Dynamics of Signaling Pathways: Mechanism and Function

信号通路的动力学：机制和功能

• 批准号: 7807436
• 负责人: Galit Lahav
• 金额: $ 33.39万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7807436
• 关键词: Affect; Apoptosis; Apoptotic; Biological; Cell Cycle; Cell Cycle Arrest; Cell Death; Cells; Cellular Stress; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA biosynthesis; Data; Development; Event; Flow Cytometry; Fluorescence Microscopy; Funding; Gene Targeting; Genes; Genetic Transcription; Human; Individual; Kinetics; Life; Malignant Neoplasms; Mass Spectrum Analysis; Methods; Mitosis; Modification; Monitor; Mutation; Outcome; Output; Pathway interactions; Pattern; Pediatric Hospitals; Physiologic pulse; Population; Post-Translational Protein Processing; Regulation; Role; Shapes; Signal Pathway; Signal Transduction; Sorting - Cell Movement; Stimulus; Stress; System; TP53 gene; Testing; Time; Width; cell growth; cellular imaging; chemical genetics; follow-up; insight; interest; irradiation; kinase inhibitor; mutant; novel strategies; p53 Signaling Pathway; parent grant; prevent; protein protein interaction; prototype; public health relevance; response; single cell analysis; small molecule; time use; tool; transcription factor

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.

描述（由申请人提供）：尽管p53信号通路可能是人类细胞中研究最佳的途径之一，但有许多与其调节和功能有关的开放问题。我们对途径中特定蛋白质蛋白相互作用的广泛了解，但是我们对输入（损伤和应力信号）和输出（从细胞生长到细胞死亡的细胞结局）之间的总体关系以及决定这些关系的反应的动力学知之甚少。我在这里要求的补充资金将允许我的实验室对这些问题采取两种新颖的方法。在AIM 1中，我们将跟进使用长期单细胞成像进行的最近发现，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在DNA复制或有丝分裂过程中自发损害。我们将询问是什么触发了P53自发脉冲，什么控制其形状和时机。我们将使用开发的系统来量化活细胞中双链DNA断裂的量，以确定p53自发脉冲在细胞生长和分裂过程中是否与自发性DNA损伤相关。我们将使用化学和遗传扰动来测试哪种调节剂启动和控制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，并开发出理解其功能的方法。这些自发的p53脉冲可能在防止无数细胞中DNA损伤的形成中具有重要作用，这是p53活性的先前未欣赏的特征。我们有初步数据表明，在非压力条件下p53脉冲不会激活p21，只有一部分细胞会激活p21，以响应照射。我们怀疑这是由于这些细胞中p53的翻译后修饰的差异。在AIM 2中，我们将使用化学和遗传扰动来迫使或防止特定的p53修饰，并使用活细胞成像测试对P21动力学和对细胞命运的影响。我们还将在第一脉冲和第二个脉冲期间确定p53的翻译后修饰，并将使用流式细胞仪进行分类和分离细胞，从而激活p21与没有的细胞。然后，我们将使用质谱法来确定这些不同人群中发生了哪些p53修改。这将使我们首次采用公正的方法，将p53修饰的组合与其特定靶基因的激活联系起来。 公共卫生相关性：我们的研究将为p53途径的控制和操纵提供新的见解，这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，并为单个活着的人类细胞中其他信号通路的动力学提供了一个原型，以用于分析，描述和理解。