Decoding Hydrogen Peroxide Signaling at Cellular Membranes

项目总结 真核细胞使用不同的机制来感知和响应环境并维持组织 动态平衡。细胞已经进化出策略，以补充稳定的活性氧物种，如氢 过氧化氢(H_2O_2)作为非转录信号分子以便快速反应和适应 环境变化。已经出现了许多关于过氧化氢信号影响的例子，包括 植物对人类免疫反应的非生物胁迫。H_2O_2信号转导与靶标的后续调控 因此，蛋白质是一种重要但仍未得到充分认识的生物控制机制。过氧化氢调节的细胞 信号在很大程度上依赖于蛋白质半胱氨酸残基中氧化还原敏感的硫醇开关的存在， 其中，这些开关的反应性高度依赖于局部的过氧化氢浓度。我们以前的 对上皮细胞的研究表明，细胞内过氧化氢浓度的关键决定因素包括生成 过氧化氢，由细胞膜表面酶，如NADPH氧化酶和跨细胞通透性 水通道蛋白(AQP)通道可以促进膜的形成。许多细胞过程，如 先天性免疫信号、囊泡运输和迁移已被证明受过氧化氢的调节，但 细胞膜是如何通过过氧化氢实现特定和特权信号的，目前还不完全清楚。 因此，我们建议进行研究，旨在建立与过氧化氢有关的一般规则和紧急概念 膜上的信号。我们的研究将包括四个主要的调查领域，寻求解决i)如何 质膜对过氧化氢的通透性是否影响氧化还原信号和调控？II.)双氧水是如何 信号和随后对蛋白质的调节改变了细胞内基本的囊泡运输途径？三)如何 H_2O_2信号发生在囊泡膜上吗？IV.)细胞内H_2O_2的空间调控如何调节细胞内 细胞的定向迁移？为了解决这些问题，我们将应用和开发高分辨率 定量荧光成像跟踪膜上过氧化氢信号的空间和时间动态， 结合蛋白质组学方法来鉴定目标修饰的半胱氨酸。进一步的研究将调查 氧化修饰如何改变靶蛋白的结构、功能和定位，构建一种 了解过氧化氢信号是如何从细胞膜传递出去的。未来的研究将以此为基础 发现指导和操纵用于治疗人类疾病的过氧化氢信号的战略的框架。 与这些研究相结合的是开发新的工具和方法来研究过氧化氢信号 可广泛应用于该领域研究的膜。

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.