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.

描述（由申请人提供）：Ceruloplasmin（CP）是血浆和其他一些体液中的主要含Cu的蛋白质。 GPI链接形式被束缚在某些细胞的表面。对该蛋白质的主要兴趣是它通过与铁蛋白的相互作用而成为细胞Fe释放的介体的作用。但是，有充分的证据表明，它具有其他功能，包括对自由基的保护并将Cu从血液中传递给细胞，这些血液可能通过“受体”来使我的细胞表面上发现的CP发生。这里提出的研究的目的是确定CP是否将CU直接传递给细胞以及通过这种机制传递。我们假设该机制将包括与Cu转运蛋白1（CTR1）和Cu/Fe还原酶（细胞色素B561或STEAP家族）的结合，并且这不仅导致CU从CP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64cu标记的小鼠CP，以与表达CTR1的小鼠细胞系一起使用；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）来产生人类CP，以与CTR1已有并且尚未被siRNA撞倒的人类细胞系一起使用。我们将首先确定CP-CU实际上是由培养细胞（内部化）的，我们已经有强有力的初步证据。各种方法将用于证明内部化，包括低pH洗涤和细胞表面的胰胰蛋白酶化，在细胞质中从CP中检测64CU，通过使用细胞内荧光铜探针监测CU进入，并营救SOD1或挽救细胞色素C氧化酶活性（或通过与HoloCP孵育缺乏细胞中CCS Cu伴侣蛋白水平的下调）。我们还将在交付过程中测试APOCP的形成，并检验CP与CTR1相互作用和还原酶以摄取的摄取的假设。还将通过筛选细胞来表达可能的候选物，并通过化学交联，并通过siRNA敲低/过表达验证，并证明过量的非放射性CU（II）和Fe（III）防止从64cu摄取64CU，从而鉴定了还原酶。 CP。交联蛋白将通过激光碎片和TOF-TOF质谱分离和鉴定，并通过过表达和敲低的功能确认其身份。在CTR1 NULL和WT成纤维细胞中的平行研究有望鉴定这些细胞中未知的CP-CU摄取系统。我们的研究结果应提供明确的证据，表明以及是否意味着等离子体CP Delvers Cu cu到哺乳动物细胞，从而扩大我们对这种独特血浆蛋白功能的了解，而不是其在FE代谢和运输中的作用。 公共卫生相关性：铜是一个非常重要的化学元素，它支持许多特定蛋白的活性，内部和外部人类有机体的细胞，从胶原蛋白到黑色素，肾上腺素和呼吸，再到铁从细胞流出。 Ceruloplasmin是血液中最丰富的含铜蛋白，并且具有多个看似不同的功能。大多数对Ceruloplasmin的研究都集中在其在铁稳态中的作用。该提案的重点是检验ceruloplasmin是直接将铜递送到细胞的重要转运蛋白的假设。它还旨在确定铜从Ceruloplasmin的转移是如何发生的，因此我们了解可能出问题的是什么以及如何避免这种问题以维持健康的细胞。