Mapping Protein Social Network Dynamics with Photoproximity Profiling Platforms

项目摘要 感知蜂窝环境中的动态变化并将信息转换为重新连接的能力 生物分子间的相互作用构成了细胞信号转导的主干。尽管有很大的兴趣和 投资于能够检测和定量蛋白质-蛋白质和其他蛋白质-生物分子的方法 交互作用，最常用的方法只映射非生理环境中的交互作用 在细胞外，许多对研究中的相互作用起作用的重要因素都消失了。这些方法 尤其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。 环境才能形成和发挥作用。因此，需要新的方法来定量定位蛋白质。 生活系统中的“社交网络”。在这里，我们建议开发和验证几种互补光- 依赖的邻近图谱平台能够检测高密度的活细胞中的蛋白质相互作用动力学 空间和时间分辨率，以及对蜂窝环境的最小扰动。我们将完成 这一目标通过三个相互关联的目标实现，这三个目标得到了初步数据和我们之前发表的 使用细胞内近距离成像平台。首先，我们将合成并测试可调的 光接近化学探测器在细胞内绘制纳米尺度的蛋白质复合体的地图。同时，我们 建议测试可能更高效的催化近距离成像平台，以提高分辨率 细胞内低丰度的大分子复合体。最后，我们建议将这些平台应用于研究 通过整合的抗氧化剂和氧化还原应激动态感知细胞内代谢和氧化还原应激的变化 未折叠的蛋白质反应通路。这些接近轮廓将使得能够起草第一个定量的， 细胞内综合应激反应的综合图谱，它将确定对 癌症、衰老和神经生成障碍等疾病。此外，这些方法和接近度轮廓 这里开发的也将广泛地用于生物界应用于各种问题 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.