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.     

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.     

The inhibition of microsomal triglyceride transfer protein activity in rat hepatoma cells promotes proteasomal and nonproteasomal degradation of apoprotein b100

In the human hepatic cell line, HepG2, apolipoprotein B100 (apoB100) degradation is increased by inhibiting lipid transfer mediated by the microsomal triglyceride transfer protein (MTP) and is predominantly accomplished by the ubiquitin-proteasome pathway. In the current study, we determined whether this degradative pathway was restricted to HepG2 cells or was of more general importance in hepatic apoB100 metabolism. Rat hepatoma McArdle RH7777 cells (McA), compared to HepG2 cells, secrete a large fraction of apoB100 associated with VLDL particles, as does the normal mammalian liver. In McA cells studied under basal conditions, the proteasome inhibitor lactacystin (LAC) increased apoB100 recovery, indicating that the role of the proteasome in apoB100 metabolism is not restricted to HepG2 cells. When apoB100 lipidation was blocked by an inhibitor of MTP (MTPI), recovery of cellular apoB100 was markedly reduced, but LAC was only partially ( approximately 50%) effective in reversing the induced degradation. This partial effectiveness of LAC may have represented either (1) incomplete inhibition by LAC of its preferred target, the chymotrypsin-like activity of the proteasome, (2) the presence of an apoB100 proteolytic activity of the proteasome resistant to LAC, or (3) a nonproteasomal proteolytic pathway of apoB100 degradation. By studying immunoisolated proteasomes and McA cells treated with LAC and/or MTPI and a variety of protease inhibitors, we determined that the proteasomal component of apoB100 degradation was entirely attributable to the chymotrypsin-like catalytic activity, but only accounted for part of apoB100 degradation induced by MTPI. The nonproteasomal apoB100 degradative pathway was nonlysosomal and resistant to E64d, DTT, and peptide aldehydes such as MG132 or ALLN but was partially sensitive to the serine protease inhibitor APMSF. Furthermore, when the protein trafficking inhibitor, brefeldin A, was used to block endoplasmic reticulum (ER) to Golgi transport in MTPI-treated McA cells, degradative activity resistant to LAC was increased, suggesting that the nonproteasomal pathway is associated with the ER.     

Ubiquitin-proteasome system mediates heme oxygenase-1 degradation through endoplasmic reticulum-associated degradation pathway

The present study investigated the cellular mechanism underlying the degradation of heme oxygenase-1 (HO-1), an endoplasmic reticulum (ER)-anchored protein. The turnover of HO-1 induced in vascular smooth muscle cells (VSMCs) was significantly attenuated by proteasome inhibitors, suggesting the involvement of a proteasome-mediated pathway. High molecular weight ubiquitin conjugates were co-immunoprecipitated with HO-1 from VSMCs after proteasome inhibition, and HO-1 ubiquitination was confirmed in HEK293 cells overexpressing His-tagged HO-1 and HA-tagged ubiquitin. Endogenous p97, an ATPase, and Ufd1, both implicated as essential components in the ER-associated degradation pathway (ERAD), were co-eluted with His-tagged HO-1 from metal affinity resin. Knockdown of either p97 or Ufd1 in HEK293 cells using specific siRNA significantly prolonged the half-life of endogenously induced HO-1 and slowed the degradation of ubiquitinated HO-1. HO-1 ubiquitination in HEK293 cells was enhanced by zinc chloride, but suppressed with a zinc chelator (N,N,N',N'-tetrakis(2-pyridylmethyl)ethyl-enediamine), suggesting the involvement of a RING-E3 ligase in this process. Collectively, these data indicate that HO-1 protein turnover is regulated by the ubiquitin-proteasome system through the ERAD pathway.     

S-nitrosylation of IRP2 regulates its stability via the ubiquitin-proteasome pathway

Nitric oxide (NO) is an important signaling molecule that interacts with different targets depending on its redox state. NO can interact with thiol groups resulting in S-nitrosylation of proteins, but the functional implications of this modification are not yet fully understood. We have reported that treatment of RAW 264.7 cells with NO caused a decrease in levels of iron regulatory protein 2 (IRP2), which binds to iron-responsive elements present in untranslated regions of mRNAs for several proteins involved in iron metabolism. In this study, we show that NO causes S-nitrosylation of IRP2, both in vitro and in vivo, and this modification leads to IRP2 ubiquitination followed by its degradation in the proteasome. Moreover, mutation of one cysteine (C178S) prevents NO-mediated degradation of IRP2. Hence, S-nitrosylation is a novel signal for IRP2 degradation via the ubiquitin-proteasome pathway.     

ZRT/IRT-like Protein 14 (ZIP14) Promotes the Cellular Assimilation of Iron from Transferrin

ZIP14 is a transmembrane metal ion transporter that is abundantly expressed in the liver, heart, and pancreas. Previous studies of HEK 293 cells and the hepatocyte cell lines AML12 and HepG2 established that ZIP14 mediates the uptake of non-transferrin-bound iron, a form of iron that appears in the plasma during pathologic iron overload. In this study we investigated the role of ZIP14 in the cellular assimilation of iron from transferrin, the circulating plasma protein that normally delivers iron to cells by receptor-mediated endocytosis. We also determined the subcellular localization of ZIP14 in HepG2 cells. We found that overexpression of ZIP14 in HEK 293T cells increased the assimilation of iron from transferrin without increasing levels of transferrin receptor 1 or the uptake of transferrin. To allow for highly specific and sensitive detection of endogenous ZIP14 in HepG2 cells, we used a targeted knock-in approach to generate a cell line expressing a FLAG-tagged ZIP14 allele. Confocal microscopic analysis of these cells detected ZIP14 at the plasma membrane and in endosomes containing internalized transferrin. HepG2 cells in which endogenous ZIP14 was suppressed by siRNA assimilated 50% less iron from transferrin compared with controls. The uptake of transferrin, however, was unaffected. We also found that ZIP14 can mediate the transport of iron at pH 6.5, the pH at which iron dissociates from transferrin within the endosome. These results suggest that endosomal ZIP14 participates in the cellular assimilation of iron from transferrin, thus identifying a potentially new role for ZIP14 in iron metabolism.     

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.     

Proteasome involvement in the degradation of the G(q) family of Galpha subunits

Metabolically unstable proteins are involved in a multitude of regulatory networks, including those that control cell signaling, the cell cycle and in many responses to physiological stress. In the present study, we have determined the stability and characterized the degradation process of some members of the G(q) class of heterotrimeric G proteins. Pulse-chase experiments in HEK293 cells indicated a rapid turnover of endogenously expressed Galpha(q) and overexpressed Galpha(q) and Galpha(16) subunits. Pretreatment with proteasome inhibitors attenuated the degradation of both G alpha subunits. In contrast, pretreatment of cells with inhibitors of lysosomal proteases and nonproteasomal cysteine proteases had very little effect on the stability of the proteins. Significantly, the turnover of these proteins is not affected by transient activation of their associated receptors. Fractionation studies showed that the rates of Galpha(q) and Galpha16 degradation are accelerated in the cytosol. In fact, we show that a mutant Galpha(q) which lacks its palmitoyl modification site, and which is localized almost entirely in the cytoplasm, has a marked increase in the rate of degradation. Taken together, these results suggest that the G(q) class proteins are degraded through the proteasome pathway and that cellular localization and/or other protein interactions determine their stability.     

ER stress induces alternative nonproteasomal degradation of ER proteins but not of cytosolic ones

Inhibition of protein folding in the endoplasmic reticulum (ER) causes ER stress, which triggers the unfolded protein response (UPR). To decrease the biosynthetic burden on the ER, the UPR inhibits in its initial stages protein synthesis. At later stages it upregulates components of ER-associated degradation (ERAD) and of the ubiquitin/proteasome system, which targets ER as well as cytosolic proteins for disposal. Here we report that, at later stages, the UPR also activates an alternative nonproteasomal pathway of degradation, which is resistant to proteasome inhibitors and is specific for ER substrates (assessed with uncleaved precursor of asialoglycoprotein receptor H2a and unassembled CD3delta) and not for cytosolic ones (p53). To mimic the initial inhibition of translation during UPR, we incubated cells with cycloheximide. After this treatment, degradation of ERAD substrates was no longer effected by proteasomal inhibition, similarly to the observed outcome of UPR. The degradation also became insensitive to abrogation of ubiquitination in a cell line carrying a thermosensitive E1 ubiquitin activating enzyme mutant. Of all protease inhibitors tested, only the metal chelator o-phenanthroline could block this nonproteasomal degradation. Preincubation of o-phenanthroline with Mn2+ or Co2+, but not with other cations, reversed the inhibition. Our results suggest that, upon inhibition of translation, an alternative nonproteasomal pathway is activated for degradation of proteins from the ER. This involves a Mn2+/Co2+-dependent metalloprotease or other metalloprotein. The alternative pathway selectively targets ERAD substrates to reduce the ER burden, but does not affect p53, the levels of which remain dependent on proteasomal control.     

Pro-caspase-3 overexpression sensitises ovarian cancer cells to proteasome inhibitors

The ubiquitin-proteasome pathway plays a critical role in the degradation of several proteins involved in the cell cycle. Dysregulation of this pathway leads to inhibition of cellular proliferation and the induction of apoptosis. Ubiquitination and its downstream consequences have been investigated intensively as targets for the development of drugs for tumour therapy. Here we have investigated the mechanism of apoptosis induced by the proteasome inhibitors MG-132, lactacystin and calpain inhibitor I (ALLN), in the HEK 293 cell line and the ovarian cancer cell lines SKOV3 and OVCAR3. We have found strong caspase-3-like and caspase-6-like activation upon treatment of HEK 293 cells with MG-132. Using a tricistronic expression vector based on a tetracycline-responsive system we generated stable SKOV3 nd OVCAR3 cell lines with inducible expression of pro-caspase-3. Induction of pro-caspase-3 expression in normally growing cells does not induce apoptosis. However, in the presence of the proteasome inhibitors MG-132, lactacystin or ALLN we found that cells overexpressing pro-caspase-3 are rapidly targeted for apoptosis. Our results demonstrate that pro-caspase-3 can sensitise ovarian cancer cells to proteasome inhibitor-induced apoptosis, and a combination of these approaches might be exploited for therapy of ovarian and other cancers.