Post-translational modification proteomics in 4D: Chemoenzymatic tools to map the dynamic spatial organization of eukaryotic signaling pathways

项目概要/摘要 空间组织和时间动态是真核细胞信号通路的固有特性。 蛋白质亚细胞定位的动态变化是通过翻译后修饰来编程的 蛋白质降解是生物学上的一个重要过程，如磷酸化和蛋白质水解，它们响应于生物刺激而发生。真核 信号传导途径依赖于将PTM引入特定蛋白质以快速改变蛋白质功能， 定位，使细胞能够对变化的内部或环境条件作出反应。但尽管 由于空间组织和时间动态在生物信号传导中的重要性，目前的技术无法 提供一个系统水平的实验映射的动态亚细胞定位后， 改性蛋白质为了应对这一挑战，我们建议开发新的PTM蛋白质组学方法， 亚细胞空间和时间分辨率通过工程遗传靶向，PTM选择性接近 标记酶。这些酶将在特定的亚细胞位置标记后修饰的蛋白质， 使其能够通过基于质谱的蛋白质组学实验进行富集和分析， 具有空间和时间分辨率的PTM。我们将首先集中在两个普遍的PTM，蛋白水解和 磷酸化，这两者在许多与人类相关的生物信号传导途径中发挥关键作用。 健康和疾病。我们将应用蛋白质工程方法来开发三种不同类型的酶 用于蛋白水解neo-C末端、磷酸丝氨酸/磷酸苏氨酸的时空分辨捕获，以及 磷酸酪氨酸。我们将利用这些工具来剖析蛋白质水解的时空动力学 在凋亡过程中;在生长因子信号传导过程中的磷酸化;以及蛋白质水解和 在细胞决定生与死的过程中磷酸化。生物学途径和状态， 我们将开发的工具几乎是无限的，确保他们将有一个广泛的， 对整个生物医学科学的变革性影响。完成拟议的工作将改变我们的 了解细胞信号如何在空间和时间上展开，并有可能揭示新的 在PTM为基础的信号通路的治疗干预的范例。

Project summary/abstract Spatial organization and temporal dynamics are inherent properties of eukaryotic cell signaling pathways. Dynamic changes in the subcellular localization of proteins are programmed by post-translational modifications (PTMs), such as phosphorylation and proteolysis, which occur in response to biological stimuli. Eukaryotic signaling pathways rely on the introduction of PTMs to specific proteins to rapidly change protein function and localization, enabling cells to respond to changing internal or environmental conditions. However, despite the importance of spatial organization and temporal dynamics in biological signaling, current technologies are unable to provide a systems-level experimental mapping of the dynamic subcellular localization of post-translationally modified proteins. To meet this challenge, we propose to develop new methods for PTM proteomics with subcellular spatial and temporal resolution by engineering genetically targetable, PTM-selective proximity labeling enzymes. These enzymes will tag post-translationally modified proteins in specific subcellular locations, enabling their enrichment and analysis with mass spectrometry-based proteomics experiments for mapping PTMs with spatial and temporal resolution. We will initially focus on two pervasive PTMs, proteolysis and phosphorylation, both of which play critical roles in numerous biological signaling pathways relevant to human health and disease. We will apply protein engineering approaches to develop three distinct classes of enzymes for spatiotemporally resolved capture of proteolytic neo-C termini, phosphoserine/phosphothreonine, and phosphotyrosine, respectively. We will deploy these tools to dissect the spatiotemporal dynamics of proteolysis during apoptosis; phosphorylation during growth factor signaling; and crosstalk between proteolysis and phosphorylation during the cellular decision between life and death. The biological pathways and states that can be probed with the tools that we will develop are nearly limitless, ensuring that they will have a broad and transformative impact across the biomedical sciences. Completion of the proposed work will transform our understanding of how cellular signaling unfolds across space and time and has the potential to reveal new paradigms for therapeutic intervention in PTM-based signaling pathways.