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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.

铜是一种重要的营养物质，在氧气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，并且是铁的运输所必需的，铁是血红蛋白内氧运输的关键。尽管铜在氧代谢中很重要，但人们对低氧(低氧)对铜稳态的影响知之甚少。低氧是一种生理应激，导致许多常见疾病的病理，因此细胞感知和响应低氧的机制对人类健康至关重要。利用基于CRISPR的创新的铜稳态调节基因敲除筛选，von Hippel Lindau(VHL)基因突变被发现刺激ATP7B的表达，ATP7B是一种主要在肝细胞中表达的铜转运蛋白。VHL是氧感应的主要调节因子，我们证明了ATP7B在培养细胞和小鼠肝脏中的表达受到缺氧的强烈诱导。ATP7B是将铜插入铜蓝蛋白的关键，铜蓝蛋白是一种铁氧合酶，已知在铁出口到血浆中的作用。在这项提议中，我们将通过ATP7B促进铜蓝蛋白介导的铁进入血浆的能力来检验ATP7B是缺氧诱导的红细胞生成所必需的这一假设。已知ATP7B基因的突变会导致肝豆状核变性，这是一种致命的肝脏铜负荷过高的疾病。我们证明，低氧诱导肝脏表达另一种铜转运蛋白ATP7A，它是ATP7B的功能同源物。在这项建议中，我们将检验低氧诱导的ATP7A可以减轻肝铜超载和肝病理改变的假设。为了增加我们研究的翻译潜力，临床批准的缺氧模拟药物roxadustat的治疗效果也将在ATP7B-/-小鼠身上进行测试，以探讨其作为治疗威尔逊病的新疗法的潜力。最后，基于我们的基因敲除筛查的成功，我们将进行基于CRISPR的创新的基因激活筛查，以寻找铜稳态的新型调节因子。为了验证这些假设，我们的建议将1)调查铜在低氧适应反应中的作用；2)在肝豆状核变性动物模型中测试低氧的治疗潜力；3)使用创新的基因激活屏幕识别哺乳动物铜稳态的新成分。