Iron transport in K562 cells: a kinetic study using native gel electrophoresis and 59Fe autoradiography

AbstractThe exact mechanisms of iron transport from endosomes to the target iron containing cellular proteins are currently unknown. To investigate this problem, we used the gradient gel electrophoresis and the sensitive detection of 59Fe by autoradiography to detect separate cellular iron compounds and their iron kinetics. Cells of human leukemic line K562 were labeled with [59Fe]transferrin for 30–600 s and cellular iron compounds in cell lysates were analyzed by native electrophoretic separation followed by 59Fe autoradiography. Starting with the first 30 s of iron uptake, iron was detectable in a large membrane bound protein complex (Band I) and in ferritin. Significant amounts of iron were also found in labile iron compound(s) with the molecular weight larger than 5000 as judged by ultrafiltration. Iron kinetics in these compartments was studied. Band I was the only compound with the kinetic properties of an intermediate. Transferrin, transferrin receptor and additional proteins of the approximate molecular weights of 130 000, 66 000 and 49 000 were found to be present in Band I. The labile iron compounds and ferritin behaved kinetically as end products. No evidence for low molecular weight transport intermediates was found. These results suggest that intracellular iron transport is highly compartmentalized, that iron released from endosomal transferrin passes to its cellular targets in a direct contact with the endosomal membrane complex assigned as Band I. The nature of the labile iron pool and its susceptibility to iron chelation is discussed

Prion Protein Modulates Cellular Iron Uptake: A Novel Function with Implications for Prion Disease Pathogenesis

Converging evidence leaves little doubt that a change in the conformation of prion protein (PrPC) from a mainly α-helical to a β-sheet rich PrP-scrapie (PrPSc) form is the main event responsible for prion disease associated neurotoxicity. However, neither the mechanism of toxicity by PrPSc, nor the normal function of PrPC is entirely clear. Recent reports suggest that imbalance of iron homeostasis is a common feature of prion infected cells and mouse models, implicating redox-iron in prion disease pathogenesis. In this report, we provide evidence that PrPC mediates cellular iron uptake and transport, and mutant PrP forms alter cellular iron levels differentially. Using human neuroblastoma cells as models, we demonstrate that over-expression of PrPC increases intra-cellular iron relative to non-transfected controls as indicated by an increase in total cellular iron, the cellular labile iron pool (LIP), and iron content of ferritin. As a result, the levels of iron uptake proteins transferrin (Tf) and transferrin receptor (TfR) are decreased, and expression of iron storage protein ferritin is increased. The positive effect of PrPC on ferritin iron content is enhanced by stimulating PrPC endocytosis, and reversed by cross-linking PrPC on the plasma membrane. Expression of mutant PrP forms lacking the octapeptide-repeats, the membrane anchor, or carrying the pathogenic mutation PrP102L decreases ferritin iron content significantly relative to PrPC expressing cells, but the effect on cellular LIP and levels of Tf, TfR, and ferritin is complex, varying with the mutation. Neither PrPC nor the mutant PrP forms influence the rate or amount of iron released into the medium, suggesting a functional role for PrPC in cellular iron uptake and transport to ferritin, and dysfunction of PrPC as a significant contributing factor of brain iron imbalance in prion disorders

Bunecny transport zeleza.

Iron, present in numerous enzymes and proteins, is indispensable for life, yet, present in loosely bound forms, it can catalyze the formation of oxidative free radicals. Resulting tissue damage is thought to be causally related to plethora of diseases including arthritis, atherosclerosis and cancer. Despite the progress in delineating the iron-dependent regulation of iron proteins in the past decade, accurate description of intracellular iron transport has been difficult, mainly because of the lack of appropriate techniques for the separation, detection, and quantitation of the cellular iron - containing compounds. Iron is bound to proteins by relatively weak bonds and nondissociative methods are needed to preserve iron compounds intact during their separation. This thesis aims to broaden our knowledge in the field of intracellular iron transport using metabolic labeling of the cells with radioactive iron isotopes and employing mild non-denaturating separation techniques. The implications of this studies have validity for a broad range of topics from the development of new iron chelators to mechanisms of iron absorption.Summary and text also in EnglishAvailable from STL, Prague, CZ / NTK - National Technical LibrarySIGLECZCzech Republi