Copper Uptake from Plasma Ceruloplasmin

描述（由申请人提供）：铜蓝蛋白（Ceruloplasmin, Cp）是血浆和其他体液中的主要含铜蛋白。一种与GPI相连的形式被拴在一些细胞的表面。对这种蛋白的主要兴趣是它通过与铁转运蛋白相互作用作为细胞铁释放的介质。然而，有强有力的证据表明，它还有其他功能，包括防止自由基，并将铜从血液中输送到细胞中，这可能是通过在细胞表面发现的Cp“受体”发生的。本文提出的研究目的是明确确定Cp是否直接将Cu传递给细胞以及通过何种机制发生。我们推测其机制可能包括与Cu转运体1 （CTR1）和Cu/Fe还原酶（细胞色素b561或Steap家族）的结合，这不仅导致Cu从Cp中释放，而且形成apop。为了验证这些想法，我们将在体内生产64cu标记的小鼠Cp，用于表达和不表达Ctr1的小鼠细胞系；并通过在昆虫细胞中克隆和表达（或从人类肝癌细胞的分泌物中获得）来产生标记的人类Cp，用于CTR1被siRNA敲除或未被敲除的人类细胞系。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化，从细胞质中检测Cp中的64Cu，使用细胞内荧光铜探针监测Cu的进入，以及通过用holoCp孵卵来拯救缺陷细胞中的SOD1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apop的形成，并测试Cp与CTR1和还原酶相互作用以发生摄取的假设。还原酶将通过筛选表达可能候选的细胞，并通过化学交联，通过siRNA敲低/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64Cu。交联蛋白将通过激光碎片化和TOF-TOF质谱分离和鉴定，并通过过表达和敲低在功能上确认其身份。在Ctr1 null和WT成纤维细胞中的平行研究有望确定这些细胞中未知的Cp-Cu摄取系统。我们的研究结果将为血浆Cp是否以及通过何种方式将Cu运送到哺乳动物细胞提供明确的证据，从而扩大我们对这种独特血浆蛋白在铁代谢和运输中的作用之外的功能的了解。

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.