Department of Physics, Syracuse University, 201 Physics Building, Syracuse, New York 13244-1130, USA.
Department of Physics, Syracuse University, 201 Physics Building, Syracuse, New York 13244-1130, USA; Department of Biomedical and Chemical Engineering, Syracuse University, 329 Link Hall, Syracuse, NY 13244, USA; The BioInspired Institute, Syracuse University, Syracuse, NY 13244, USA.
Biochim Biophys Acta Biomembr. 2023 Aug;1865(6):184173. doi: 10.1016/j.bbamem.2023.184173. Epub 2023 May 19.
GTP-bound RAS interacts with its protein effectors in response to extracellular stimuli, leading to chemical inputs for downstream pathways. Significant progress has been made in measuring these reversible protein-protein interactions (PPIs) in various cell-free environments. Yet, acquiring high sensitivity in heterogeneous solutions remains challenging. Here, using an intermolecular fluorescence resonance energy transfer (FRET) biosensing approach, we develop a method to visualize and localize HRAS-CRAF interactions in living cells. We demonstrate that the EGFR activation and the HRAS-CRAF complex formation can be concurrently probed in a single cell. This biosensing strategy discriminates EGF-stimulated HRAS-CRAF interactions at the cell and organelle membranes. In addition, we provide quantitative FRET measurements for assessing these transient PPIs in a cell-free environment. Finally, we prove the utility of this approach by showing that an EGFR-binding compound is a potent inhibitor of HRAS-CRAF interactions. The outcomes of this work form a fundamental basis for further explorations of the spatiotemporal dynamics of various signaling networks.
GTP 结合态 RAS 与它的蛋白效应物相互作用，以响应细胞外刺激，从而为下游途径提供化学输入。在各种无细胞环境中测量这些可逆蛋白-蛋白相互作用（PPIs）已经取得了显著进展。然而，在异质溶液中获得高灵敏度仍然具有挑战性。在这里，我们使用分子间荧光共振能量转移（FRET）生物传感方法，开发了一种在活细胞中可视化和定位 HRAS-CRAF 相互作用的方法。我们证明了可以在单个细胞中同时探测 EGFR 的激活和 HRAS-CRAF 复合物的形成。这种生物传感策略可以区分细胞和细胞器膜上 EGF 刺激的 HRAS-CRAF 相互作用。此外，我们提供了定量 FRET 测量结果，用于评估无细胞环境中的这些瞬时 PPIs。最后，我们通过证明一种 EGFR 结合化合物是 HRAS-CRAF 相互作用的有效抑制剂，证明了这种方法的实用性。这项工作的结果为进一步探索各种信号网络的时空动态奠定了基础。
