Excess capacity of the iron regulatory protein system

Iron regulatory proteins (IRP1 and IRP2) are master regulators of cellular iron metabolism. IRPs bind to iron-responsive elements (IREs) present in the untranslated regions of mRNAs encoding proteins of iron storage, uptake, transport, and export. Because simultaneous knockout of IRP1 and IRP2 is embryonically lethal, it has not been possible to use dual knockouts to explore the consequences of loss of both IRP1 and IRP2 in mammalian cells. In this report, we describe the use of small interfering RNA to assess the relative contributions of IRP1 and IRP2 in epithelial cells. Stable cell lines were created in which either IRP1, IRP2, or both were knocked down. Knockdown of IRP1 decreased IRE binding activity but did not affect ferritin H and transferrin receptor 1 (TfR1) expression, whereas knockdown of IRP2 marginally affected IRE binding activity but caused an increase in ferritin H and a decrease in TfR1. Knockdown of both IRPs resulted in a greater reduction of IRE binding activity and more severe perturbation of ferritin H and TfR1 expression compared with single IRP knockdown. Even though the knockdown of IRP-1, IRP-2, or both was efficient, resulting in nondetectable protein and under 5% of wild type levels of mRNA, all stable knockdowns retained an ability to modulate ferritin H and TfR1 appropriately in response to iron challenge. However, further knockdown of IRPs accomplished by transient transfection of small interfering RNA in stable knockdown cells completely abolished the response of ferritin H and TfR1 to iron challenge, demonstrating an extensive excess capacity of the IRP system.     

Self-association and ligand-induced conformational changes of iron regulatory proteins 1 and 2

Iron regulatory proteins (IRPs) regulate iron metabolism in mammalian cells. We used biophysical techniques to examine the solution properties of apo-IRP1 and apo-IRP2 and the interaction with their RNA ligand, the iron regulatory element (IRE). Sedimentation velocity and equilibrium experiments have shown that apo-IRP1 exists as an equilibrium mixture of monomers and dimers in solution, with an equilibrium dissociation constant in the low micromolar range and slow kinetic exchange between the two forms. However, only monomeric IRP1 is observed in complex with IRE. In contrast, IRP2 exists as monomer in both the apo-IRP2 form and in the IRP2/IRE complex. For both IRPs, sedimentation velocity and dynamic light-scattering experiments show a decrease of the Stokes radius upon binding of IRE. This conformational change was also observed by circular dichroism. Studies with an RNA molecule complementary to IRE indicate that, although specific base interactions can increase the stability of the protein/RNA complex, they are not essential for inducing this conformational change. The dynamic change of the IRP between different oligomeric and conformational states induced by interaction with IRE may play a role in the iron regulatory functions of IRPs.     

Alterations in the interaction between iron regulatory proteins and their iron responsive element in normal and Alzheimer's diseased brains.

Iron regulatory proteins (IRPs) are cytoplasmic mRNA binding proteins involved in intracellular regulation of iron homeostasis. IRPs regulate expression of ferritin and transferrin receptor at the mRNA level by interacting with a conserved RNA structure termed the iron-responsive element (IRE). This concordant regulation of transferrin receptors and ferritin is designed so a cell can obtain iron when it is needed, and sequester iron when it is in excess. However, we have reported that iron accumulates in the brain in Alzheimer's disease without a concomitant increase in ferritin. An increase in iron without proper sequestration can increase the vulnerability of cells to oxidative stress. Oxidative stress is a component of many neurological diseases including Alzheimer's. We hypothesized that alterations in the IRP/IRE interaction could be the site at which iron mismanagement occurs in the Alzheimer's brains. In this report we demonstrate that in normal human brain extracts, the IRP is detected as a double IRE/IRP complex by RNA band shift assay, but in 2 of 6 Alzheimer's brain (AD) extracts examined a single IRE/IRP complex was obtained. Furthermore, the mobility of the single IRE/IRP complex in Alzheimer's brain extracts is decreased relative to the double IRE/IRP complex. Western blot and RNA band super shift assay demonstrate that IRP1 is involved in the formation of the single IRE/IRP complex. In vitro analyses suggest that the stability of the doublet complex and single AD complex are different. The single complex from the AD brain are more stable. A more stable IRE/IRP complex in the AD brain could increase stability of the transferrin receptor mRNA and inhibit ferritin synthesis. At the cellular level, the outcome of this alteration in the molecular regulatory mechanism would be increased iron accumulation without an increase in ferritin; identical to the observation we reported in AD brains. The appearance of the single IRE/IRP complex in Alzheimer's brain extracts is associated with relatively high endogenous ribonuclease activity. We propose that elevated RNase activity is one mechanism by which the iron regulatory system becomes dysfunctional.     

Role of nitric oxide in cellular iron metabolism

Iron regulatory proteins (IRP1 and IRP2) control the synthesis of transferrin receptors (TfR) and ferritin by binding to iron-responsive elements (IREs) which are located in the 3 untranslated region (UTR) and the 5 UTR of their respective mRNAs. Cellular iron levels affect binding of IRPs to IREs and consequently expression of TfR and ferritin. Moreover, NO, a redox species of nitric oxide that interacts primarily with iron, can activate IRP1 RNA-binding activity resulting in an increase in TfR mRNA levels. We have shown that treatment of RAW 264.7 cells (a murine macrophage cell line) with NO⁺ (nitrosonium ion, which causes S-nitrosylation of thiol groups) resulted in a rapid decrease in RNA-binding of IRP2, followed by IRP2 degradation, and these changes were associated with a decrease in TfR mRNA levels. Moreover, we demonstrated that stimulation of RAW 264.7 cells with lipopolysaccharide (LPS) and interferon- (IFN-) increased IRP1 binding activity, whereas RNA-binding of IRP2 decreased and was followed by a degradation of this protein. Furthermore, the decrease of IRP2 binding/protein levels was associated with a decrease in TfR mRNA levels in LPS/IFN--treated cells, and these changes were prevented by inhibitors of inducible nitric oxide synthase. These results suggest that NO⁺-mediated degradation of IRP2 plays a major role in iron metabolism during inflammation.     

Iron accumulation and iron-regulatory protein activity in human hepatoma (HepG2) cells

Iron may populate distinct hepatocellular iron pools that differentially regulate expression of proteins such as ferritin and transferrin receptor (TfR) through iron-regulatory mRNA-binding proteins (IRPs), and may additionally regulate uptake and accumulation of non-transferrin-bound iron (NTBI). We examined iron-regulatory protein (IRP) binding activity and ferritin/TfR expression in human hepatoma (HepG2) cells exposed to iron at different levels for different periods. Several iron-dependent RNA-binding activities were identified, but only IRP increased with beta-mercaptoethanol. With exposures between 0 and 20 microg/ml iron, decreases in IRP binding accompanied large changes in TfR and ferritin expression, while chelation of residual iron with deferoxamine (DFO) caused a large increase in IRP binding with little additional effect on TfR or ferritin expression. Cellular iron content increased beyond 4 days of exposure to iron at 20 microg/ml, when IRP binding, TfR, and ferritin had all reached stable levels. However, iron content of the cells plateaued by 7 days, or decreased with 24 h exposure to very high concentrations (>50 microg/ml) of iron. These results indicate that iron-replete HepG2 cells exhibit a narrow range of maximal responsiveness of the IRP-regulatory mechanism, whose functional response is blunted both by excessive iron exposure and by removal of iron from a chelatable pool. HepG2 cells are able to limit iron accumulation upon higher or prolonged exposure to NTBI, apparently independent of the IRP mechanism.     

The cellular mechanisms of body iron homeostasis

Cells tightly regulate iron levels through the activity of iron regulatory proteins (IRPs) that bind to RNA motifs called iron responsive elements (IREs). When cells become iron-depleted, IRPs bind to IREs present in the mRNAs of ferritin and the transferrin receptor, resulting in diminished translation of the ferritin mRNA and increased translation of the transferrin receptor mRNA. Similarly, body iron homeostasis is maintained through the control of intestinal iron absorption. Intestinal epithelia cells sense body iron through the basolateral endocytosis of plasma transferrin. Transferrin endocytosis results in enterocytes whose iron content will depend on the iron saturation of plasma transferrin. Cell iron levels, in turn, inversely correlate with intestinal iron absorption. In this study, we examined the relationship between the regulation of intestinal iron absorption and the regulation of intracellular iron levels by Caco-2 cells. We asserted that IRP activity closely correlates with apical iron uptake and transepithelial iron transport. Moreover, overexpression of IRE resulted in a very low labile or reactive iron pool and increased apical to basolateral iron flux. These results show that iron absorption is primarily regulated by the size of the labile iron pool, which in turn is regulated by the IRE/IRP system.     

Expression of iron-related proteins during infection by bovine herpes virus type-1

Bovine herpesvirus 1 (BHV-1), a dsDNA animal virus, is an economically important pathogen of cattle and the aetiological agent of many types of disease. The efficient replication of a DNA virus is strictly dependent on iron since this metal plays a crucial role in the catalytic center of viral ribonucleotide reductase. Consequently, iron metabolism is an important area for virus/host interaction and a large body of evidence suggests that viral infection is potentially influenced by the iron status of the host. The aim of the present study was to address the effects of BHV-1 on iron metabolism in Madin-Darby bovine kidney (MDBK) cells at different times of post-infection. For this purpose, cell viability, iron regulatory proteins (IRPs) activity and levels, transferrin receptor 1 (TfR-1), ferritin expression and LIP were evaluated. Our data demonstrate that a productive BHV-1 infection in MDBK cells determines an overall decrease of IRPs RNA-binding activity without affecting their expression. As consequence of this modulation, an increased ferritin mRNA translation and a decreased TfR-1 mRNA translation were also observed. Moreover, the LIP level was decreased following viral infection. These results are consistent with the hypothesis that by reducing the iron up-take and by enhancing the sequestration of free iron, animal cells will limit the iron availability for virus proliferation. Therefore, the results presented herein support the view that iron metabolism could be critical for the interaction between DNA viruses, such as BHV-1, and mammalian cells. Delineation of the interplay among pathogen and host may provide new antimicrobial agents.