Dynamics of Signaling Pathways: Mechanism and Function

描述(申请人提供)：尽管P53信号通路可能是人类细胞中研究最好的通路之一，但关于其调节和功能仍有许多悬而未决的问题。我们对该途径中特定的蛋白质-蛋白质相互作用了解很多，但对输入(损伤和应激信号)和输出(从细胞生长到细胞死亡的细胞结果)之间的整体关系以及决定这些关系的反应动力学了解很少。我在这里要求的补充资金将使我的实验室能够采取两种新颖的方法来解决这些问题。在目标1中，我们将跟进最近的一项发现，该发现使用长期单细胞成像，即在没有外部DNA损伤的情况下，p53显示自发脉冲。这些脉冲似乎与细胞周期有关，因此我们怀疑它们反映了DNA复制或有丝分裂过程中的自发损伤。我们将询问是什么触发了P53自发脉冲，以及是什么控制了它们的形状和时间。我们将使用我们开发的一个系统来量化活细胞中双链DNA断裂的数量，以确定P53自发脉冲是否与细胞生长和分裂期间的自发DNA损伤相关。我们将使用化学和遗传扰动来测试哪些调节器启动和控制P53自发脉冲。了解P53自发脉冲的调节将为我们提供操纵它们的工具，并开发理解它们功能的方法。这些自发的P53脉冲可能在防止非应激细胞中DNA损伤的积累方面起到重要作用--这是以前没有意识到的P53‘S活性的一个特征。我们有初步数据表明，在非应激条件下，P53脉冲不会激活p21，只有一部分细胞会对辐射做出反应，激活p21。我们怀疑这是由于这些细胞中P53的翻译后修饰的差异。在目标2中，我们将使用化学和遗传扰动来强制或阻止特定的p53修饰，并使用活细胞成像测试对p21动力学和细胞命运的影响。我们还将确定在第一次和第二次脉冲期间p53的翻译后修饰，并将使用流式细胞术来分类和分离激活p21的细胞与不激活p21的细胞。然后，我们将使用质谱仪来确定在这些不同的人群中发生了哪些p53修饰。这将使我们第一次能够采取一种公正的方法，将p53修饰的组合与其特定目标基因的激活联系起来。 公共卫生相关性：我们的研究将为控制和操纵P53途径提供新的见解，P53途径可能是保护人类细胞免受癌症发展的最重要途径。这些研究将对p53的生物学机制和功能有更深入的了解，并将为分析、描述和理解单个活细胞中其他信号通路的动力学提供一个原型。

DESCRIPTION (provided by applicant): Although the p53 signaling pathway is perhaps one of the best studied pathways in the human cell, there are many open questions related to its regulation and function. We understand a great deal about specific protein- protein interactions in the pathway, but we understand little about the overall relationship between input (damage and stress signals) and output (cellular outcomes ranging from cell growth to cell death), and the kinetics of the response that determines these relationships. The supplementary funding I request here would allow my lab to take two novel approaches to these questions. In Aim 1, we will follow up on a recent discovery, made using long-term single-cell imaging, that p53 shows spontaneous pulses in the absence of external DNA damage. These pulses appear to be connected to the cell cycle and we therefore suspect that they reflect spontaneous damage during DNA replication or mitosis. We will ask what triggers p53 spontaneous pulses and what controls their shape and timing. We will use a system we have developed for quantifying the amount of double-stranded DNA breaks in live cells to determine whether p53 spontaneous pulses correlate with spontaneous DNA damage during cellular growth and division. We will use chemical and genetic perturbation to test which regulators initiate and control p53 spontaneous pulses. Understanding the regulation of p53 spontaneous pulses will give us the tools to manipulate them, and to develop approaches to understand their function. It is possible that these spontaneous p53 pulses have an important role in preventing the build-up of DNA damage in unstressed cells - a previously unappreciated feature of p53's activity. We have preliminary data showing that p53 pulses in non-stressed conditions do not activate p21 and only a subset of the cells activates p21 in response to irradiation. We suspect that this is due to differences in the post-translational modifications on p53 in these cells. In Aim 2 we will use chemical and genetic perturbations to force, or prevent specific p53 modifications and test the effect on p21 dynamics and on cell fate using live cell imaging. We will also determine the post-translational modification of p53 during the first and second pulse and will use flow cytometry to sort and isolate cells that activate p21 versus cells that do not. We will then use mass spectrometry to determine which p53 modifications occur in these different populations. This will allow us, for the first time, to take an unbiased approach for connecting combinations of p53 modifications with the activation of its specific target genes. PUBLIC HEALTH RELEVANCE: Our study will provide new insights into the control and manipulation of the p53 pathway, perhaps the most important pathway protecting human cells against the development of cancer. These studies will give a deeper understanding of the biological mechanisms and function of p53, and will provide a prototype for the analysis, description, and understanding of the dynamics of other signaling pathways in single living human cells.