Autophagy,ferritin and iron chelation

Ferritin is an iron storage molecule in vertebrates that stores iron in a redox inactive form. Ferritin is synthesized in response to high cellular iron levels and is degraded and iron released when iron demand is increased. Previously we determined that the turnover of ferritin occurs via the proteasome when the iron exporter ferroportin is expressed, and via the lysosome when the iron chelator deferoxamine is given to cells. Deferoxamine is used to treat hemochromatosis, a disease of iron accumulation that can be either genetic or acquired.

Autophagy provides a mechanism by which cytosolic proteins gain access to the lumen of lysosomes. Our results suggest that entry of ferritin into lysosomes is highly specific and not a consequence of generalized engulfment of cytosolic compartments by lysosomes. Entry of ferritin is also independent of the presence of LAMP-2, which suggests that ferritin entry does not result from chaperone-mediated autophagy. In summary, in this study we identify a new route that links ferritin degradation to activation of autophagy. The identification of this pathway will help to understand the molecular events that lead to activation of deferoxamine-mediated ferritin degradation and may contribute to the design of new therapeutic strategies for iron chelation therapy.     

Ferroportin-mediated mobilization of ferritin iron precedes ferritin degradation by the proteasome

Ferritin is a cytosolic molecule comprised of subunits that self-assemble into a nanocage capable of containing up to 4500 iron atoms. Iron stored within ferritin can be mobilized for use within cells or exported from cells. Expression of ferroportin (Fpn) results in export of cytosolic iron and ferritin degradation. Fpn-mediated iron loss from ferritin occurs in the cytosol and precedes ferritin degradation by the proteasome. Depletion of ferritin iron induces the monoubiquitination of ferritin subunits. Ubiquitination is not required for iron release but is required for disassembly of ferritin nanocages, which is followed by degradation of ferritin by the proteasome. Specific mammalian machinery is not required to extract iron from ferritin. Iron can be removed from ferritin when ferritin is expressed in Saccharomyces cerevisiae, which does not have endogenous ferritin. Expressed ferritin is monoubiquitinated and degraded by the proteasome. Exposure of ubiquitination defective mammalian cells to the iron chelator desferrioxamine leads to degradation of ferritin in the lysosome, which can be prevented by inhibitors of autophagy. Thus, ferritin degradation can occur through two different mechanisms.     

Ferritin accumulation under iron scarcity in Drosophila iron cells

Ferritins are highly stable, multi-subunit protein complexes with iron-binding capacities that reach 4500 iron atoms per ferritin molecule. The strict dependence of cellular physiology on an adequate supply of iron cofactors has likely been a key driving force in the evolution of ferritins as iron storage molecules. The insect intestine has long been known to contain cells that are responsive to dietary iron levels and a specialized group of “iron cells” that always accumulate iron-loaded ferritin, even when no supplementary iron is added to the diet. Here, we further characterize ferritin localization in Drosophila melanogaster larvae raised under iron-enriched and iron-depleted conditions. High dietary iron intake results in ferritin accumulation in the anterior midgut, but also in garland (wreath) cells and in pericardial cells, which together filter the circulating hemolymph. Ferritin is also abundant in the brain, where levels remain unaltered following dietary iron chelation, a treatment that depletes ferritin from the aforementioned tissues. We attribute the stability of ferritin levels in the brain to the function of the blood-brain barrier that may shield this organ from systemic iron fluctuations. Most intriguingly, our dietary manipulations demonstrably iron-depleted the iron cells without a concomitant reduction in their production of ferritin. Therefore, insect iron cells may constitute an exception from the evolutionary norm with respect to iron-dependent ferritin regulation. It will be of interest to decipher both the physiological purpose served and the mechanism employed to untie ferritin regulation from cellular iron levels in this cell type.     

细菌生产人铁蛋白的新功能及应用

铁蛋白(Ferritin)是一种广泛存在于生物体中的笼状蛋白,由24个亚基自组装形成的蛋白质外壳和铁内核两部分组成,是维持机体铁代谢平衡的重要蛋白。最新发现,人血清铁蛋白含量的变化与某些疾病相关,特别是发现利用大肠杆菌重组表达、仿生合成的磁性人铁蛋白具有双功能特性,即识别肿瘤并使其可视化。此外,铁蛋白独特的结构及理化性质使其成为理想的纳米载体,用于构筑多功能肿瘤成像和药物输送的平台。本文重点介绍人铁蛋白的新功能及其在疾病诊断和肿瘤靶向治疗中的应用前景。     

Cell sensitivity to oxidative stress is influenced by ferritin autophagy

To test the consequences of lysosomal degradation of differently iron-loaded ferritin molecules and to mimic ferritin autophagy under iron-overload and normal conditions, J774 cells were allowed to endocytose heavily iron loaded ferritin, probably with some adventitious iron (Fe-Ft), or iron-free apo-ferritin (apo-Ft). When cells subsequently were exposed to a bolus dose of hydrogen peroxide, apo-Ft prevented lysosomal membrane permeabilization (LMP), whereas Fe-Ft enhanced LMP. A 4-h pulse of Fe-Ft initially increased oxidative stress-mediated LMP that was reversed after another 3h under standard culture conditions, suggesting that lysosomal iron is rapidly exported from lysosomes, with resulting upregulation of apo-ferritin that supposedly is autophagocytosed, thereby preventing LMP by binding intralysosomal redox-active iron. The obtained data suggest that upregulation of the stress protein ferritin is a rapid adaptive mechanism that counteracts LMP and ensuing apoptosis during oxidative stress. In addition, prolonged iron starvation was found to induce apoptotic cell death that, interestingly, was preceded by LMP, suggesting that LMP is a more general phenomenon in apoptosis than so far recognized. The findings provide new insights into aging and neurodegenerative diseases that are associated with enhanced amounts of cellular iron and show that lysosomal iron loading sensitizes to oxidative stress.     

Decoupling ferritin synthesis from free cytosolic iron results in ferritin secretion

Ferritin is a multisubunit protein that is responsible for storing and detoxifying cytosolic iron. Ferritin can be found in serum but is relatively iron poor. Serum ferritin occurs in iron overload disorders, in inflammation, and in the genetic disorder hyperferritinemia with cataracts. We show that ferritin secretion results when cellular ferritin synthesis occurs in the relative absence of free cytosolic iron. In yeast and mammalian cells, newly synthesized ferritin monomers can be translocated into the endoplasmic reticulum and transits through the secretory apparatus. Ferritin chains can be translocated into the endoplasmic reticulum in an in?vitro translation and membrane insertion system. The insertion of ferritin monomers into the ER occurs under low-free-iron conditions, as iron will induce the assembly of ferritin. Secretion of ferritin chains provides a mechanism that limits ferritin nanocage assembly and ferritin-mediated iron sequestration in the absence of the translational inhibition of ferritin synthesis.     

铁自噬与铁死亡及其相关疾病

铁是血红素、线粒体呼吸链复合体和各种生物酶的重要辅助因子,参与氧气运输、氧化还原反应和代谢物合成等生物过程。铁蛋白(ferritin)是一种铁存储蛋白质,通过储存和释放铁来维持机体内铁平衡。铁自噬(ferritinophagy)作为一种选择性自噬方式,介导铁蛋白降解释放游离铁,参与细胞内铁含量的调控。适度铁自噬维持细胞内铁含量稳定,但铁自噬过度会释放出大量游离铁。通过芬顿 (Fenton)反应催化产生大量的活性氧(reactive oxygen species, ROS),发生脂质过氧化造成细胞受损。因此,铁自噬在维持细胞生理性铁稳态中发挥至关重要的作用。核受体共激活因子4 (nuclear receptor co-activator 4, NCOA4)被认为是铁自噬的关键调节因子,与铁蛋白靶向结合,并传递至溶酶体中降解释放游离铁,其介导的铁自噬构成了铁代谢的重要组成部分。最新研究表明,NCOA4受体内铁含量、自噬、溶酶体和低氧等因素的调控。NCOA4介导的铁蛋白降解与铁死亡(ferroptosis)有关。铁死亡是自噬性细胞死亡过程。铁自噬通过调节细胞铁稳态和细胞ROS生成,成为诱导铁死亡的上游机制,与贫血、神经退行性疾病、癌症、缺血/再灌注损伤与疾病的发生发展密切相关。本文针对NCOA4介导的铁自噬通路在铁死亡中的功能特征,探讨NCOA4在这些疾病中的作用,可能为相关疾病的治疗提供启示。     

Ferritin-stimulated lipid peroxidation,lysosomal leak,and macroautophagy promote lysosomal “metastability” in primary hepatocytes determining in vitro cell survival

Several pathologies are associated with elevated levels of serum ferritin, for which growth inhibitory properties have been reported; however, the underlying mechanisms are still poorly defined. Previously we have described cytotoxic properties of isoferritins released from primary hepatocytes in vitro, which induce apoptosis in an iron and oxidative stress-dependent mode. Here we show that this ferritin species stimulates endosome clustering and giant endosome formation in primary hepatocytes accompanied by enhanced lysosomal membrane permeability (LMP). In parallel, protein modification by lipid peroxidation-derived 4-hydroxynonenal (HNE) is strongly promoted by ferritin, the HNE-modified proteins (HNE-P) showing remarkable aggregation. Emphasizing the prooxidant context, GSH is rapidly depleted and the GSH/GSSG ratio is substantially declining in ferritin-treated cells. Furthermore, ferritin triggers a transient upregulation of macroautophagy which is abolished by iron chelation and apparently supports HNE-P clearance. Macroautophagy inhibition by 3-methyladenine strongly amplifies ferritin cytotoxicity in a time- and concentration-dependent mode, suggesting an important role of macroautophagy on cellular responses to ferritin endocytosis. Moreover, pointing at an involvement of lysosomal proteolysis, ferritin cytotoxicity and lysosome fragility are aggravated by the protease inhibitor leupeptin. In contrast, EGF which suppresses ferritin-induced cell death attenuates ferritin-mediated LMP. In conclusion, we propose that HNE-P accumulation, lysosome dysfunction, and macroautophagy stimulated by ferritin endocytosis provoke lysosomal “metastability” in primary hepatocytes which permits cell survival as long as in- and extrinsic determinants (e.g., antioxidant availability, damage repair, EGF signaling) keep the degree of lysosomal destabilization below cell death-inducing thresholds.     

Ferritin forms dynamic oligomers to associate with microtubules in vivo: implication for the role of microtubules in iron metabolism

Ferritin, a ubiquitously distributed iron storage protein, has been reported to interact with microtubules in vitro (Hasan et al., 2005, FEBS journal 272:822-831). Here, we demonstrate that ferritin binds with the microtubules in an oligomeric form and that the microtubule-bound ferritin contains more than two-fold amount of iron compared to the unbound ferritin fraction in vitro. Indirect immunofluorescence microscopy showed that a significant fraction of the ferritin molecules colocalized with the microtubules as oligomers in a wide variety of cell lines. These findings are consistent with the immediate oligomerization of rhodamine-labeled ferritin, microinjected in living human hepatoma cells. Ferritin oligomers were dynamic in the cytoplasm, and an anti-microtubule drug significantly inhibited their intracellular movement. Treatment of cells with an iron donor, ferric ammonium citrate, remarkably increased the number of cells containing ferritin oligomers. On the other hand, when the cells, such as mouse neuroblastoma cells, were deprived of iron, ferritin oligomers were localized in the microtubule dense, neurite shafts, but were disappeared from the microtubule deficient neurite tips. These data indicate that the microtubules provide a scaffold for the cytoplasmic distribution and transport of the iron-rich ferritin and implicate the role of microtubules in iron metabolism.     

Iron starvation regulates the type III secretion system in Bordetella bronchiseptica

The type III secretion system (T3SS) plays a key role in the exertion of full virulence by Bordetella bronchiseptica. However, little is known about the environmental stimuli that induce expression of T3SS genes. Here, it is reported that iron starvation is a signal for T3SS gene expression in B. bronchiseptica. It was found that, when B. bronchiseptica is cultured under iron-depleted conditions, secretion of type III secreted proteins is greater than that in bacteria grown under iron-replete conditions. Furthermore, it was confirmed that induction of T3SS-dependent host cell cytotoxicity and hemolytic activity is greatly enhanced by infection with iron-depleted Bordetella. In contrast, production of filamentous hemagglutinin is reduced in iron-depleted Bordetella. Thus, B. bronchiseptica controls the expression of virulence genes in response to iron starvation.     

Ferritinophagy drives uropathogenic Escherichia coli persistence in bladder epithelial cells

Autophagy is a cellular recycling pathway, which in many cases, protects host cells from infections by degrading pathogens. However, uropathogenic Escherichia coli (UPEC), the predominant cause of urinary tract infections (UTIs), persist within the urinary tract epithelium (urothelium) by forming reservoirs within autophagosomes. Iron is a critical nutrient for both host and pathogen, and regulation of iron availability is a key host defense against pathogens. Iron homeostasis depends on the shuttling of iron-bound ferritin to the lysosome for recycling, a process termed ferritinophagy (a form of selective autophagy). Here, we demonstrate for the first time that UPEC shuttles with ferritin-bound iron into the autophagosomal and lysosomal compartments within the urothelium. Iron overload in urothelial cells induces ferritinophagy in an NCOA4-dependent manner causing increased iron availability for UPEC, triggering bacterial overproliferation and host cell death. Addition of even moderate levels of iron is sufficient to increase and prolong bacterial burden. Furthermore, we show that lysosomal damage due to iron overload is the specific mechanism causing host cell death. Significantly, we demonstrate that host cell death and bacterial burden can be reversed by inhibition of autophagy or inhibition of iron-regulatory proteins, or chelation of iron. Together, our findings suggest that UPEC persist in host cells by taking advantage of ferritinophagy. Thus, modulation of iron levels in the bladder may provide a therapeutic avenue to controlling UPEC persistence, epithelial cell death, and recurrent UTIs.     

Ferritin: a novel mechanism for delivery of iron to the brain and other organs

Traditionally, transferrin has been considered the primary mechanism for cellular iron delivery, despite suggestive evidence for additional iron delivery mechanisms. In this study we examined ferritin, considered an iron storage protein, as a possible delivery protein. Ferritin consists of H- and L-subunits, and we demonstrated iron uptake by ferritin into multiple organs and that the uptake of iron is greater when the iron is delivered via H-ferritin compared with L-ferritin. The delivery of iron via H-ferritin but not L-ferritin was significantly decreased in mice with compromised iron storage compared with control, indicating that a feedback mechanism exists for H-ferritin iron delivery. To further evaluate the mechanism of ferritin iron delivery into the brain, we used a cell culture model of the blood-brain barrier to demonstrate that ferritin is transported across endothelial cells. There are receptors that prefer H-ferritin on the endothelial cells in culture and on rat brain microvasculature. These studies identify H-ferritin as an iron transport protein and suggest the presence of an H-ferritin receptor for mediating iron delivery. The relative amount of iron that could be delivered via H-ferritin could make this protein a predominant player in cellular iron delivery. blood-brain barrier; iron transport; H-ferritin     

A human mitochondrial ferritin encoded by an intronless gene

Ferritin is a ubiquitous protein that plays a critical role in regulating intracellular iron homoeostasis by storing iron inside its multimeric shell. It also plays an important role in detoxifying potentially harmful free ferrous iron to the less soluble ferric iron by virtue of the ferroxidase activity of the H subunit. Although excess iron is stored primarily in cytoplasm, most of the metabolically active iron in cells is processed in mitochondria. Little is yet known of how these organelles regulate iron homeostasis and toxicity. Here we report an unusual intronless gene on chromosome 5q23.1 that encodes a 242-amino acid precursor of a ferritin H-like protein. This 30-kDa protein is targeted to mitochondria and processed to a 22-kDa subunit that assembles into typical ferritin shells and has ferroxidase activity. Immunohistochemical analysis showed that it accumulates in high amounts in iron-loaded mitochondria of erythroblasts of subjects with impaired heme synthesis. This new ferritin may play an important role in the regulation of mitochondrial iron homeostasis and heme synthesis.     

Artesunate Induces Cell Death in Human Cancer Cells via Enhancing Lysosomal Function and Lysosomal Degradation of Ferritin

Artesunate (ART) is an anti-malaria drug that has been shown to exhibit anti-tumor activity, and functional lysosomes are reported to be required for ART-induced cancer cell death, whereas the underlying molecular mechanisms remain largely elusive. In this study, we aimed to elucidate the molecular mechanisms underlying ART-induced cell death. We first confirmed that ART induces apoptotic cell death in cancer cells. Interestingly, we found that ART preferably accumulates in the lysosomes and is able to activate lysosomal function via promotion of lysosomal V-ATPase assembly. Furthermore, we found that lysosomes function upstream of mitochondria in reactive oxygen species production. Importantly, we provided evidence showing that lysosomal iron is required for the lysosomal activation and mitochondrial reactive oxygen species production induced by ART. Finally, we showed that ART-induced cell death is mediated by the release of iron in the lysosomes, which results from the lysosomal degradation of ferritin, an iron storage protein. Meanwhile, overexpression of ferritin heavy chain significantly protected cells from ART-induced cell death. In addition, knockdown of nuclear receptor coactivator 4, the adaptor protein for ferritin degradation, was able to block ART-mediated ferritin degradation and rescue the ART-induced cell death. In summary, our study demonstrates that ART treatment activates lysosomal function and then promotes ferritin degradation, subsequently leading to the increase of lysosomal iron that is utilized by ART for its cytotoxic effect on cancer cells. Thus, our data reveal a new mechanistic action underlying ART-induced cell death in cancer cells.     

Iron uptake mediated by binding of H-ferritin to the TIM-2 receptor in mouse cells

Ferritin binds specifically and saturably to a variety of cell types, and recently several ferritin receptors have been cloned. TIM-2 is a specific receptor for H ferritin (HFt) in the mouse. TIM-2 is a member of the T cell immunoglobulin and mucin domain containing (TIM) protein family and plays an important role in immunity. The expression of TIM-2 outside of the immune system indicates that this receptor may have broader roles. We tested whether ferritin binding to TIM-2 can serve as an iron delivery mechanism. TIM-2 was transfected into normal (TCMK-1) mouse kidney cells, where it was appropriately expressed on the cell surface. HFt was labeled with (55)Fe and (55)Fe-HFt was incubated with TIM-2 positive cells or controls. (55)Fe-HFt uptake was observed only in TIM-2 positive cells. HFt uptake was also seen in A20 B cells, which express endogenous TIM-2. TIM-2 levels were not increased by iron chelation. Uptake of (55)Fe-HFt was specific and temperature-dependent. HFt taken up by TIM-2 positive cells transited through the endosome and eventually entered a lysosomal compartment, distinguishing the HFt pathway from that of transferrin, the classical vehicle for cellular iron delivery. Iron delivered following binding of HFt to TIM-2 entered the cytosol and became metabolically available, resulting in increased levels of endogenous intracellular ferritin. We conclude that TIM-2 can function as an iron uptake pathway.     

Ferritin and ferritin isoforms II: protection against uncontrolled cellular proliferation, oxidative damage and inflammatory processes

Ferritin is a major iron storage protein involved in the regulation of iron availability. Each ferritin molecule comprises 24 subunits. Various combinations of H-subunits and L-subunits make up the 24-subunit protein structure and these ferritin isoforms differ in their H-subunit to L-subunit ratio, as well as in their metabolic properties. Ferritin is an acute-phase protein and its expression is up-regulated in conditions such as uncontrolled cellular proliferation, in any condition marked by excessive production of toxic oxygen radicals, and by infectious and inflammatory processes. Under such conditions ferritin up-regulation is predominantly stimulated by increased reactive oxygen radical production and by cytokines. The major function of ferritin in these conditions is to reduce the bio-availability of iron in order to stem uncontrolled cellular proliferation and excessive production of reactive oxygen radicals. Ferritin is not, however, indiscriminately up-regulated in these conditions as a marked shift towards a predominance in H-subunit rich ferritins occurs. Preliminary indications are that, while the L-subunit primarily fulfils the conventional iron storage role, the H-subunit functions primarily as rapid regulator of iron availability, and perhaps indirectly as regulator of other cellular processes. It is suggested that the optimum differential expression of the two subunits differ for different cells and under different conditions and that the expression of appropriate isoferritins offers protection against uncontrolled cellular proliferation, oxidative stress and against side effects of infectious and inflammatory conditions.