Mechanism of Copper Uptake from Blood Plasma Ceruloplasmin by Mammalian Cells

Abstract

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.

Contribution of Transferrin and Ceruloplasmin Neurotransmission and Oxidant/Antioxidant Status to the Effects of Everolimus: A Case Series

“…Everolimus treatment improved autism symptoms in all four cases with increased serum Cp and Tf levels. As described above, mTOR activity regulates iron and copper homeostasis through modulating Tf as an iron mediator [6] and Cp as a copper mediator [7]. Thus, the present findings suggest that everolimus-induced attenuation of mTOR hyperactivity results in increased serum Tf and Cp levels to induce the homeostatic balance between mTOR activity.…”

“…Furthermore, increased intracellular copper levels suppress mTOR signaling [5]. Thus, mTOR activity may regulate iron and copper homeostasis with Tf acting as an iron mediator [6] and ceruloplasmin (Cp) as a copper mediator [7]. Oxidized low-density lipoprotein (oxLDL) suppresses the P13K/AKT/mTOR signaling pathway as the oxidative stress marker [8].…”

Therapeutic Potential of Everolimus on Core Autism Symptoms and Increasing Serum Ceruloplasmin and Transferrin Levels in a Pubescent Boy with Tuberous Sclerosis

“…Drawing these strands together, pharmacological effects of everolimus may be related to increases in serum levels of antioxidants. Cp is an important serum antioxidant [42] and the main copper binding protein in blood plasma [43]. Previous studies have reported that increased intracellular copper levels suppressed mTOR signaling [44], and that copper treatment down regulated mTOR signaling [45].…”

Hypoxia imaging and theranostic potential of [64Cu][Cu(ATSM)] and ionic Cu(II) salts: a review of current evidence and discussion of the retention mechanisms

“…2. Starting with ionic copper(II) in blood, either from direct injection of ionic copper compounds or through dismantling of copper(II) complexes, copper(II) is quickly reduced to Cu(I) by reductases [102][103][104][105], immediately binding to copper-binding proteins. On the other hand, tumor hypoxia stimulates CTR-1 expression [29,94], which then binds to Cu(I) and is transported into the tumor cells.…”