Mitochondria-Nuclear Crosstalk in Hypoxic Gene Induction

描述（由申请人提供）：细胞、组织和生物体感知和响应氧气水平变化的能力通常对它们的生存至关重要。细胞对缺氧的适应就是一个很好的例子。间歇性缺氧发作与多种人体病理生理状态（如睡眠呼吸暂停、中枢性低通气综合征和血管闭塞）有关；它们可以改变新陈代谢，诱导血管生成，并影响炎症反应。一些研究报道，对缺氧的适应需要诱导大量的核基因，线粒体呼吸链在哺乳动物和酵母细胞中一些缺氧基因的诱导中起重要作用。我们研究的总体目标是了解线粒体在这一过程中的作用。这些研究结合了蛋白质组学、基因组学和遗传策略来分析酵母中的缺氧基因诱导。此外，将对培养的哺乳动物细胞进行平行研究。目的1将使用基因谱分析方法鉴定酵母线粒体呼吸链控制下的缺氧核基因。目的2将重点关注线粒体依赖的缺氧基因诱导途径本身。遗传操作将用于鉴定和排序酵母基因，这是必不可少的诱导线粒体依赖的缺氧核基因。目的3将评估线粒体产生的活性氧和一氧化氮在酵母缺氧基因诱导中的重要性，重点了解蛋白酪氨酸硝化和蛋白羰基化在缺氧信号传导中的作用。目的4关注哺乳动物细胞中的缺氧基因诱导。它解决了两个有争议或未完全探索的问题。首先，它将使用在酵母中已经成功的方法来询问哺乳动物细胞在暴露于缺氧或缺氧时是否会经历短暂的氧化或亚硝化应激，如果是，识别在缺氧或缺氧条件下羰基化或酪氨酸硝化的蛋白质。其次，它将用于RNAi敲除实验，以评估这些羰基化或硝化蛋白在哺乳动物细胞缺氧信号传导中的重要性。细胞对缺氧反应的失败可导致多种病理状态（如贫血、心肌梗死、视网膜病变和肿瘤生长）。对缺氧信号通路的了解可能会导致这些疾病的新疗法，并可能有助于我们对人类衰老的理解。

DESCRIPTION (provided by applicant): The ability of cells, tissues, and organisms to sense and respond to changes in oxygen levels is often crucial to their survival. Cellular adaptation to hypoxia is a good example of this. Intermittent episodes of hypoxia are associated with a variety of human patho-physiological states (e.g., sleep apnea, central hypoventilation syndrome, and vascular occlusion); these can alter metabolism, induce angiogenesis, and affect inflammatory responses. Several studies have reported that adaptation to hypoxia requires the induction of a large number of nuclear genes and that the mitochondrial respiratory chain plays an important role in the induction of some of these hypoxic genes in mammalian and yeast cells. The overall goal of our studies is to understand the role of the mitochondrion in this process. These studies combine proteomic, genomic, and genetic strategies to analyze hypoxic gene induction in yeast. In addition, parallel studies will be done with mammalian cells in culture. Aim 1 will use gene profiling methods to identify hypoxic nuclear genes that are under the control of the mitochondrial respiratory chain in yeast. Aim 2 will focus on the mitochondrial - dependent hypoxic gene induction pathway itself. Genetic manipulation will be used to identify and order yeast genes that are essential for the induction of mitochondrial-dependent hypoxic nuclear genes. Aim 3 will evaluate the importance of mitochondrially-generated reactive oxygen species and nitric oxide in yeast hypoxic gene induction, with emphasis on understanding the role of protein tyrosine nitration and protein carbonylation in hypoxic signaling. Aim 4 focuses on hypoxic gene induction in mammalian cells. It addresses two questions that are controversial or incompletely explored. First, it will use methodologies that have been successful in yeast to ask if mammalian cells experience transient oxidative or nitrosative stress when exposed to hypoxia or anoxia and, if so, identify proteins carbonylated or tyrosine nitrated under anoxic or hypoxic conditions. Second, it will use in RNAi knock down experiments to assess the importance of these carbonylated or nitrated proteins in hypoxic signaling in mammalian cells. The failure of cells to respond properly to hypoxia can lead to a variety of pathological states (e.g., anemia, myocardial infarction, retinopathy, and the growth of tumors). An understanding of hypoxic signaling pathways may lead to new therapies for these diseases and may help with our understanding of human aging.