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

 描述（由申请人提供）：内在疾病控制p53和其他癌症相关IDP的功能PI的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因子，通过蛋白质-蛋白质相互作用和翻译后修饰（PTM）激活。p53的缺失或突变可以显著增加对癌症的易感性。p53也是一种内在无序蛋白（IDP）。IDP是高度动态的，不形成稳定的三级结构，并包含可变量的瞬时二级结构。IDP结构域是PTM的热点，它们经常通过偶联折叠和结合介导蛋白质-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合物结合结构的限定水平的瞬时二级结构。这些残余结构水平可以通过调节在耦合折叠和结合反应期间发生的构象熵的变化来调节与其他蛋白质的结合亲和力。我们最近在《自然化学生物学》上发表的文章表明，在体外和活细胞内，无序的p53转录激活结构域（p53 β）中的残余螺旋度水平控制着与E3泛素连接酶Mdm 2的结合亲和力。游离p53蛋白的剩余螺旋度水平受Mdm 2结合位点两侧保守脯氨酸的控制。将这些脯氨酸突变为丙氨酸导致更高的p53螺旋度和更强的Mdm 2结合。这种更强的Mdm 2结合消除了PTM的作用，导致DNA损伤后p53的更快降解。较低水平的p53降低靶基因表达并阻止细胞周期停滞。我们的研究结果表明，精确水平的内在障碍和残余螺旋是必要的调节p53信号网络和改变的障碍水平可以修改磷酸化和其他PTM的影响。其他研究小组的研究表明，PTM可以改变疾病的内在水平。内在紊乱和PTM状态的水平一起允许IDP结构域动态地响应细胞网络中的信号传导变化。我们建议改变p53内在紊乱的水平，并确定对激活动力学和靶基因表达的影响。我们还将确定内在障碍如何与PTM结合来控制蛋白质-蛋白质相互作用。最后，我们将研究其他癌症相关IDP的内在紊乱水平如何控制结构和功能。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PTM结合以控制蛋白质-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阵列和报告基因分析来监测p53突变体的激活动力学和靶基因表达。为了研究内在无序如何与PTM结合来控制蛋白质-蛋白质相互作用以及内在无序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.