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

 描述(申请人提供)：内在障碍控制P53和其他与癌症相关的IDPs的功能PI‘s Daughdrill/Chen项目摘要/摘要--P53是一种肿瘤抑制因子和细胞周期调节器，由蛋白质相互作用和翻译后修饰(PTM)激活。P53的缺失或突变会极大地增加癌症的易感性。P53也是一种固有的无序蛋白(IDP)。IDPs是高度动态的，不形成稳定的三级结构，并且含有数量可变的瞬时二级结构。IDP结构域是PTMS的研究热点，经常通过偶联折叠和结合来介导蛋白质与蛋白质之间的相互作用。与其他蛋白质相互作用的IDP域可以包含与其复杂结合结构相似的特定水平的瞬时二级结构。这些水平的残基结构可以通过调节在耦合的折叠和结合反应中发生的构象熵的变化来调节与其他蛋白质的结合亲和力。我们最近发表在《自然化学生物学》上的文章表明，在体外和活细胞内，无序的p53转录激活结构域(P53TAD)中的残留螺旋水平控制着与E3泛素连接酶MDM2的结合亲和力。游离p53TAD的残留螺旋度水平受MDM2结合位点两侧保守的Pro控制。将这些脯氨酸突变为丙氨酸导致更高的p53TAD螺旋度和更强的MDM2结合。这种更强的MDM2结合消除了PTMS的作用，从而导致DNA损伤后P53的更快降解。较低的P53水平会减少靶基因的表达，防止细胞周期停滞。我们的结果表明，精确水平的内在紊乱和残留螺旋对于调控P53信号网络是必要的，改变紊乱水平可以改变磷酸化和其他PTM的效果。来自其他研究小组的研究表明，PTMS可以改变内在的紊乱水平。内在无序和PTM状态的共同作用使IDP域能够动态地对蜂窝网络中的信号变化做出反应。我们建议改变P53的内在紊乱水平，并确定其对激活动力学和靶基因表达的影响。我们还将确定内在紊乱如何与PTMS结合来控制蛋白质之间的相互作用。最后，我们将调查其他与癌症相关的国内流离失所者的内在紊乱水平如何控制结构和功能。为了实现这些目标，设计了以下特定目标：目的1)确定内在紊乱如何控制P53的功能，目的2)确定内在紊乱如何与PTMS结合来控制蛋白质-蛋白质相互作用，以及目的3)确定内在紊乱如何控制结合亲和力和结合动力学。为了检验这些目标 我们将使用单细胞荧光显微镜、qPCR阵列和报告分析来监测p53突变体的激活动力学和靶基因表达。为了研究本征无序如何与PTMS结合来控制蛋白质-蛋白质相互作用，以及本征无序如何控制结合亲和力和结合动力学，我们将主要使用核磁共振光谱、等温滴定量热法和停流动力学。

 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.