Molecular Mechanisms of the Hypoxic Response

缺氧反应的分子机制

基本信息

批准号：
8217211
负责人：
FRANK S LEE
金额：
$ 25.95万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-04-01 至 2015-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8217211
关键词：
Adult Anemia Biological Models Bone Marrow Cardiovascular system Chronic Kidney Failure Clinical Collaborations Complex Complication Coronary artery Coupled Disease End stage renal failure Enhancers Erythrocytes Erythrocytoses Erythropoietin Family Functional disorder Gene Expression Regulation Gene Targeting Genes Genetic Genetic Transcription Genetically Engineered Mouse Germ-Line Mutation Glycolysis Glycoproteins Hormones Human Hydroxylation Hypoxia Hypoxia Inducible Factor In Vitro Inherited Kidney Knockout Mice Knowledge Lead Life Liver Mammalian Cell Mediating Methods Missense Mutation Modeling Modification Molecular Mus Mutation Names Oxygen Oxygen measurement, partial pressure, arterial Pathway interactions Patients Physiology Post-Translational Protein Processing Procollagen-Proline Dioxygenase Production Protein Isoforms Proteins Rare Diseases Red Cell Mass result Regulation Role Site Tertiary Protein Structure Therapeutic Ubiquitin angiogenesis bHLH-PAS factor HLF base cerebrovascular chemotherapy egg glucose uptake human disease hypoxia inducible factor 1 in vitro Assay in vivo interest mouse model multicatalytic endopeptidase complex neoplastic oxygen-regulated proteins professor protein degradation public health relevance research study response tissue oxygenation transcription factor

项目摘要

DESCRIPTION (provided by applicant): The master regulator of the mammalian transcriptional response to hypoxia is the transcription factor Hypoxia Inducible Factor (HIF), the subunit of which is regulated at the level of protein turnover in an oxygen-sensitive manner. Under normoxic conditions, Prolyl Hydroxylase Domain protein (PHD) site- specifically hydroxylates HIF-(, which in turn targets HIF-( for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, this posttranslational modification, which is inherently oxygen dependent, is inhibited, thereby allowing stabilization of HIF-(. HIF then upregulates a battery of genes involved in cellular, local, and systemic responses to hypoxia. The prototypical HIF target gene is that encoding for Erythropoietin (EPO), a glycoprotein hormone that regulates red blood cell mass in response to changes in oxygen tension. Thus, understanding HIF regulation will have implications for understanding and treating disorders of red blood cell mass regulation, such as anemia, which in turn is a significant complication seen in many clinical settings, including end stage renal disease and chemotherapy. More generally, hypoxia is a central feature of many human diseases, including coronary artery, cerebrovascular, and neoplastic disease, and therefore knowledge regarding HIF regulation will also impact our understanding of these diseases. There are three HIF-( isoforms (HIF-1(, HIF-2(, and HIF-3() and three Prolyl Hydroxylase Domain proteins (PHD1, PHD2, PHD3) that can hydroxylate them, raising the critical question of which isoforms are important for human physiology and pathophysiology. In collaboration with Professor Terence Lappin's group, we have identified a family with hereditary erythrocytosis (increased red blood cell mass) due to a G537W missense mutation in the HIF2A gene, and another family with erythrocytosis due to a P317R missense mutation in the PHD2 gene. These studies provide the first identification of hereditary mutations in any HIF or in any PHD isoform, and establish two new genetic causes of erythrocytosis. We have subsequently identified additional mutations in both genes. Our Specific Aims are to (1) study new erythrocytosis-associated HIF-2( and PHD2 mutations using in vitro assays in order to bolster our hypothesis that these proteins critically control EPO, (2) employ a Hif2a knockin mouse to model the human G537W missense mutation and examine functional consequences in vivo of dysregulation of Hif2-(, and (3) employ both a Phd2 knockin mouse for the P317R mutation, and a global conditional Phd2 knockout mouse to examine the mechanism by which Phd2 regulates red cell mass. Collectively, we anticipate that these studies will substantially increase our understanding of EPO regulation and, more broadly, our understanding of the mammalian oxygen sensing pathway. PUBLIC HEALTH RELEVANCE: This project seeks to identify and characterize the molecular pathway that leads to the control of red blood cell mass, and more generally, the response to low oxygen tension. The proposed studies focus on two proteins named Hypoxia Inducible Factor-2 and Prolyl Hydroxylase Domain protein 2 that have been implicated in controlling the hormone, Erythropoietin, that determines red cell mass. The proposed experiments will have implications for treating diseases such as anemia, in which red blood cell mass is abnormally low.

描述（申请人提供）：哺乳动物对缺氧转录反应的主要调节因子是转录因子缺氧诱导因子（HIF），其亚基以氧敏感方式在蛋白质周转水平上受到调节。在含氧量正常的条件下，脯氨酰羟化酶结构域蛋白 (PHD) 位点特异性羟基化 HIF-(，进而以 HIF-( 为目标，通过泛素蛋白酶体途径进行降解。在缺氧条件下，这种本质上依赖于氧的翻译后修饰是抑制，从而使 HIF-( 稳定。然后，HIF 上调一系列涉及细胞、局部和全身对缺氧反应的基因。典型的 HIF 靶基因是它编码促红细胞生成素 (EPO)，这是一种糖蛋白激素，可调节红细胞质量以响应氧张力的变化。因此，了解 HIF 调节将对理解和治疗红细胞质量调节疾病（例如贫血）产生影响。反过来，缺氧是许多临床环境中常见的严重并发症，包括终末期肾病和化疗。更一般地说，缺氧是许多人类疾病的核心特征，包括冠状动脉、脑血管和肿瘤疾病，因此也是有关 HIF 的知识。监管也将影响我们对这些疾病的理解。存在三种 HIF-( 亚型（HIF-1(、HIF-2( 和 HIF-3()）和三种脯氨酰羟化酶结构域蛋白（PHD1、PHD2、PHD3））可以将它们羟基化，这就提出了一个关键问题：哪些亚型是对人类生理学和病理生理学很重要。我们与 Terence Lappin 教授的团队合作，发现了一个患有遗传性红细胞增多症（红色增加）的家族。由于 HIF2A 基因中的 G537W 错义突变而导致红细胞增多症，以及由于 PHD2 基因中的 P317R 错义突变而导致红细胞增多症的家族。这些研究首次鉴定了任何 HIF 或任何 PHD 同种型中的遗传性突变。确定了红细胞增多症的两个新的遗传原因。我们随后确定了这两个基因的其他突变。我们的具体目标是（1）研究与红细胞增多症相关的新基因。使用体外测定的 HIF-2( 和 PHD2 突变，以支持我们的假设，即这些蛋白质关键控制 EPO，(2) 使用 Hif2a 敲入小鼠来模拟人类 G537W 错义突变，并检查 Hif2- 失调的体内功能后果（，以及（3）使用 P317R 突变的 Phd2 敲入小鼠和全局条件性 Phd2 敲除小鼠来检查 Phd2 调节红色的机制细胞团。总的来说，我们预计这些研究将大大增加我们对 EPO 调节的理解，更广泛地，我们对哺乳动物氧传感途径的理解。公共健康相关性：该项目旨在识别和表征导致红细胞质量控制的分子途径，更普遍的是，对低氧张力的反应。拟议的研究重点关注两种名为缺氧诱导因子 2 和脯氨酰羟化酶结构域蛋白 2 的蛋白质，它们与控制决定红细胞质量的促红细胞生成素激素有关。拟议的实验将对治疗贫血等红细胞质量异常低的疾病产生影响。