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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.