Distinct mechanisms of ferritin delivery to lysosomes in iron-depleted and iron-replete cells

Ferritin is a cytosolic protein that stores excess iron, thereby protecting cells from iron toxicity. Ferritin-stored iron is believed to be utilized when cells become iron deficient; however, the mechanisms underlying the extraction of iron from ferritin have yet to be fully elucidated. Here, we demonstrate that ferritin is degraded in the lysosome under iron-depleted conditions and that the acidic environment of the lysosome is crucial for iron extraction from ferritin and utilization by cells. Ferritin was targeted for degradation in the lysosome even under iron-replete conditions in primary cells; however, the mechanisms underlying lysosomal targeting of ferritin were distinct under depleted and replete conditions. In iron-depleted cells, ferritin was targeted to the lysosome via a mechanism that involved autophagy. In contrast, lysosomal targeting of ferritin in iron-replete cells did not involve autophagy. The autophagy-independent pathway of ferritin delivery to lysosomes was deficient in several cancer-derived cells, and cancer-derived cell lines are more resistant to iron toxicity than primary cells. Collectively, these results suggest that ferritin trafficking may be differentially regulated by cell type and that loss of ferritin delivery to the lysosome under iron-replete conditions may be related to oncogenic cellular transformation.     

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.     

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。     

Quantitative analysis of immunogold labelling for ferritin in liver from control and iron-overloaded rats

Summary The distribution of ferritin antigenicity in control and iron-loaded rat hepatocytes was investigated with an immunogold-ferritin antibody technique. Antibody to horse spleen ferritin showed immunoreactivity as determined by dot blotting with immunogold/silver staining with purified rat liver ferritin but not with rat haemosiderin. The initial site of ferritin degradation was studied by analysing the density of gold labelling in the cytosol and lysosomes in combination with pre-embedding acid phosphatase cytochemistry.Immunoreactive ferritin was present in the cytosol, cytosolic clusters and lysosomes of normal hepatocytes. After iron-loading, the labelling density increased over tenfold in parenchymal cell cytosol with a smaller increase in Kupffer cells. Ferritin clusters contained substantially more immunoreactive ferritin than equivalent areas of lysosomes or cytosol. Analysis of the labelling density in hepatocyte lysosomes showed that, despite a striking increase in iron content, one-quarter of the lysosomes showed less immunolabelled ferritin than the cytosol. The existence of a wide range of ferritin labelling densities in the lysosomes with a large proportion unlabelled suggests that the ferritin protein shell is not degraded at a significant rate either in the cytosol or in clusters but only after incorporation into lysosomes.     

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.     

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.     

Dedicated SNAREs and specialized TRIM cargo receptors mediate secretory autophagy

Autophagy is a process delivering cytoplasmic components to lysosomes for degradation. Autophagy may, however, play a role in unconventional secretion of leaderless cytosolic proteins. How secretory autophagy diverges from degradative autophagy remains unclear. Here we show that in response to lysosomal damage, the prototypical cytosolic secretory autophagy cargo IL‐1β is recognized by specialized secretory autophagy cargo receptor TRIM16 and that this receptor interacts with the R‐SNARE Sec22b to recruit cargo to the LC3‐II⁺ sequestration membranes. Cargo secretion is unaffected by downregulation of syntaxin 17, a SNARE promoting autophagosome–lysosome fusion and cargo degradation. Instead, Sec22b in combination with plasma membrane syntaxin 3 and syntaxin 4 as well as SNAP‐23 and SNAP‐29 completes cargo secretion. Thus, secretory autophagy utilizes a specialized cytosolic cargo receptor and a dedicated SNARE system. Other unconventionally secreted cargo, such as ferritin, is secreted via the same pathway.     

The effects of ascorbic acid on the intracellular metabolism of iron and ferritin

An important property of ascorbic acid is its ability to increase the availability of storage iron to chelators. To examine the mechanism of this effect, K562 cells were incubated with ascorbate, attaining an intracellular level of 1 nmol/10(7) cells. In contrast to the reductive mobilization of iron seen with isolated ferritin, ascorbate stabilized iron preincorporated into cellular ferritin. Biosynthetic labeling with [35S]methionine demonstrated that ascorbate also retarded the degradation of the ferritin protein shell. Ferritin is normally degraded in lysosomes. The lysosomal protease inhibitors leupeptin and chloroquine produced a qualitatively similar stabilization of ferritin. Ascorbate did not act as a general inhibitor of proteolysis, however, since it did not effect hemoglobin degradation in these cells. The stabilization of cellular ferritin by ascorbate was accompanied by an expansion of the pool of chelatable iron.     

Intracellular fate of ferritin in HeLa cells following microinjection

It is known that following iron overload newly synthesized ferritin molecules accumulate in lysosomes. However, the way in which these molecules enter the lysosomes has not been clarified. In order to assess if these molecules can be taken up by lysosomes from the cell sap, i.e., by way of autophagy, ferritin was introduced into HeLa cells through microinjection with a glass capillary. The fate of the ferritin was studied after varying intervals with the electron microscope. Shortly after microinjection ferritin molecules could be observed in the cell sap. After both 1 and 2 h, they were found in clusters and still mainly in the cell sap. After 4 h, ferritin molecules were present not only in the cell sap and in autophagic vacuoles but also in occasional secondary lysosomes. After 12 h, they were seen mainly in lysosomes, undergoing degradation. In no instance were ferritin molecules translocated into other organelles such as mitochondria, Golgi apparatus, or endoplasmic reticulum. The present study demonstrates that ferritin can be introduced into cells by glass capillary microinjection without cell damage. From its initial location in the cell sap ferritin is taken up into the lysosomal vacuome. Autophagy is considered to be the principal mechanism for the transfer of the ferritin molecules into lysosomes.     

Subcellular localization of ferritin and iron taken up by rat hepatocytes.

The subcellular localization of ferritin and its iron taken up by rat hepatocytes was investigated by sucrose-density-gradient ultracentrifugation of cell homogenates. After incubation of hepatocytes with 125I-labelled [59Fe]ferritin, cells incorporate most of the labels into structures equilibrating at densities where acid phosphatase and cytochrome c oxidase are found, suggesting association of ferritin and its iron with lysosomes or mitochondria. Specific solubilization of lysosomes by digitonin treatment indicates that, after 8 h incubation, most of the 125I is recovered in lysosomes, whereas 59Fe is found in mitochondria as well as in lysosomes. As evidenced by gel chromatography of supernatant fractions, 59Fe accumulates with time in cytosolic ferritin. To account for these results a model is proposed in which ferritin, after being endocytosed by hepatocytes, is degraded in lysosomes, and its iron is released and re-incorporated into cytosolic ferritin and, to a lesser extent, into mitochondria.     

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     

Lysosome membrane lipid microdomains: novel regulators of chaperone-mediated autophagy

Chaperone-mediated autophagy (CMA) is a selective mechanism for the degradation of soluble cytosolic proteins in lysosomes. The limiting step of this type of autophagy is the binding of substrates to the lysosome-associated membrane protein type 2A (LAMP-2A). In this work, we identify a dynamic subcompartmentalization of LAMP-2A in the lysosomal membrane, which underlies the molecular basis for the regulation of LAMP-2A function in CMA. A percentage of LAMP-2A localizes in discrete lysosomal membrane regions during resting conditions, but it exits these regions during CMA activation. Disruption of these regions by cholesterol-depleting agents or expression of a mutant LAMP-2A excluded from these regions enhances CMA activity, whereas loading of lysosomes with cholesterol significantly reduces CMA. Organization of LAMP-2A into multimeric complexes, required for translocation of substrates into lysosomes via CMA, only occurs outside the lipid-enriched membrane microdomains, whereas the LAMP-2A located within these regions is susceptible to proteolytic cleavage and degradation. Our results support that changes in the dynamic distribution of LAMP-2A into and out of discrete microdomains of the lysosomal membrane contribute to regulate CMA.     

Analysis of iron-containing compounds in different compartments of the rat liver after iron loading

Summary The livers of iron-loaded rats were fractionated and a cytosolic fraction, a lysosomal fraction, a siderosomal fraction and haemosiderin were obtained. All iron-containing compounds from these fractions were isolated and their morphology, Fe/P ratios, iron core diameter and peptide content were compared. The cytosolic fraction contained ferritin (CF) and a slower sedimenting, light ferritin (CLF). The lysosomal fraction also contained ferritin (LF) and a slower sedimenting light ferritin (LLF). The siderosomal fraction contained ferritin (SF), a faster sedimenting non-ferritin iron compound (SIC) and haemosiderin (HS). SIC and HS did not resemble ferritin as much as the other products did, but were found to be water-insoluble aggregates. The Fe/P ratios of CF and CLF were lower than the Fe/P ratios of LF and LLF and these in turn had lower Fe/P ratios than SF, SIC and HS. The iron core diameter of the cytosolic ferritin was increased after lysosomal uptake. The iron core diameters of the siderosomal products were smaller. CLF, CF, LF, LLF and SF contained one kind of subunit of approximately 20.5 kDa. SIC and HS contained other peptides in addition to the 20.5-kDa subunit. The results indicate that storage of ferritin molecules is not limited to the cytosolic compartment, but is also the case in the lysosomes. Extensive degradation of the ferritin molecule seems to be confined to the siderosomes.     

Age-related decline in chaperone-mediated autophagy

Intracellular protein degradation rates decrease with age in many tissues and organs. In cultured cells, chaperone-mediated autophagy, which is responsible for the selective degradation of cytosolic proteins in lysosomes, decreases with age. In this work we use lysosomes isolated from rat liver to analyze age-related changes in the levels and activities of the main components of chaperone-mediated autophagy. Lysosomes from "old" (22-month-old) rats show lower rates of chaperone-mediated autophagy, and both substrate binding to the lysosomal membrane and transport into lysosomes decline with age. A progressive age-related decrease in the levels of the lysosome-associated membrane protein type 2a that acts as a receptor for chaperone-mediated autophagy was responsible for decreased substrate binding in lysosomes from old rats as well as from late passage human fibroblasts. The cytosolic levels and activity of the 73-kDa heat-shock cognate protein required for substrate targeting to lysosomes were unchanged with age. The levels of lysosome-associated hsc73 were increased only in the oldest rats. This increase may be an attempt to compensate for reduced activity of the pathway with age.     

Pathophysiology of chaperone-mediated autophagy

In contrast to the classically described “in bulk” lysosomal degradation, the first evidence for selective degradation of cytosolic proteins in lysosomes was presented more than 20 years ago. Throughout this time, we have gained a better understanding about this process, now known as chaperone-mediated autophagy (CMA). The identification of new substrates for CMA and novel components, in both the cytosol and the lysosomes, along with better insights on how CMA is regulated, have all helped to shape the possible physiological roles of CMA. We review here different intracellular functions of CMA that arise from its unique characteristics when compared to other forms of autophagy. In view of these functions, we discuss the relevance of the changes in CMA activity in aging and in different pathological conditions.     

Ascorbic acid inhibits lysosomal autophagy of ferritin

Ascorbic acid retards ferritin degradation in K562 erythroleukemia cells leading to an increase in the availability of cellular iron (Bridges, K. R., and Hoffman, K. E. (1986) J. Biol. Chem. 261, 14273-14277). To explore the mechanism of this effect, the influence of ascorbate on subcellular ferritin distribution was examined. Cellular ferritin was pulse-labeled with 59Fe for 2 h, after which the cells were hypotonically lysed and fractionated on an 8% Percoll density gradient. Immediately after the labeling, all of the ferritin was in the cytoplasmic fractions at the top of the gradient. When the labeling was followed by a 24-h period of growth, a portion of the ferritin shifted to the lysosome-associated fractions at the bottom of the gradient, consistent with lysosomal autophagy of cytoplasmic ferritin. When ascorbate was added to the culture medium during the 24-h incubation, the magnitude of the shift was reduced. This process was also examined by size-fractionation of the contents of labeled cells using a Sepharose CL-6B column. Immediately after labeling, ferritin emerged from the column in two peaks, indicating the existence of both ferritin monomer and aggregates within the cytoplasm. After a 24-h period of growth, the monomer peak disappeared, while a new ferritin peak coincident with lysosomes emerged again, indicative of lysosomal autophagy of ferritin. In cells cultured with ascorbate for 24-h, there was a marked attenuation of the shift of ferritin to the lysosomal fractions. The monomer peak disappeared, as in the controls, but there was instead, an accumulation of ferritin as cytoplasmic aggregates. The total ferritin content of the ascorbate-treated cells was increased by 4-fold over that of the control. These experiments indicate that ascorbate blocks the degradation of cytoplasmic ferritin by reducing lysosomal autophagy of the protein. The access to the cell of the potentially toxic iron stored within the ferritin molecule is thereby increased.     

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。