的水平允许IDP域对细胞网络的信号变化动态响应。我们建议改变p53中内在障碍的水平，并确定对激活动力学和靶基因表达的影响。我们还将确定内在疾病如何与PTM结合以控制蛋白质 - 蛋白质相互作用。最后，我们将研究其他与癌症相关的IDPS控制结构和功能中的内在疾病水平。以下特定目的旨在实现这些目标：目标1）确定内在障碍如何控制p53的功能，目标2）确定内在疾病与PTM与PTMS结合以控制蛋白质 - 蛋白质相互作用，而目标3）确定内在障碍如何控制结合亲和力和结合动力学。测试这些目标我们将使用单细胞荧光显微镜，QPCR阵列和报告基因测定法监测p53突变体的激活动力学和靶基因表达。为了研究内在疾病如何与PTM结合以控制蛋白质 - 蛋白质相互作用以及内在障碍如何控制结合亲和力和结合动力学，我们将主要使用NMR光谱，等温滴定量热法和停止流动动力学。