Mechanism of Copper Uptake from Blood Plasma Ceruloplasmin by Mammalian Cells.
Mechanism of Copper Uptake from Blood Plasma Ceruloplasmin by Mammalian Cells.
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DOI：
10.1371/journal.pone.0149516
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Linder MC
中科院分区：
文献类型：
--
作者：
Ramos D;Mar D;Ishida M;Vargas R;Gaite M;Montgomery A;Linder MC
铜蓝蛋白是血浆中主要的铜结合蛋白，由于其在细胞铁外流中的作用而引起人们的特别关注，但它还有其他功能。在这里，我们测试了一种假设，即它通过与细胞表面还原酶和转运蛋白相互作用，释放其铜以供细胞摄取，从而产生载角蛋白。用双房室中生长的紧密连接的乳腺上皮细胞单层(PMC42)和由HepG2细胞分泌的纯化的人64Cu标记的铜蓝蛋白，证实了铜蓝蛋白对铜的摄取和跨皮转运。单分子层在16小时内几乎吸收了所有的64铜，并将一半分泌到顶液(乳汁)中。此作用可被Ag(I)部分抑制。从注射64铜的小鼠血浆中提纯的铜蓝蛋白中的64铜在小鼠胚胎成纤维细胞(MEF)中3-6h内呈线性累积。在CTR1+/+细胞中的比率略高于CTR1-/-细胞，在2℃时低3倍。铜蓝蛋白衍生的64Cu不能通过广泛的洗涤或胰酶处理而被去除，大部分在胞浆中被回收。实际细胞铜(用炉原子吸收测定)在暴露于全角蓝蛋白24小时后显著增加。这伴随着培养上清液中HALO向载角蛋白的转化，在无细胞的孵育过程中不会发生。四种不同的内吞抑制剂均不能阻止铜蓝蛋白对64铜的摄取。高浓度的非放射性Cu(II)-或Fe(III)-NTA(细胞表面还原酶底物)或Cu(I)-NTA(竞争转运蛋白摄取)几乎消除了从铜蓝蛋白中摄取64Cu。MEF具有细胞表面还原酶活性，表达Steap2(但不表达Steap3、Steap4或dCytB)。然而，六天的siRNA治疗不足以减少活性或摄取。我们认为铜蓝蛋白是一种循环铜转运蛋白，可能与细胞表面的Steap2相互作用，形成载铜蛋白，铜(I)通过CTR1和未知的铜摄取转运蛋白进入细胞。
Ceruloplasmin, the main copper binding protein in blood plasma, has been of particular interest for its role in efflux of iron from cells, but has additional functions. Here we tested the hypothesis that it releases its copper for cell uptake by interacting with a cell surface reductase and transporters, producing apoceruloplasmin. Uptake and transepithelial transport of copper from ceruloplasmin was demonstrated with mammary epithelial cell monolayers (PMC42) with tight junctions grown in bicameral chambers, and purified human 64Cu-labeled ceruloplasmin secreted by HepG2 cells. Monolayers took up virtually all the 64Cu over 16h and secreted half into the apical (milk) fluid. This was partly inhibited by Ag(I). The 64Cu in ceruloplasmin purified from plasma of 64Cu-injected mice accumulated linearly in mouse embryonic fibroblasts (MEFs) over 3-6h. Rates were somewhat higher in Ctr1+/+ versus Ctr1-/- cells, and 3-fold lower at 2°C. The ceruloplasmin-derived 64Cu could not be removed by extensive washing or trypsin treatment, and most was recovered in the cytosol. Actual cell copper (determined by furnace atomic absorption) increased markedly upon 24h exposure to holoceruloplasmin. This was accompanied by a conversion of holo to apoceruloplasmin in the culture medium and did not occur during incubation in the absence of cells. Four different endocytosis inhibitors failed to prevent 64Cu uptake from ceruloplasmin. High concentrations of non-radioactive Cu(II)- or Fe(III)-NTA (substrates for cell surface reductases), or Cu(I)-NTA (to compete for transporter uptake) almost eliminated uptake of 64Cu from ceruloplasmin. MEFs had cell surface reductase activity and expressed Steap 2 (but not Steaps 3 and 4 or dCytB). However, six-day siRNA treatment was insufficient to reduce activity or uptake. We conclude that ceruloplasmin is a circulating copper transport protein that may interact with Steap2 on the cell surface, forming apoceruloplasmin, and Cu(I) that enters cells through CTR1 and an unknown copper uptake transporter.