Copper Uptake from Plasma Ceruloplasmin

从血浆铜蓝蛋白中摄取铜

• 批准号: 8367816
• 负责人: MARIA C LINDER
• 金额: $ 31.07万
• 依托单位: CALIFORNIA STATE UNIVERSITY FULLERTON
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8367816
• 关键词: Amniotic Fluid; Appearance; Binding; Bioinformatics; Blood; Blood Cells; Body Fluids; Carrier Proteins; Cell Surface Proteins; Cell membrane; Cell surface; Cells; Ceruloplasmin; Characteristics; Chemicals; Collagen; Complex; Copper; Cultured Cells; Cytosol; Detection; Docking; Down-Regulation; Elements; Embryo; Endoplasmic Reticulum; Epinephrine; Epithelial Cells; Erythrocytes; Extracellular Fluid; Family; Fibroblasts; Gel; Heart; Hepatocyte; Histidine; Homeostasis; Human; Human Cell Line; Insecta; Ions; Iron; Kinetics; Knowledge; Label; Lasers; Lead; Light; Link; Mammalian Cell; Mammary gland; Mass Spectrum Analysis; Mediator of activation protein; Melanins; Membrane; Membrane Transport Proteins; Metabolism; Metals; Milk; Modeling; Molecular Chaperones; Monitor; Mouse Cell Line; Mus; Organism; Other Body Fluids and Secretions; Oxidoreductase; Personal Communication; Physiological; Placenta; Plasma; Plasma Proteins; Procedures; Process; Proteins; Radioactive; Radiolabeled; Rattus; Reaction; Research; Respiration; Rest; Role; Screening procedure; Small Interfering RNA; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Study models; Surface; System; Testing; Tight Junctions; Time; Tissues; ceruloplasmin receptor; crosslink; cytochrome b561; cytochrome c oxidase; design; electron donor; expression cloning; hepatoma cell; in vivo; innovation; interest; knock-down; link protein; metal transporting protein 1; monolayer; overexpression; prevent; radiotracer; sensor; uptake

DESCRIPTION (provided by applicant): Ceruloplasmin (Cp) is the main Cu-containing protein in the blood plasma and some other body fluids. A GPI- linked form is tethered to the surface of some cells. The main interest in this protein has been its role as a mediator of cellular Fe release through interaction with ferroportin. However, there is strong evidence it has additional functions that include protection against radicals and delivering Cu to cells from the blood that may occur through "receptors" for Cp found on my cell surfaces. The objectives of the research here proposed are to determine definitively whether or not Cp delivers Cu directly to cells and by what mechanisms this occurs. We hypothesize that the mechanism would include binding to Cu transporter 1 (CTR1) and a Cu/Fe reductase (of the cytochrome b561 or Steap family), and that this results not only in release of Cu from Cp but the formation of apoCp. To test these ideas we will produce 64Cu-labeled mouse Cp in vivo, for use with mouse cell lines that do and do not express Ctr1; and produce labeled human Cp by cloning and expression in insect cells (or obtaining it from the secretions of human hepatoma cells) for use with human cell lines in which CTR1 has and has not been knocked down with siRNA. We will first determine that Cp-Cu is actually taken up (internalized) by the cultured cells, for which we already have strong preliminary evidence. Various approaches will be used to demonstrate internalization, including low pH washes and trypsinization of the cell surface, detection of 64Cu from Cp in the cytosol, monitoring of Cu entry by use of intracellular fluorescent copper probes, and rescue of SOD1 or cytochrome c oxidase activity (or down-regulation of CCS Cu chaperone protein levels) in deficient cells by incubation with holoCp. We will also test for formation of apoCp during the delivery process, and test the hypothesis that Cp interacts with both CTR1 and a reductase for uptake to occur. The reductase will be identified by screening cells for expression of likely candidates, and through chemical cross-linking, with verification by siRNA knockdown/overexpression, and demonstrating that excess non-radioactive Cu (II) and Fe (III) prevent uptake of 64Cu from Cp. Cross-linked proteins will be separated and identified by laser fragmentation and TOF-TOF mass spectrometry, and their identity functionally confirmed through overexpression and knockdown. Parallel studies in Ctr1 null and WT fibroblasts are expected to identify an unknown Cp-Cu uptake system in these cells. The results of our studies should provide definitive evidence on whether and by what means plasma Cp delvers Cu to mammalian cells, thus expanding our knowledge of the functions of this unique plasma protein beyond its role in Fe metabolism and transport. PUBLIC HEALTH RELEVANCE: Copper is a very important chemical element supporting the activities of numerous specific proteins, inside and outside of the cells of the human organism, from collagen to melanin, adrenaline and respiration, to the flow of iron out of cells. Ceruloplasmin is the most abundant copper-containing protein in the blood, and has multiple seemingly different functions. Most research on ceruloplasmin has focused on its role in iron homeostasis. This proposal focuses on testing the hypothesis that ceruloplasmin is an important transport protein for the delivery of copper directly to cells; it is also designed to determine exactly how transfer of copper from ceruloplasmin occurs, so we understand what may go wrong and how such problems might be circumvented to maintain healthy cells.

性状（由申请方提供）：血浆铜蓝蛋白（Cp）是血浆和其他体液中主要的含铜蛋白。GPI连接的形式被拴在一些细胞的表面。对这种蛋白质的主要兴趣是其通过与膜铁转运蛋白相互作用作为细胞Fe释放的介体的作用。然而，有强有力的证据表明，它具有额外的功能，包括保护免受自由基的侵害，并将Cu从血液中输送到细胞中，这些功能可能通过在细胞表面发现的Cp“受体”发生。本文提出的研究目标是明确确定Cp是否直接将Cu传递到细胞以及通过何种机制发生。我们假设该机制包括与Cu转运蛋白1（CTR 1）和Cu/Fe还原酶（细胞色素b561或Steap家族）结合，这不仅导致Cu从Cp中释放，还导致apoCp的形成。为了测试这些想法，我们将在体内产生64 Cu标记的小鼠Cp，用于表达和不表达Ctr 1的小鼠细胞系;并通过在昆虫细胞中克隆和表达（或从人肝癌细胞的分泌物中获得）产生标记的人Cp，用于其中CTR 1已经和没有被siRNA敲低的人细胞系。我们将首先确定Cp-Cu实际上被培养的细胞吸收（内化），对此我们已经有了强有力的初步证据。将使用各种方法来证明内化，包括低pH洗涤和细胞表面的胰蛋白酶消化、检测胞质溶胶中来自Cp的64 Cu、使用细胞内荧光铜探针监测Cu进入以及通过与holoCp孵育来拯救缺陷细胞中的SOD 1或细胞色素c氧化酶活性（或下调CCS Cu伴侣蛋白水平）。我们还将测试在递送过程中apoCp的形成，并测试Cp与CTR 1和还原酶相互作用以发生摄取的假设。将通过筛选细胞表达可能的候选物，并通过化学交联，通过siRNA敲减/过表达进行验证，并证明过量的非放射性Cu（II）和Fe（III）阻止从Cp中摄取64 Cu来鉴定还原酶。将通过激光裂解和TOF-TOF质谱法分离和鉴定交联蛋白，并通过过表达和敲低功能性地确认其身份。在Ctr 1 null和WT成纤维细胞中的平行研究预计将在这些细胞中识别未知的Cp-Cu摄取系统。我们的研究结果应提供明确的证据，是否以及通过什么手段血浆Cp delvers铜到哺乳动物细胞，从而扩大我们的知识，这种独特的血浆蛋白的功能超出其在铁代谢和运输的作用。 公共卫生关系：铜是一种非常重要的化学元素，支持人体组织细胞内外许多特定蛋白质的活动，从胶原蛋白到黑色素，肾上腺素和呼吸，到铁从细胞中流出。铜蓝蛋白是血液中含量最丰富的含铜蛋白，具有多种看似不同的功能。大多数关于铜蓝蛋白的研究集中在其在铁稳态中的作用。该提案的重点是测试铜蓝蛋白是将铜直接输送到细胞的重要转运蛋白的假设;它还旨在确定铜从铜蓝蛋白转移的确切方式，因此我们了解可能出现的问题以及如何规避这些问题以保持健康的细胞。