Decoding Hydrogen Peroxide Signaling at Cellular Membranes

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，如氢， 过氧化物（H2O2）作为非转录信号分子，以快速响应和适应 环境变化。已经出现了许多H2O2信号传导影响的例子，范围从 植物的非生物胁迫对人类免疫反应的影响。H2O2信号传导和随后的靶向调节 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。H2O2调节细胞 信号传导很大程度上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， 其中这些开关的反应性高度依赖于局部H2O2浓度。我们以前的 对上皮细胞的研究表明，细胞内H2O2浓度的关键决定因素包括生成 通过细胞膜表面酶如NADPH氧化酶和细胞间渗透性， 水通道蛋白（AQP）通道可促进细胞膜。许多细胞过程，如 先天免疫信号传导、囊泡运输和迁移已被证明受H2O2调节，但 细胞膜如何允许H2O2的特异性和特权信号仍然不完全清楚。 因此，我们提出的研究，旨在建立一般规则和紧急概念有关的H2O2 膜上的信号我们的研究将包括四个主要的调查领域，旨在解决i）如何 质膜对H2O2的渗透性是否影响氧化还原信号和调节？（二）H2O2如何 信号和随后的蛋白质调节改变了细胞中基本的囊泡运输途径？㈢如何 H2O2信号发生在囊泡膜上吗？（四）细胞内H2O2的空间控制如何调节细胞内H2O2的释放？ 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 定量荧光成像以跟踪膜处H2O2信号的空间和时间动力学， 结合蛋白质组学方法来鉴定靶向修饰的半胱氨酸。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， 了解H2O2信号如何从细胞膜传递。未来的研究将以此为基础 该框架旨在揭示指导和操纵H2O2信号以治疗人类疾病的策略。 与这些研究相结合，将开发新的工具和方法来研究H2O2信号， 膜，可以广泛应用于该领域的研究。

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.