STRUCTURE, FUNCTION, AND REGULATION OF COPPER AND IRON PROTEINS

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We have been studying the structure, function and regulation of mammalian proteins associated with Cu and Fe, particularly ferritin (involved in iron storage) and ceruloplasmin (involved in Cu transport). In the next few years, MBRS students are likely to be studying aspects of one of the following projects: (a) The structure, function and regulation of two major isoforms of ceruloplasmin (Cp) found in rat and human plasma; (b) the structure and function of milk Cp, which we have postulated is a significant source of Cu for the newborn; and (c) the potential stimulation of ferritin synthesis and secretion by lymphokines released in inflammation, which we have postulated involves a new form of ferritin mRNA translated on membrane-bound polyribosomes. In (a), the amino acid composition, N-terminal sequence, apparent molecular weight, and carbohydrate and copper content of two major Cp isoforms in rat and human serum will be determined and compared; as will be their relative abundance in various physiological states (inflammation, cancer and estrogen treatment), and their relative effectiveness in donating Cu to cultured cells and to tissues after intravenous infusion. In (b), we will quantify and structurally analyse Cp in the milk from various species (but especially the pig), and test in rats and pigs the hypothesis that milk Cp is an especially available source of Cu for the newborn, delivered via specific intestinal receptors. Concerning (c), we will test the hypothesis that a ferritin mRNA having no iron-regulatory element (IRE) in its 5'UTR (but having a signal sequence) increases in concentration in the liver in response to inflammatory lymphokines, and is translated on ER- bound polyribosomes, resulting in increased secretion of "serum-type" ferritin into the blood. This will be studied in tissue culture, with rat hepatoma cells and IL-I, IL-6 and TNF, using immunoprecipitation and immunoassays (to detect, quantify and characterize the ferritin secreted), cDNA hybridization of RNA from free and membrane bound polyribosomes (to observe whether ferritin mRNA is driven to the ER-bound polyribosomes, as in vivo), and in vitro translation in the presence and absence of IRE- binding protein and Fe chelators (to examine whether and IRE is present in the mRNA from the bound polyribosomes) ,and whether Fe is mediating the response to lymphokine. We expect these studies will show that ceruloplasmin and ferritin have additional and unexpected functions relating to development and disease.