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：
10614637
负责人：
MICHAEL J. PETRIS
金额：
$ 42.55万
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/10614637
关键词：
Address Altitude Anemia Animal Model Asthma Attenuated CRISPR-mediated transcriptional activation CRISPR/Cas technology Cell Line Cell Nucleus Cells Ceruloplasmin Chronic Kidney Failure Chronic Obstructive Pulmonary 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 HIF1A gene 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 clinical translation 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 translational potential 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）是一种必需的营养素，在氧气传输和利用中起着至关重要的作用。铜直接通过氧化磷酸化在氧气消耗中起作用，这对于铁的运输是必需的，这对于血红蛋白内的氧气转运至关重要。尽管铜在氧代谢中的重要性，但关于降低氧气水平（低氧）对铜稳态的影响知之甚少。缺氧是一种生理压力，有助于许多常见疾病的病理，因此细胞感知和对低氧反应的机制对人类健康至关重要。发现，使用基于CRISPR的创新基因敲除屏幕来对铜稳态的新调节剂，发现von Hippel-Lindau（VHL）基因中的突变刺激了ATP7B的表达，ATP7B是一种主要在肝细胞中表达的铜转运蛋白。 VHL是氧气传感的主要调节剂，我们证明ATP7B表达是由培养细胞和小鼠肝脏中缺氧强烈诱导的。 ATP7B对于将铜插入Ceruloplasmin（一种在铁出口到等离子体中的作用）至关重要。在此提案中，我们将通过促进Ceruloplasmin介导的铁出口到血浆中的能力来检验假设，即低氧诱导的红细胞生成需要ATP7B。已知ATP7B基因中的突变会导致Wilson病，Wilson病是肝铜超负荷的致命疾病。我们证明缺氧诱导替代铜转运蛋白ATP7A的肝表达，该转运蛋白是ATP7B的功能同源物。在此提案中，我们将检验以下假设：缺氧诱导的ATP7A可以减弱Wilson病的ATP7B - / - 小鼠模型中的肝铜过载和肝脏病理学。为了提高我们的研究的转化潜力，还将在ATP7B - / - 小鼠中测试临床认可的低氧拟模拟药物Roxadustat的治疗功效，以解决其作为威尔逊病的新型治疗方法的重新使用的潜力。最后，基于我们的淘汰赛屏幕的成功，我们将对铜稳态的新型调节剂进行创新的基因基因激活屏幕。为了检验这些假设，我们的建议将1）研究铜在适应性反应中的作用； 2）使用创新的基因激活筛选，测试Wilson病动物模型中缺氧的治疗潜力和3）确定哺乳动物铜稳态的新成分。