Mapping Protein Social Network Dynamics with Photoproximity Profiling Platforms

项目摘要 感知细胞环境的动态变化并将这些信息转化为 生物分子相互作用形成细胞信号转导的骨架。尽管兴趣浓厚， 对能够检测和定量蛋白质-蛋白质和其它蛋白质-生物分子方法的投资 相互作用，最常用的方法仅映射非生理环境中的相互作用 在细胞外，许多重要的因素有助于研究中的相互作用丢失。这些方法 特别不适合研究依赖于细胞结构和化学物质的细胞中的信号事件。 环境，以形成和发挥作用。因此，需要新的方法来定量绘制蛋白质图谱 “社交网络”的概念。在这里，我们建议开发和验证几个互补光- 依赖性邻近分析平台，其能够检测具有高活性的活细胞中的蛋白质相互作用动力学 空间和时间分辨率，以及对细胞环境的最小扰动。要全面完成 通过三个相互关联的目标实现这一目标，这些目标得到了初步数据和我们先前发表的 与细胞内光邻近性分析平台一起工作。首先，我们将合成和测试可调 光致接近化学探针，用于在细胞内绘制纳米尺度的蛋白质复合物。同时，我们 建议测试潜在的更有效的催化光邻近性分析平台，以提高 低丰度的大分子复合物。最后，我们建议应用这些平台进行研究 通过整合的抗氧化剂动态感知细胞内改变的代谢和氧化还原应激， 未折叠蛋白反应途径。这些邻近情况将有助于起草第一份定量报告， 细胞综合应激反应的全面地图，这将确定干预点， 疾病，如癌症、衰老和神经再生障碍。此外，所述方法和邻近度简档 在此开发的也将是广泛有用的生物界的应用程序，以不同的问题， 细胞内信号转导

Project Summary The ability to sense dynamic changes in the cellular environment and translate that information into rewired biomolecular interactions forms the backbone of cellular signal transduction. Despite significant interest and investment in methods capable of detecting and quantifying protein-protein and other protein-biomolecule interactions, the most commonly employed methods solely map interactions in non-physiologic environments outside of cells where many important factors contributing to the interactions under study are lost. These methods are particularly poorly suited to study signaling events in cells that rely on the cellular architecture and chemical environment in order to form and function. Therefore, new methods are needed to quantitatively map protein “social networks” inside of living systems. Here we propose to develop and validate several complementary light- dependent proximity profiling platforms capable of detecting protein interaction dynamics in live cells with high spatial and temporal resolution, as well as minimal perturbation to the cellular environment. We will accomplish this goal through three interconnected aims that are supported by preliminary data and our previously published work with an intracellular photoproximity profiling platform. First, we will synthesize and test tunable photoproximity chemical probes to map protein complexes at nanometer scale inside of cells. In parallel, we propose to test potentially more efficient catalytic photoproximity profiling platforms for increased resolution of low abundance macromolecular complexes inside of cells. Finally, we propose to apply these platforms to study the dynamic sensing of altered metabolic and redox stress inside cells through the integrated antioxidant and unfolded protein response pathways. These proximity profiles will enable drafting of the first quantitative, comprehensive maps of the integrated stress response in cells, which will identify points of intervention for diseases such as cancer, aging and neurogenerative disorders. Furthermore, the methods and proximity profiles developed herein will also be widely useful to the biological community for application to diverse questions in intracellular signal transduction.