Nitric Oxide Signaling in Hypoxia and Immunity in Drosophila

描述（由申请人提供）：缺氧在最大的健康问题中占有重要地位。它是由伴随心肌梗塞和中风的缺血引起的损伤的原因，并且它在限制肿瘤生长以及减弱重要的化学疗法的作用方面起着核心作用。虽然我们对缺氧诱导因子（HIF）对缺氧转录的调节机制有了一定的了解，但对缺氧反应的其他模式仍然知之甚少。细胞中氧气的主要消耗者--线粒体如何适应氧气供应的不足，它又如何向细胞的其他部分发出信号，要求它们适应这些不足？由线粒体产生的活性氧被认为是将氧应激传递给细胞的责任，但尚未建立信号产生、转导和作用的精确途径。认识到问题的复杂性和问题的基本性质，似乎模式生物中强大的遗传学可以做出重大贡献。我们发现，一氧化氮（NO）介导的即时反应，缺氧果蝇，包括逮捕胚胎在一个可逆的假死状态。我们一直致力于发展一个强大的和大大简化的实验环境中，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，揭示了缺氧免疫的联系，并给了我们我们所寻求的简化。免疫报告信号的NO的作用，和缺氧的反应，可以在培养的细胞服从强大的RNAi屏幕重演。在本研究中，我们将结合联合收割机的体内研究和细胞培养模型的分析来研究NO介导的信号传导。体内研究将探讨感染如何诱导NOS，以及NO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，似乎起源于线粒体，并将跟踪这种信号的转化和运输，因为它被传递到细胞质。最后，我们将确定线粒体感知缺氧并发出线粒体内剧烈变化信号的基因和途径，并将线粒体变化与线粒体发出的信号之间的耦合，这些信号对细胞的生存和人类的福祉产生巨大影响。这些研究将使我们对缺氧反应的主要组成部分有一个机械的理解，有助于理解生物多样性对缺氧的耐受性，并为我们提供操纵缺氧敏感性的方法，以潜在地有益于重要健康问题。最常见的危及生命的健康问题，心肌梗塞和中风，通过中断氧气供应造成损害，但我们对由此产生的急性缺氧的生物反应知之甚少。我们将在一个强大的模型遗传系统果蝇中剖析对缺氧的反应机制。我们的发现有可能影响心肌梗死和中风的治疗，并可能对维持移植器官和癌症治疗等领域产生影响。

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.