Nitric Oxide Signaling in Hypoxia and Immunity in Drosophila

描述(由申请人提供)：低氧是最大的健康问题中的重要因素。它是心肌梗死和卒中引起的缺血损伤的罪魁祸首，它在限制肿瘤生长和钝化重要化疗药物的作用方面发挥着核心作用。虽然我们对低氧诱导因子(HIF)对转录的低氧调控有一个机制的了解，但对缺氧的其他反应模式仍然知之甚少。线粒体是细胞中氧气的主要消耗者，它如何适应氧气供应的不足，以及它如何向细胞的其他部分发出信号，要求适应这些不足？线粒体产生的活性氧物种被认为是负责向细胞传递氧应激的物质，但信号产生、转导和作用的确切途径尚未建立。认识到问题的复杂性和问题的根本性质，模式生物中可用的强大遗传学似乎可以做出重大贡献。我们发现，一氧化氮(NO)介导了果蝇对缺氧的即时反应，包括使处于可逆暂停生命状态的胚胎停止生长。我们致力于开发一个强大的、大大简化的实验环境，在其中我们可以剖析NO信号机制。我们发现，NO调节果蝇的先天免疫反应，揭示了缺氧和免疫之间的联系，并给了我们所寻求的简化。免疫记者发出NO作用的信号，而对缺氧的反应可以在经过强大的RNAi筛选的培养细胞中重现。在这个方案中，我们将结合体内研究和细胞培养模型的分析来研究NO介导的信号转导。体内研究将探索感染如何诱导一氧化氮合酶，以及一氧化氮信号如何促进免疫反应。在果蝇S2细胞中，我们将研究缺氧诱导的NO信号的基础，该信号似乎起源于线粒体，并将跟踪该信号在被输送到细胞质时的转换和运输。最后，我们将定义线粒体感知缺氧并向线粒体内的剧烈变化发出信号的基因和途径，以及线粒体变化和线粒体发出的信号之间的耦合是否会对细胞的生存和人类的福祉产生如此巨大的影响。这些研究将使我们从机制上了解低氧反应的一个主要组成部分，有助于理解生物对低氧耐受性的多样性，并为我们提供操纵低氧敏感性的方法，以潜在地有益于重要的健康问题。公共卫生相关性最常见的威胁生命的健康问题--心肌梗死和中风，通过中断氧气供应而造成损害，但我们对由此导致的急性缺氧的生物反应知之甚少。我们将剖析一个强大的模型遗传系统--果蝇--对缺氧的反应机制。我们的发现有可能影响心脏梗死和中风的治疗，并可能对支持器官移植和癌症治疗等不同领域产生影响。

DESCRIPTION (provided by applicant): Hypoxia figures importantly in the biggest of health issues. It is responsible for the damage caused by the ischemia accompanying cardiac infarct and stroke, and it plays a central role in limiting tumor growth as well as blunting the actions of important chemotherapies. While we have a mechanistic understanding of hypoxic regulation of transcription by Hypoxia Inducible Factor (HIF), other modes of response to oxygen deprivation are still poorly understood. How does the mitochondrion, the major consumer of oxygen in the cell, adjust to shortfalls in oxygen supply, and how does it signal to the rest of the cell, demanding accommodations to these shortfalls? Reactive oxygen species produced by mitochondria have been ascribed the responsibility of communicating oxygen stress to the cell, but precise pathways of signal generation, transduction and action have not been established. Recognizing the complexity of the issues and the fundamental nature of the questions, it seems that the powerful genetics available in model organisms could make a major contribution. We found that nitric oxide (NO) mediates immediate responses to hypoxia in Drosophila, including an arrest of embryos in a reversible state of suspended animation. We have devoted ourselves to the development of a powerful and greatly simplified experimental context in which we can dissect the NO-signaling mechanisms. Our finding that NO regulates innate immune responses in Drosophila revealed a hypoxia-immunity connection, and gave us the simplification we sought. Immune reporters signal the action of NO, and the response to hypoxia can be recapitulated in cultured cells amenable to powerful RNAi screens. In this proposal, we will combine in vivo studies and analysis of cell culture models to investigate NO-mediated signaling. In vivo studies will probe how infection induces NOS, and how NO signaling contributes to the immune response. In Drosophila S2 cells, we will examine the basis of hypoxia-induced NO signaling that appears to originate in the mitochondria, and will trace the transformation and transport of this signal as it is conveyed to the cytoplasm. Finally, we will define the genes and pathways by which mitochondria sense hypoxia and signal the dramatic changes within the mitochondria, and will the coupling between mitochondrial changes and the signals emanating from the mitochondria that have such huge impacts on survival of cells and the well-being of man. These studies will give us a mechanistic understanding of a major component of the hypoxia response, contribute to the understanding of the biological diversity in tolerance to hypoxia, and give us approaches to manipulate hypoxia sensitivity to potentially benefit issues of important health concern. PUBLIC HEALTH RELEVANCE The most common life-threatening health problems, cardiac infarct and stroke, cause damage by interrupting oxygen supply, yet we understand little about the biological responses to the resulting acute hypoxia. We will dissect the mechanisms of response to hypoxia in a powerful model genetic system, Drosophila. Our findings have the potential to influence treatment of cardiac infarct and stroke, and could have an impact on areas as diverse as sustaining organs for transplantation, and cancer therapies.