Iron homeostasis is essential for maintaining the physiological requirement for iron while preventing iron overload. Multicopper ferroxidases regulate the oxidation of Fe(II) to Fe(III), circumventing the generation of harmful hydroxyl-free radicals. Ceruloplasmin is the major multicopper ferroxidase in blood; however, hephaestin, a membrane-bound ceruloplasmin homolog, has been implicated in the export of iron from duodenal enterocytes into blood. These ferroxidases supply transferrin, the iron-carrier protein in plasma, with Fe(III). Transferrin circulates through blood and delivers iron to cells via the transferrin receptor pathway. Due to the insoluble and reactive nature of free Fe(III), the oxidation of Fe(II) upon exiting the duodenal enterocyte may require an interaction between the ferroxidase and transferrin. In Chapter 3, the putative interaction of transferrin with ceruloplasmin and a soluble form of recombinant hephaestin was investigated. Utilizing native polyacrylamide gel electrophoresis, covalent cross-linking and surface plasmon resonance, a stable interaction between the two proteins was not detected. The lack of interaction between hephaestin and transferrin prompted the investigation into the localization of hephaestin in the human small intestine. Hephaestin has been reported to have both intracellular and extracellular locations in murine tissue. In the Appendix, the location of hephaestin in human tissue was investigated using a novel polyclonal antibody. Hephaestin was localized to the basolateral membrane and an intracellular location of the enterocyte, as well as a novel location in the myenteric plexus of the duodenum. The delivery of iron to cells via the transferrin receptor pathway is well established; however, little is known about the interaction of transferrin with the transferrin receptor at the molecular level. In Chapters 4 and 5, surface plasmon resonance was employed to further characterize the binding event between transferrin and the transferrin receptor. It was found that mutations affecting iron release in transferrin did not impact receptor binding. However, when N-lobe residues predicted to form contacts with the transferrin receptor were targeted, significant changes in the transferrin receptor binding kinetics and affinity were observed.

Iron homeostasis is essential for maintaining the physiological requirement for iron while preventing iron overload. Multicopper ferroxidases regulate the oxidation of Fe(II) to Fe(III), circumventing the generation of harmful hydroxyl-free radicals. Ceruloplasmin is the major multicopper ferroxidase in blood; however, hephaestin, a membrane-bound ceruloplasmin homolog, has been implicated in the export of iron from duodenal enterocytes into blood. These ferroxidases supply transferrin, the iron-carrier protein in plasma, with Fe(III). Transferrin circulates through blood and delivers iron to cells via the transferrin receptor pathway. Due to the insoluble and reactive nature of free Fe(III), the oxidation of Fe(II) upon exiting the duodenal enterocyte may require an interaction between the ferroxidase and transferrin. In Chapter 3, the putative interaction of transferrin with ceruloplasmin and a soluble form of recombinant hephaestin was investigated. Utilizing native polyacrylamide gel electrophoresis, covalent cross-linking and surface plasmon resonance, a stable interaction between the two proteins was not detected. The lack of interaction between hephaestin and transferrin prompted the investigation into the localization of hephaestin in the human small intestine. Hephaestin has been reported to have both intracellular and extracellular locations in murine tissue. In the Appendix, the location of hephaestin in human tissue was investigated using a novel polyclonal antibody. Hephaestin was localized to the basolateral membrane and an intracellular location of the enterocyte, as well as a novel location in the myenteric plexus of the duodenum. The delivery of iron to cells via the transferrin receptor pathway is well established; however, little is known about the interaction of transferrin with the transferrin receptor at the molecular level. In Chapters 4 and 5, surface plasmon resonance was employed to further characterize the binding event between transferrin and the transferrin receptor. It was found that mutations affecting iron release in transferrin did not impact receptor binding. However, when N-lobe residues predicted to form contacts with the transferrin receptor were targeted, significant changes in the transferrin receptor binding kinetics and affinity were observed.

Iron homeostasis is essential for maintaining the physiological requirement for iron while preventing iron overload. Multicopper ferroxidases regulate the oxidation of Fe(II) to Fe(III), circumventing the generation of harmful hydroxyl-free radicals. Ceruloplasmin is the major multicopper ferroxidase in blood; however, hephaestin, a membrane-bound ceruloplasmin homolog, has been implicated in the export of iron from duodenal enterocytes into blood. These ferroxidases supply transferrin, the iron-carrier protein in plasma, with Fe(III). Transferrin circulates through blood and delivers iron to cells via the transferrin receptor pathway. Due to the insoluble and reactive nature of free Fe(III), the oxidation of Fe(II) upon exiting the duodenal enterocyte may require an interaction between the ferroxidase and transferrin. In Chapter 3, the putative interaction of transferrin with ceruloplasmin and a soluble form of recombinant hephaestin was investigated. Utilizing native polyacrylamide gel electrophoresis, covalent cross-linking and surface plasmon resonance, a stable interaction between the two proteins was not detected. The lack of interaction between hephaestin and transferrin prompted the investigation into the localization of hephaestin in the human small intestine. Hephaestin has been reported to have both intracellular and extracellular locations in murine tissue. In the Appendix, the location of hephaestin in human tissue was investigated using a novel polyclonal antibody. Hephaestin was localized to the basolateral membrane and an intracellular location of the enterocyte, as well as a novel location in the myenteric plexus of the duodenum. The delivery of iron to cells via the transferrin receptor pathway is well established; however, little is known about the interaction of transferrin with the transferrin receptor at the molecular level. In Chapters 4 and 5, surface plasmon resonance was employed to further characterize the binding event between transferrin and the transferrin receptor. It was found that mutations affecting iron release in transferrin did not impact receptor binding. However, when N-lobe residues predicted to form contacts with the transferrin receptor were targeted, significant changes in the transferrin receptor binding kinetics and affinity were observed. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Iron is an essential element for human organism, because it cooperates as a cofactor of enzymes in many metabolic pathways. Iron is a component of hemoglobin, and thus it is indispensable for the oxygen transport to tissues. It can exist as a ferrous or ferric form. However, ferrous iron paticipates in reactions in which highly reactive hydroxyl group can be formed. This product is harmful for the organism. Non-heme iron is taken up to the circulation through duodenal enterocyte. Iron excretion is carried out only by desquamation of the enterocytes or by bleeding. Therefore, iron intake must be strictly regulated. Iron overloading is observed in some chronic diseases (hereditary hemochromatosis, alcohol liver disease). In contrary, iron depletion can be a case of iron deficiency anemia. The aim of this master thesis is to determine the expression of iron transport molecules in duodenum in chronic diseases which originate due to disturbances of iron intake regulation. We determine the expression of molecules of iron transport (DMT1, Dcytb, ferroportin, hephaestin) on mRNA level by qPCR and on protein level by western blot. The level of serum hepcidin was determined by ELISA. Our results show an increased expression of mRNA of transporters DMT1 and ferroportin as well as ferrireductase Dcytb and ferroxidase...