Novel roles of copper in adaptive responses to hypoxia

铜在缺氧适应性反应中的新作用

• 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
• 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

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，在氧的运输和利用中起着至关重要的作用。铜通过氧化磷酸化直接作用于氧的消耗，并且是运输铁所必需的，铁是血红蛋白内氧运输的关键。尽管铜在氧代谢中的重要性，但关于氧水平降低（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，因此细胞感知和响应缺氧的机制对人类健康至关重要。使用创新的基于CRISPR的敲除筛选铜稳态的新型调节剂，发现von Hippel Lindau（VHL）基因的突变刺激ATP 7 B的表达，ATP 7 B是一种主要在肝细胞中表达的铜转运蛋白。VHL是一个主调节氧传感器，我们证明，ATP 7 B的表达强烈诱导缺氧培养的细胞和小鼠肝脏。ATP 7 B对于将铜插入血浆铜蓝蛋白中是必不可少的，血浆铜蓝蛋白是一种已知在铁输出到血浆中起作用的铁氧化酶。在这个建议中，我们将测试的假设，ATP 7 B是需要缺氧诱导的红细胞生成通过其能力，以促进铜蓝蛋白介导的铁输出到血浆中。已知ATP 7 B基因的突变会导致肝豆状核变性，这是一种致命的肝铜超载疾病。我们证明，缺氧诱导肝脏表达的替代铜转运蛋白，ATP 7A，这是一个功能同源的ATP 7 B。在这个提议中，我们将测试的假设，缺氧诱导的ATP 7A可以减轻肝铜超载和肝脏病理的ATP 7 B-/-小鼠模型的威尔逊病。为了提高我们研究的转化潜力，还将在ATP 7 B-/-小鼠中测试临床批准的低氧模拟药物Roxadustat的治疗效果，以解决其重新用作Wilson病新治疗的潜力。最后，基于我们敲除筛选的成功，我们将进行一项创新的基于CRISPR的基因激活筛选，以寻找铜稳态的新型调节剂。为了验证这些假设，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组分。