Decoding Hydrogen Peroxide Signaling at Cellular Membranes (Equipment Supplement)

项目摘要 真核细胞采用不同的机制来感知和响应它们的环境并维持组织 体内平衡细胞已经进化出策略来吸收稳定的活性氧，如过氧化氢 （H2O2）作为非转录信号分子，以快速响应和适应环境 变化已经出现了H2O2信号传导影响的许多例子，从非生物胁迫到植物生长， 植物对人体免疫反应的影响因此，H2O2信号传导和随后的靶蛋白调节是 一种重要但仍未被充分认识的生物控制机制。H2O2调节的细胞信号传导主要是 依赖于蛋白质半胱氨酸残基中氧化还原敏感性巯基开关的存在， 这些开关高度依赖于局部H2O2浓度。我们之前在上皮细胞中的研究 显示细胞H2O2浓度的关键决定因素包括细胞膜产生H2O2， 表面酶如NADPH氧化酶和细胞膜的渗透性， 水通道蛋白（Aquaporin，AQP）许多细胞过程如先天免疫信号传导， 已经证明，H2O2可以调节囊泡的运输和迁移，但细胞膜如何允许 对于H2O2的特异性和特权信号传导仍然不完全了解。因此，我们建议研究 旨在建立与膜上H2O2信号相关的一般规则和新兴概念。我们的研究 将包括四个主要的调查领域，寻求解决i）质膜渗透性如何， H2O2影响氧化还原信号和调节？ii.）H2O2如何信号和随后的蛋白质调节 改变细胞中重要的囊泡运输途径？iii）H2O2信号如何在囊泡中发生 膜？（四）细胞H2O2的空间控制如何调节细胞的定向迁移？解决 这些问题，我们将应用和发展高分辨率定量荧光成像，以跟踪空间 H2O2信号在膜上的时间动态，结合蛋白质组学方法， 靶向修饰的半胱氨酸。进一步的研究将探讨氧化修饰如何改变靶蛋白 结构，功能和定位，构建H2O2信号如何传递的机制理解 从细胞膜中分离出来未来的研究将建立在这个框架，以揭示战略，以指导和 操纵H2O2信号用于治疗人类疾病。与这些研究相结合的将是 新的工具和方法来研究H2O2信号在膜，可广泛应用于研究， 领域

PROJECT SUMMARY Eukaryotic cells employ different mechanisms to sense and respond to their environment and maintain tissue homeostasis. Cells have evolved strategies to co-opt stable reactive oxygen species such as hydrogen peroxide (H2O2) as non-transcriptional signaling molecules in order to rapidly respond and adapt to environmental changes. Numerous examples of the influence of H2O2 signaling have emerged, ranging from abiotic stress in plants to immune responses in humans. H2O2 signaling and subsequent regulation of target proteins is therefore an important but still underappreciated biological control mechanism. H2O2-regulated cell signaling is largely dependent on the presence of redox-sensitive thiol switches in protein cysteine residues, where the reactivity of these switches is highly dependent on the local H2O2 concentration. Our previous studies in epithelial cells have shown that key determinants of cellular H2O2 concentrations include generation of H2O2, by cell membrane surface enzymes such as the NADPH oxidases and permeability across cellular membranes which can be facilitated by Aquaporin (AQP) channels. A number of cellular processes such as innate immune signaling, vesicular trafficking and migration have been shown to be regulated by H2O2, but how cellular membranes allow for specific and privileged signaling by H2O2 remains incompletely understood. We therefore propose studies that aim to establish general rules and emergent concepts related to H2O2 signals at membranes. Our studies will encompass four major areas of inquiry that seek to address i) How does plasma membrane permeability to H2O2 influence redox signaling and regulation? ii.) How do H2O2 signals and subsequent regulation of proteins alter essential vesicular trafficking pathways in the cell? iii) How does H2O2 signaling occur at vesicular membranes? iv.) How does spatial control of cellular H2O2 regulate the directional migration of cells? To address these questions, we will apply and develop high resolution quantitative fluorescence imaging to follow the spatial and temporal dynamics of H2O2 signals at membranes, in combination with proteomic approaches to identify target modified cysteines. Further studies will investigate how oxidative modifications alter target protein structure, function and localization, constructing a mechanistic understanding of how H2O2 signals are relayed from cellular membranes. Future studies will build on this framework to uncover strategies to direct and manipulate H2O2 signals for treatment of human disease. Integrated to these studies will be the development of novel tools and approaches to study H2O2 signals at membranes that can be broadly applied for research in this field.