Novel roles of copper in adaptive responses to hypoxia

铜在缺氧适应性反应中的新作用

基本信息

批准号：
10345243
负责人：
MICHAEL J. PETRIS
金额：
$ 44.41万
依托单位：
UNIVERSITY OF MISSOURI-COLUMBIA
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-30 至 2026-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10345243
关键词：
Address Altitude Anemia Animal Model Asthma Attenuated CRISPR/Cas technology Cell Line Cell Nucleus Cells Ceruloplasmin Chronic Kidney Failure Chronic Obstructive Airway Disease Clinical Clustered Regularly Interspaced Short Palindromic Repeats Complex Congestive Heart Failure Copper Cultured Cells Diabetes Mellitus Disease Diving Electron Transport Erythrocytes Erythroid Progenitor Cells Erythropoiesis Exertion Gene Activation Genes Genetic Genetic Transcription Goals Health Hemoglobin Hepatic Hepatocyte Hepatolenticular Degeneration Homeostasis Homologous Gene Human Hypoxia Iron Knock-out Liver Liver diseases Malignant Neoplasms Mammals Mediating Messenger RNA Metabolism Mitochondria Modeling Molecular Mus Mutation Nutrient Oxidative Phosphorylation Oxygen Oxygen Consumption Pathology Pharmaceutical Preparations Physiological Plasma Play Process Production Proteins Regulation Research Role Sleep Apnea Syndromes Stress Testing Tissues Treatment Efficacy VHL gene VHL protein Wilson disease protein base gain of function gain of function mutation genome-wide innovation iron metabolism metal transporting protein 1 mimetics mouse model normoxia novel oxygen transport response success therapeutic evaluation tissue oxygenation transcription factor ubiquitin-protein ligase

项目摘要

Copper (Cu) is an essential nutrient that plays vital roles in oxygen transport and utilization. Copper functions directly in the consumption of oxygen via oxidative phosphorylation and is required for the transport of iron which is a vital for oxygen transport within hemoglobin. Despite the importance of copper in oxygen metabolism, little is known regarding the effects of reduced oxygen levels (hypoxia) on copper homeostasis. Hypoxia is a physiological stress that contributes to the pathology of many common diseases, thus the mechanisms by which cells sense and respond to hypoxia is of fundamental importance to human health. Using an innovative CRISPR-based knockout screen for novel regulators of copper homeostasis, mutations in the von Hippel Lindau (VHL) gene were found to stimulate the expression of ATP7B, a copper transporter primarily expressed in hepatocytes. VHL is a master regulator of oxygen sensing and we demonstrate that ATP7B expression is strongly induced by hypoxia in cultured cells and in the liver of mice. ATP7B is essential for inserting copper into the ceruloplasmin, a ferroxidase with known roles in iron export into the plasma. In this proposal, we will test the hypothesis that ATP7B is required for hypoxia-induced erythropoiesis through its ability to facilitate ceruloplasmin-mediated iron export into the plasma. Mutations in the ATP7B gene are known to cause Wilson disease, a lethal disorder of hepatic copper overload. We demonstrate that hypoxia induces hepatic expression of an alternative copper transporter, ATP7A, which is a functional homologue of ATP7B. In this proposal, we will test the hypothesis that hypoxia-induced ATP7A can attenuate hepatic copper overload and liver pathology in the ATP7B-/- mouse model of Wilson disease. To increase the translational potential of our studies, the therapeutic efficacy of a clinically approved hypoxia-mimetic drug Roxadustat will also be tested in ATP7B-/- mice to address its potential for repurposing as a novel treatment for Wilson disease. Finally, based on the demonstrated success of our knockout screen, we will perform an innovative CRISPR- based gene activation screen for novel regulators of copper homeostasis. To test these hypotheses, our proposal will 1) investigate the roles of copper in adaptive responses to hypoxia; 2) test the therapeutic potential of hypoxia in animal models of Wilson disease and 3) identify novel components of mammalian copper homeostasis using an innovative gene activation screen.

铜 (Cu) 是一种必需营养素，在氧气运输和利用中发挥着重要作用。铜通过氧化磷酸化直接参与氧的消耗，并且是铁的运输所必需的，而铁对于血红蛋白内的氧运输至关重要。尽管铜在氧代谢中很重要，但人们对氧水平降低（缺氧）对铜稳态的影响知之甚少。缺氧是一种生理应激，导致许多常见疾病的病理学，因此细胞感知和响应缺氧的机制对人类健康至关重要。使用基于 CRISPR 的创新敲除筛选新型铜稳态调节因子，发现 von Hippel Lindau (VHL) 基因的突变可以刺激 ATP7B（一种主要在肝细胞中表达的铜转运蛋白）的表达。 VHL 是氧传感的主要调节器，我们证明 ATP7B 表达是由培养细胞和小鼠肝脏中的缺氧强烈诱导的。 ATP7B 对于将铜插入铜蓝蛋白至关重要，铜蓝蛋白是一种铁氧化酶，在铁输出到血浆中发挥着已知的作用。在本提案中，我们将测试以下假设：ATP7B 通过促进铜蓝蛋白介导的铁输出至血浆的能力，是缺氧诱导的红细胞生成所必需的。已知 ATP7B 基因突变会导致威尔逊病，这是一种致命的肝铜超载疾病。我们证明缺氧会诱导另一种铜转运蛋白 ATP7A 的肝脏表达，ATP7A 是 ATP7B 的功能同源物。在本提案中，我们将测试缺氧诱导的 ATP7A 可以减轻威尔逊病 ATP7B-/- 小鼠模型中的肝铜超载和肝脏病理学的假设。为了增加我们研究的转化潜力，临床批准的拟缺氧药物 Roxadustat 的治疗效果也将在 ATP7B-/- 小鼠中进行测试，以解决其重新用作威尔逊病新疗法的潜力。最后，基于我们的敲除筛选所取得的成功，我们将对新型铜稳态调节剂进行基于 CRISPR 的创新基因激活筛选。为了检验这些假设，我们的建议将：1）研究铜在缺氧适应性反应中的作用； 2) 在威尔逊病动物模型中测试缺氧的治疗潜力，3) 使用创新的基因激活筛选来识别哺乳动物铜稳态的新成分。