Fluorogen Activating Peptide-Based FRET to Quantify FceRI Activation Mechanisms

 描述(申请人提供)：肥大细胞激活的一个关键角色是高亲和力的IgE受体，FceRI。虽然对FceRI通过多价抗原的交联所启动的事件链有很多了解，但我们对蛋白质动力学如何促进信号传递的理解存在着根本的差距。我们的长期目标是了解蛋白质相互作用的动态和随机行为如何影响信号传播。这一建议的目的是直接量化FceRI信号早期事件中蛋白质的相互作用和周围膜环境的变化。我们的中心假设是FceRI信号复合体形成的时空协调调节了细胞反应的强度和持续时间。这项拟议研究的基本原理是，了解蛋白质-蛋白质相互作用动力学在信号中的作用是理解细胞如何塑造免疫反应的强度和质量的下一步。这项提议的目标是开发基于荧光素激活肽(FAP)技术的新型FRET测量，为测量活细胞中蛋白质-蛋白质和蛋白质-脂质相互作用提供改进的信号检测。这一新的方法将补充我们正在进行的项目，使我们能够解决有关FceRI信号动力学的其他问题，这些问题是传统方法无法解决的。这种方法是创新的，因为我们将开发在分子水平上对生化事件进行成像的方法，使我们能够获得有关使用传统生物化学技术无法确定的信号事件的动态信息。这项拟议的研究意义重大，因为我们提议获得的定量信息以前从未被直接测量过，并将为细胞生物学领域带来新的视角。此外，基于FAP的FRET方法将广泛适用于其他细胞信号通路。我们获得的有关FceRI信令的信息将有助于填补我们关于FceRI如何启动信令的知识空白。最终， 我们希望这些信息将为针对蛋白质相互作用和定位的药物设计开辟新的途径。

 DESCRIPTION (provided by applicant): A key player in mast cell activation is the high affinity IgE receptor, FceRI. While much is known about the chain of events initiated by crosslinking of FceRI through multivalent antigen, there is a fundamental gap in our understanding of how protein dynamics facilitate signaling. Our long term goal is to understand how the dynamic and stochastic behavior of protein-protein interactions influences signal propagation. The objective of this proposal is to directly quantify protein interactions and changes in the surrounding membrane environment during early events in FceRI signaling. Our central hypothesis is that the spatiotemporal coordination of FceRI signaling complex formation modulates the strength and duration of the cellular response. The rationale for the proposed research is that understanding the role of protein-protein interaction dynamics in signaling is the next step in understanding how the cell shapes the strength and quality of an immune response. The goal of this proposal is to develop novel FRET measurements based on fluorogen activating peptide (FAP) technology that will provide improved signal detection for measuring protein-protein and protein-lipid interactions in living cells. This new methodology will complement our ongoing projects by allowing us to address additional questions about FceRI signaling dynamics that are simply unaddressable with conventional methods. The approach is innovative because we will develop methodology for imaging biochemical events at the molecular level that will allow us to obtain dynamic information about signaling events that cannot be determined using traditional biochemistry techniques. The proposed research is significant because the quantitative information that we propose to obtain has not been directly measured before and will bring new perspectives to the cell biology community. Furthermore the FAP-based FRET methods will be broadly applicable to other cell signaling pathways. The information we gain about FceRI signaling will help to fill the gaps in our knowledge of how FceRI initiates signaling. Ultimately, we expect that this information will open new avenues for drug design that target protein interactions and localization.