HOIL-1 is not required for iron-mediated IRP2 degradation in HEK293 cells

Iron regulatory protein 2 (IRP2) binds to iron-responsive elements (IREs) to regulate the translation and stability of mRNAs encoding several proteins involved in mammalian iron homeostasis. Increases in cellular iron stimulate the polyubiquitylation and proteasomal degradation of IRP2. One study has suggested that haem-oxidized IRP2 ubiquitin ligase-1 (HOIL-1) binds to a unique 73-amino acid (aa) domain in IRP2 in an iron-dependent manner to regulate IRP2 polyubiquitylation and degradation. Other studies have questioned the role of the 73-aa domain in iron-dependent IRP2 degradation. We investigated the potential role of HOIL-1 in the iron-mediated degradation of IRP2 in human embryonic kidney 293 (HEK293) cells. We found that transiently expressed HOIL-1 and IRP2 interact via the 73-aa domain, but this interaction is not iron-dependent, nor does it enhance the rate of IRP2 degradation by iron. In addition, stable expression of HOIL-1 does not alter the iron-dependent degradation or RNA-binding activity of endogenous IRP2. Reduction of endogenous HOIL-1 by siRNA has no affect on the iron-mediated degradation of endogenous IRP2. These data demonstrate that HOIL-1 is not required for iron-dependent degradation of IRP2 in HEK293 cells, and suggest that a HOIL-1 independent mechanism is used for IRP2 degradation in most cell types.     

Redox control of iron regulatory protein 2 stability

Iron regulatory protein 2 (IRP2) is a critical switch for cellular and systemic iron homeostasis. In iron-deficient or hypoxic cells, IRP2 binds to mRNAs containing iron responsive elements (IREs) and regulates their expression. Iron promotes proteasomal degradation of IRP2 via the F-box protein FBXL5. Here, we explored the effects of oxygen and cellular redox status on IRP2 stability. We show that iron-dependent decay of tetracycline-inducible IRP2 proceeds efficiently under mild hypoxic conditions (3% oxygen) but is compromised in severe hypoxia (0.1% oxygen). A treatment of cells with exogenous H(2)O(2) protects IRP2 against iron and increases its IRE-binding activity. IRP2 is also stabilized during menadione-induced oxidative stress. These data demonstrate that the degradation of IRP2 in iron-replete cells is not only oxygen-dependent but also sensitive to redox perturbations.     

Effect of hypoxia on the binding and subcellular distribution of iron regulatory proteins

Iron regulatory proteins 1 and 2 (IRP1, IRP2) are key determinants of uptake and storage of iron by the liver, and are responsive to oxidative stress and hypoxia potentially at the level of both protein concentration and mRNA-binding activity. We examined the effect of hypoxia (1% O₂) on IRP1 and IRP2 levels (Western blots) and mRNA-binding activity (gel shift assays) in human hepatoma HepG2 cells, and compared them with HEK 293 cells, a renal cell line known to respond to hypoxia. Total IRP binding to an iron responsive element (IRE) mRNA probe was increased several fold by hypoxia in HEK 293 cells, maximally at 4–8 h. An earlier and more modest increase (1.5- to 2-fold, peaking at 2 h and then declining) was seen in HepG2 cells. In both cell lines, IRP1 made a greater contribution to IRE-binding activity than IRP2. IRP1 protein levels were increased slightly by hypoxia in HEK 293 but not in HepG2 cells. IRP1 was distributed between cytosolic and membrane-bound fractions, and in both cells hypoxia increased both the amount and IRE-binding activity of the membrane-associated IRP1 fraction. Further density gradient fractionation of HepG2 membranes revealed that hypoxia caused an increase in total membrane IRP1, with a shift in the membrane-bound fraction from Golgi to an endoplasmic reticulum (ER)-enriched fraction. Translocation of IRP to the ER has previously been shown to stabilize transferrin receptor mRNA, thus increasing iron availability to the cell. Iron depletion with deferoxamine also caused an increase in ER-associated IRP1. Phorbol ester caused serine phosphorylation of IRP1 and increased its association with the ER. The calcium ionophore ionomycin likewise increased ER-associated IRP1, without affecting total IRE-binding activity. We conclude that IRP1 is translocated to the ER by multiple signals in HepG2 cells, including hypoxia, thereby facilitating its role in regulation of hepatic gene expression. Electronic supplementary material The online version of this article () contains supplementary material, which is available to authorized users.     

Iron regulatory protein 2 turnover through a nonproteasomal pathway

Iron regulatory protein 2 (IRP2) controls the synthesis of many proteins involved in iron metabolism, and the level of IRP2 itself is regulated by varying the rate of its degradation. The proteasome is known to mediate degradation, with specificity conferred by an iron-sensing E3 ligase. Most studies on the degradation of IRP2 have employed cells overexpressing IRP2 and also rendered iron deficient to further increase IRP2 levels. We utilized a sensitive, quantitative assay for IRP2, which allowed study of endogenous IRP2 degradation in HEK293A cells under more physiologic conditions. We found that under these conditions, the proteasome plays only a minor role in the degradation of IRP2, with almost all the IRP2 being degraded by a nonproteasomal pathway. This new pathway is calcium-dependent but is not mediated by calpain. Elevating the cellular level of IRP2 by inducing iron deficiency or by transfection causes the proteasomal pathway to account for the major fraction of IRP2 degradation. We conclude that under physiological, iron-sufficient conditions, the steady-state level of IRP2 in HEK293A cells is regulated by the nonproteasomal pathway.     

Effect of hypoxia on the expression of iron regulatory proteins 1 and the mechanisms involved

Iron is essential for many biological processes, including oxygen delivery, and its supply is tightly regulated. Iron regulatory proteins (IRPs, IRP1 and IRP2) are master regulators of cellular iron metabolism. Hypoxia triggers a broad range of gene responses that are primarily mediated by hypoxia-inducible factor-1 (HIF-1). In this study, we have shown that hypoxia could not only upregulate the expression of hypoxia inducible factor-1 but also downregulate the expression of IRP1. However, the molecular mechanisms that govern the IRP1 response to hypoxia are not known. Herein we suggested that HIF/HRE system was an essential link between IRP1 and hypoxia. The HRE of IRP1 5'-regulation regions could combine with HIF-1 in vitro. Dual-luciferase reporter assay showed that IRP1 was directly downregulated by HIF/HRE system.     

Involvement of heme regulatory motif in heme-mediated ubiquitination and degradation of IRP2

Iron regulatory protein 2 (IRP2), a regulator of iron metabolism, is modulated by ubiquitination and degradation. We have shown that IRP2 degradation is triggered by heme-mediated oxidation. We report here that not only Cys201, an invariant residue in the heme regulatory motif (HRM), but also His204 is critical for IRP2 degradation. Spectroscopic studies revealed that Cys201 binds ferric heme, whereas His204 is a ferrous heme binding site, indicating the involvement of these residues in sensing the redox state of the heme iron and in generating the oxidative modification. Moreover, the HRM in IRP2 has been suggested to play a critical role in its recognition by the HOIL-1 ubiquitin ligase. Although HRMs are known to sense heme concentration by simply binding to heme, the HRM in IRP2 specifically contributes to its oxidative modification, its recognition by the ligase, and its sensing of iron concentration after iron is integrated into heme.