Mechanisms of Homeostatic Control of Copper in Tissues

组织中铜的稳态控制机制

• 批准号: 8137344
• 负责人: Lawrence Wilson Gray
• 金额: $ 4.18万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-22 至 2012-09-21
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8137344
• 关键词: ATP phosphohydrolase; ATP7A protein; African; Asians; Bile fluid; Binding; Bioavailable; Biochemical; Biological Assay; Biological Markers; Caucasians; Caucasoid Race; Cell Line; Cell physiology; Cells; Characteristics; Chelation Therapy; Childhood; Cirrhosis; Clinical Trials; Copper; Copper Chelation; Data; Detection; Diagnosis; Diagnostic; Disease; Down-Regulation; Early Diagnosis; Enzymes; Equilibrium; Excretory function; Failure; Gene Mutation; Genes; Goals; Gray unit of radiation dose; Hepatic; Hepatocyte; Hepatolenticular Degeneration; Homeostasis; Kidney; Kinetics; Lead; Life; Liver; Measures; Menkes Kinky Hair Syndrome; Mental disorders; Messenger RNA; MicroRNAs; Modeling; Molecular; Molecular Weight; Mus; Mutation; Nature; Neurologic; Northern Blotting; Patients; Physiology; Population; Proteins; Public Health; Pump; Regulation; Research; Resolution; Screening procedure; Series; Small Interfering RNA; Source; Spinal Cord Diseases; Staging; Testing; Time; Tissues; Trace Elements; Up-Regulation; Urine; Western Blotting; Wilson disease protein; absorption; base; copper-transporting ATPase; disease diagnosis; insight; kidney cell; mouse model; nervous system disorder; novel; response; success; tandem mass spectrometry; trafficking; uptake; urinary

DESCRIPTION (provided by applicant): The long-term goal of this research is to elucidate the systemic and cellular processes that control copper homeostasis. Copper is an essential trace element. The misbalance of copper, either through poor absorption or overload results in severe disorders such as Menkes disease, progressive ataxic myelopathy, and Wilson's disease (WD). WD is caused by mutations in the ATP7B gene. ATP7B is a copper- transporting ATPase, that pumps copper out of the body and into the bile. ATP7B inactivation causes the accumulation of toxic levels of copper In the body, especially in the liver which can lead to cirrohosis and failure. WD patients also suffer from neurological and pyschiatric abnormalities. If WD is diagnosed early it can be treated with life-long treatment of copper-chelation therapy. Since, all the manifestations presented in Wilson's disease are not unique, diagnosing the disease is a challenge. Even though we now understand some general aspects of copper regulation, we have yet to explain some of the characteristic features of WD. Specifically, (1) why do WD patients have increased urinary copper? (2) What is the nature of the urinary copper? (3) How is the kidney able to balance copper so well? My recent discovery of a novel small copper-carrier (SCC) in the urine of WD patients can help fill these gaps. Specific Aim I: To characterize SCC in the urine of the Wilson's disease mouse (Atp7b-/-). To do this, I will use a series of high-resolution chromatographic steps to purify SCC to homogeneity and characterize it by tandem mass-spectrometry. Specific Aim II: To characterize the relationship between CTR1 levels in ATP7b-/- hepatoctyes and SCC in the urine. I hypothesize that CTR1 down-regulation leads to increased SCC in the urine. To test this I will measure CTR1 mRNA and protein (by real-Time PCR and Western blot) in the liver at different stages of the disease and compare it to amounts of SCC over the same time period Specific Aim III: To determine the molecular basis of ATP7A protein upregulation in Atp7b-/- kidney. I hypothesize that copper delivery by SCC, ATP7B inactivation, and decrease in miR-107 lead to upregulation of ATP7A. To test this, I will perform copper uptake, siRNA, and northern blot analyses. Relevance to public health: Understanding these questions would provide better insight into how the body balances copper levels. Additionally, SCC could be a unique indicator of WD.

描述（由申请人提供）：这项研究的长期目标是阐明控制铜稳态的系统性和细胞过程。铜是必不可少的痕量元素。铜的失误是通过吸收不良或过载导致严重疾病，例如梅克斯病，进行性骨髓性骨髓病和威尔逊病（WD）。 WD是由ATP7B基因突变引起的。 ATP7B是一种运输ATPase的铜，将铜从体内泵出并进入胆汁。 ATP7B灭活会导致体内有毒铜水平的积累，尤其是在肝脏中，这可能导致肝硬化和衰竭。 WD患者还患有神经系统和胸肌异常。如果早点诊断出WD，可以通过终身治疗铜旋转疗法进行治疗。由于威尔逊氏病中的所有表现都不是独一无二的，因此诊断疾病是一个挑战。即使我们现在了解铜调节的一些一般方面，我们尚未解释WD的某些特征。具体而言，（1）为什么WD患者增加尿铜？ （2）尿铜的性质是什么？ （3）肾脏如何能够很好地平衡铜？我最近在WD患者尿液中发现了一种新型的小型铜载体（SCC）可以帮助填补这些空白。特定目的I：要在威尔逊病小鼠尿液中表征SCC（ATP7B - / - ）。为此，我将使用一系列高分辨率色谱步骤将SCC纯化为同质性，并通过串联质谱法对其进行表征。特定目标II：表征ATP7B - / - 肝菌和尿液中SCC中CTR1水平之间的关系。我假设CTR1下调导致尿液中的SCC增加。为了测试这一点，我将在疾病的不同阶段测量肝脏中的Ctr1 mRNA和蛋白质（通过实时PCR和Western印迹），并将其与同一时期特定于AIM III的SCC量进行比较：确定分子基础ATP7B - / - 肾脏中的ATP7A蛋白上调。我假设SCC，ATP7B失活以及miR-107的降低导致ATP7A的上调。为了测试这一点，我将执行铜吸收，siRNA和北印迹分析。与公共卫生的相关性：了解这些问题将为人体如何平衡铜水平提供更好的了解。另外，SCC可能是WD的独特指标。