Copper regulated synthesis, secretion and degradation of ceruloplasmin in a mouse immortalized hepatocytic cell line.
Ceruloplasmin, a blue copper oxidase circulating in serum of all vertebrates, is a glycoprotein synthesized mainly in hepatocytes and secreted into plasma with six tightly bound atoms of copper per molecule. Many aspects of the mechanisms by which synthesis and secretion of this protein are regulated by copper are still not known. In HepG2 hepatocarcinoma cells this fine regulation is not maintained; we have then utilized Met-murine-hepatocytes (MMH), isolated from the liver of transgenic mice expressing a truncated form of c-Met (hepatocyte growth factor receptor), that are immortalized but not transformed. Copper deficiency was induced by treatment of cells with bathocuproine disulphonate. Experiments of metabolic labeling with 35S-methionine-cysteine and of Western blotting followed by immunostaining, demonstrated that maturation and secretion of ceruloplasmin but not its synthesis are affected by copper availability. In this paper we have shown that in copper deficiency ceruloplasmin accumulates in a pre-Golgi compartment, in which the protein is still in a Endo H sensitive form, and where presumably copper binding to the apo-protein takes place. Moreover, we found that treatment of copper-deficient cells with the proteasomal inhibitor lactacycstin leads to immature ceruloplasmin accumulation in the cell. We have optimized conditions to induce in vitro copper deficiency and found that MMH-D3 cells represent a suitable model to study in detail the molecular mechanism of copper-regulated ceruloplasmin synthesis, secretion and degradation.
Pisu
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Istituto Nazionale di Ricerca per gli Alimenti e la Nutrizione (INRAN), Via Ardeatina 546, 00178 Rome, Italy.
Bellovino
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D
Gaetani
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D000818:Animals; D045744:Cell Line, Tumor; D002570:Ceruloplasmin / Q000096:biosynthesis* / Q000378:metabolism; D003300:Copper / Q000172:deficiency / Q000502:physiology*; D006031:Glycosylation; D006056:Golgi Apparatus; D022781:Hepatocytes / Q000378:metabolism*; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D051379:Mice; D019859:Proto-Oncogene Proteins c-met / Q000235:genetics
D003300:Copper; D002570:Ceruloplasmin; D019859:Proto-Oncogene Proteins c-met; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase