William Breuer

Iron Acquired from Transferrin by K562 Cells Is Delivered into a Cytoplasmic Pool of Chelatable Iron(II)

The release of iron from transferrin (Tf) in the acidic milieu of endosomes and its translocation into the cytosol are integral steps in the process of iron acquisition via receptor-mediated endocytosis (RME). The translocated metal is thought to enter a low molecular weight cytoplasmic pool, presumed to contain the form of iron which is apparently sensed by iron responsive proteins and is the direct target of iron chelators. The process of iron delivery into the cytoplasmic chelatable pool of K562 cells was studied in situ by continuous monitoring of the fluorescence of cells loaded with the metal-sensitive probe calcein. Upon exposure to Tf at 37°C, intracellular fluorescence decayed, corresponding to an initial iron uptake of 40 nM/min. The Tf-mediated iron uptake was profoundly inhibited by weak bases, the protonophore monensin, energy depletion, or low temperatures (<25°C), all properties characteristic of RME. Cell iron levels were affected by the slowly permeating chelator desferrioxamine only after prolonged incubations. Conversely, rapidly penetrating, lipophilic iron(II) chelators such as 2,2′-bipyridyl, evoked swift increases in cell calcein fluorescence, equivalent to sequestration of 0.2-0.5 μM cytosolic iron, depending on the degree of pre-exposure to Tf. Addition of iron(III) chelators to permeabilized 2,2′-bipyridyl-treated cells, failed to reveal significant levels of chelatable iron(III). The finding that the bulk of the in situ cell chelatable pool is comprised of iron(II) was corroborated by pulsing K562 cells with Tf-Fe, followed by addition of iron(II) and/or iron(III) chelators and extraction of chelator-Fe complexes into organic solvent. Virtually all of the accumulated Fe in the chelatable pool could be complexed by iron(II) chelators. The cytoplasmic concentration of iron(II) fluctuated between 0.3 and 0.5 μM, and its mean transit time through the chelatable pool was 1-2 h. We conclude that after iron is translocated from the endosomes, it is maintained in the cytosol as a transit pool of chelatable iron(II). The ostensible absence of chelatable iron(III) implicates the intracellular operation of vigorous reductive mechanisms.

The importance of non-transferrin bound iron in disorders of iron metabolism.

The concept of non-transferrin bound iron (NTBI) was introduced 22 years ago by Hershko et al. (Brit. J. Haematol. 40 (1978) 255). It stemmed from a suspicion that, in iron overloaded patients, the large amounts of excess iron released into the circulation are likely to exceed the serum transferrin (Tf) iron-binding capacity (TIBC), leading to the appearance of various forms of iron not bound to Tf. In accordance with this assumption, NTBI was initially looked for and detected in patients with > or = 100% Tf-saturation. As techniques for its detection became more sophisticated and sensitive, NTBI was also found in conditions where Tf was not fully saturated, leading to a revision of the original view of NTBI as a simple spillover phenomenon. In this review, we will discuss some of the properties of NTBI, methods for its detection, its significance and potential value as an indicator for therapeutic regimens of iron chelation and supplementation.

Synthesis of tritiated 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid ([3H]DIDS) and its covalent reaction with sites related to anion transport in human red blood cells.

SummaryThe potent and specific inhibitor of anion permeability, 4,4′-diisothicyanostilbene-2,2′-disulfonic acid (DIDS) was synthesized in tritiated form ([3H]DIDS) from tritiated 5-nitrotoluene-o-sulfonic acid. Its reactions with and effects on red blood cells were compared with those of a reduced form ([3H]H2DIDS), previously used as a tracer for DIDS. The rate of covalent reaction of [3H]DIDS was substantially faster than that of [3H]H2DIDS at all temperatures tested. With both agents, the rate of reaction was increased in alkaline media, although the response occurred at a lower pH with [3H]DIDS. On the other hand, the relationship of irreversible membrane binding to the degree of inhibition of sulfate fluxes was linear and virtually the same for both agents, with 100% inhibition associated with the binding of approximately 1.2×106 molecules per cell. About 90% of the binding for each probe was to a particular membrane protein, known as band 3, equivalent to about 1 mole of agent per mole of protein.

Labile iron in parenteral iron formulations and its potential for generating plasma nontransferrin‐bound iron in dialysis patients

Background Labile plasma iron (LPI) associated with iron supplementation has been implicated in complications found in dialysis patients. As LPI can potentially catalyse oxygen radical generation, we determined the presence of labile iron in the parenteral preparations and the frequency of occurrence of LPI in dialysis patients.

Cell functions impaired by frataxin deficiency are restored by drug-mediated iron relocation

Various human disorders are associated with misdistribution of iron within or across cells. Friedreich ataxia (FRDA), a deficiency in the mitochondrial iron-chaperone frataxin, results in defective use of iron and its misdistribution between mitochondria and cytosol. We assessed the possibility of functionally correcting the cellular properties affected by frataxin deficiency with a siderophore capable of relocating iron and facilitating its metabolic use. Adding the chelator deferiprone at clinical concentrations to inducibly frataxin-deficient HEK-293 cells resulted in chelation of mitochondrial labile iron involved in oxidative stress and in reactivation of iron-depleted aconitase. These led to (1) restoration of impaired mitochondrial membrane and redox potentials, (2) increased adenosine triphosphate production and oxygen consumption, and (3) attenuation of mitochondrial DNA damage and reversal of hypersensitivity to staurosporine-induced apoptosis. Permeant chelators of higher affinity than deferiprone were not as efficient in restoring affected functions. Thus, although iron chelation might protect cells from iron toxicity, rendering the chelated iron bioavailable might underlie the capacity of deferiprone to restore cell functions affected by frataxin deficiency, as also observed in FRDA patients. The siderophore-like properties of deferiprone provide a rational basis for treating diseases of iron misdistribution, such as FRDA, anemia of chronic disease, and X-linked sideroblastic anemia with ataxia.

Dynamics of the cytosolic chelatable iron pool of K562 cells

The labile iron pool of cells (LIP) constitutes the primary source of metabolic and catalytically reactive iron in the cytosol. We studied LIP homeostasis in K562 cells using the fluorescent metal‐sensitive probe calcein. Following brief exposure to iron(II) salts or to oxidative or reductive stress, LIP rose by up to 120% relative to the normal level of 350 nM. However, the rate of recovery to normal LIP level differed markedly with each treatment (respective of 27, 65–88 and ≤17 min). We show that the capacity of K562 cells to adjust LIP levels is highly dependent on the origin of the LIP increase and on the pre‐existing cellular iron status.

Downregulation of hepcidin and haemojuvelin expression in the hepatocyte cell-line HepG2 induced by thalassaemic sera

β‐Thalassaemia represents a group of diseases, in which ineffective erythropoiesis is accompanied by iron overload. In a mouse model of β‐thalassaemia, we observed that the liver expressed relatively low levels of hepcidin, which is a key factor in the regulation of iron absorption by the gut and of iron recycling by the reticuloendothelial system. It was hypothesised that, despite the overt iron overload, a putative plasma factor found in β‐thalassaemia might suppress liver hepcidin expression. Sera from β‐thalassaemia and haemochromatosis (C282Y mutation) patients were compared with those of healthy individuals regarding their capacity to induce changes the expression of key genes of iron metabolism in human HepG2 hepatoma cells. Sera from β‐thalassaemia major patients induced a major decrease in hepcidin (HAMP) and lipocalin2 (oncogene 24p3) (LCN2) expression, as well as a moderate decrease in haemojuvelin (HFE2) expression, compared with sera from healthy individuals. A significant correlation was found between the degree of downregulation of HAMP and HFE2 induced by β‐thalassaemia major sera (r = 0·852, P < 0·0009). Decreased HAMP expression was also found in HepG2 cells treated with sera from β‐thalassaemia intermedia patients. In contrast, the majority of sera from hereditary haemochromatosis patients induced an increase in HAMP expression, which correlated with transferrin (Tf) saturation (r = 0·765, P < 0·0099). Our results suggest that, in β‐thalassaemia, serum factors might override the potential effect of iron overload on HAMP expression, thereby providing an explanation for the failure to arrest excessive intestinal iron absorption in these patients.

Newly delivered transferrin iron and oxidative cell injury

© 1997 Federation of European Biochemical Societies.

On the mode of action of phlorizin as an antimalarial agent in in vitro cultures of plasmodium falciparum

Phlorizin (phloretin-2-beta-glucoside) is a drug which effectively inhibits intraerythrocytic malaria growth in in vitro cultures of Plasmodium falciparum IC50 = 16 +/- 7 microM). Work with synchronously grown cultures indicates that susceptibility to phlorizin is apparent at the trophozoite stage and onward, and that 2-8 hours exposure to the drug causes an irreversible arrest of parasite growth. The drug has also been found to inhibit pores which are induced by the parasite in the host cell membrane (IC50 = 17 +/- 2 microM) and which are apparently essential for intraerythrocytic growth. The effect on the pores is apparent soon after exposure of the cells to the drug and can be reversed, although extensive washing and incubation in culture conditions are required to achieve it. The results of this study indicate that the putative site of action of phlorizin on the pores is on the cytoplasmic surface of the host cell membrane. The drug which normally cannot permeate uninfected red cells, gains access to the cytoplasm via the pores, appearing in the host cell membrane. Those become eventually the target of phlorizin itself. The proposed mechanism of action of phlorizin on malarial growth invokes blockade of the pores, although additional effects of the drug on intraerythrocytic parasites cannot be ruled out.

Ubiquitin conjugation triggers misfolded protein sequestration into quality control foci when Hsp70 chaperone levels are limiting

Ubiquitylation of partially misfolded proteins by the yeast Doa10 E3 ligase requires the Hsp40 cochaperone Sis1, whereas the Hsp70 chaperones Ssa1 and Ssa2 are dispensable. Elimination of the Hsp70 chaperones prevents proteasomal degradation, resulting in ubiquitin-dependent sequestration of the misfolded proteins in Hsp42-positive foci.