AMD-like retinopathy associated with intravenous iron.

Iron accumulation in the retina is associated with the development of age-related macular degeneration (AMD). IV iron is a common method to treat iron deficiency anemia in adults, and its retinal manifestations have not hitherto been identified. To assess whether IV iron formulations can be retina-toxic, we generated a mouse model for iron-induced retinal damage. Male C57BL/6J mice were randomized into groups receiving IV iron-sucrose (+Fe) or 30% sucrose (-Fe). Iron levels in neurosensory retina (NSR), retinal pigment epithelium (RPE), and choroid were assessed using immunofluorescence, quantitative PCR, and the Perls' iron stain. Iron levels were most increased in the RPE and choroid while levels in the NSR did not differ significantly in +Fe mice compared to controls. Eyes from +Fe mice shared histological features with AMD, including Bruch's membrane (BrM) thickening with complement C3 deposition, as well as RPE hypertrophy and vacuolization. This focal degeneration correlated with areas of high choroidal iron levels. Ultrastructural analysis provided further detail of the RPE/photoreceptor outer segment vacuolization and Bruch's membrane thickening. Findings were correlated with a clinical case of a 43-year-old patient who developed numerous retinal drusen, the hallmark of AMD, within 11 months of IV iron therapy. Our results suggest that IV iron therapy may have the potential to induce or exacerbate a form of retinal degeneration. This retinal degeneration shares features with AMD, indicating the need for further study of AMD risk in patients receiving IV iron treatment.

Expression, purification, and characterization of recombinant human L-chain ferritin.

Ferritins form nanocage architectures and demonstrate their potential to serve as functional nanomaterials with potential applications in medical imaging and therapy. In our study, the cDNA of human L-chain ferritin was cloned into plasmid pET-28a for its overexpression in Escherichia coli. However, the recombinant human L-chain ferritin (rLF) was prone to form inclusion bodies. Molecular chaperones were co-expressed with rLF to facilitate its correct folding. Our results showed that the solubility of rLF was increased about 3-fold in the presence of molecular chaperones, including GroEL, GroES and trigger factor. Taking advantage of its N-terminal His-tag, rLF was then purified with Ni-affinity chromatography. With a yield of 10 mg/L from bacterial culture, the purified rLF was analyzed by circular dichroism spectrometry for its secondary structure. Furthermore, the rLF nanocages were characterized using dynamic light scattering and transmission electron microscopy.

Ferritin heavy chain in triple negative breast cancer: a favorable prognostic marker that relates to a cluster of differentiation 8 positive (CD8+) effector T-cell response.

Ferritin heavy chain (FTH1) is a 21-kDa subunit of the ferritin complex, known for its role in iron metabolism, and which has recently been identified as a favorable prognostic protein for triple negative breast cancer (TNBC) patients. Currently, it is not well understood how FTH1 contributes to an anti-tumor response. Here, we explored whether expression and cellular compartmentalization of FTH1 correlates to an effective immune response in TNBC patients. Analysis of the tumor tissue transcriptome, complemented with in silico pathway analysis, revealed that FTH1 was an integral part of an immunomodulatory network of cytokine signaling, adaptive immunity, and cell death. These findings were confirmed using mass spectrometry (MS)-derived proteomic data, and immunohistochemical staining of tissue microarrays. We observed that FTH1 is localized in both the cytoplasm and/or nucleus of cancer cells. However, high cytoplasmic (c) FTH1 was associated with favorable prognosis (Log-rank p = 0.001), whereas nuclear (n) FTH1 staining was associated with adverse prognosis (Log-rank p = 0.019). cFTH1 staining significantly correlated with total FTH1 expression in TNBC tissue samples, as measured by MS analysis (Rs = 0.473, p = 0.0007), but nFTH1 staining did not (Rs = 0.197, p = 0.1801). Notably, IFN γ-producing CD8+ effector T cells, but not CD4+ T cells, were preferentially enriched in tumors with high expression of cFTH1 (p = 0.02). Collectively, our data provide evidence toward new immune regulatory properties of FTH1 in TNBC, which may facilitate development of novel therapeutic targets.

Redox-active quinones induces genome-wide DNA methylation changes by an iron-mediated and Tet-dependent mechanism.

DNA methylation has been proven to be a critical epigenetic mark important for various cellular processes. Here, we report that redox-active quinones, a ubiquitous class of chemicals found in natural products, cancer therapeutics and environment, stimulate the conversion of 5 mC to 5 hmC in vivo, and increase 5 hmC in 5751 genes in cells. 5 hmC increase is associated with significantly altered gene expression of 3414 genes. Interestingly, in quinone-treated cells, labile iron-sensitive protein ferritin light chain showed a significant increase at both mRNA and protein levels indicating a role of iron regulation in stimulating Tet-mediated 5 mC oxidation. Consistently, the deprivation of cellular labile iron using specific chelator blocked the 5 hmC increase, and a delivery of labile iron increased the 5 hmC level. Moreover, both Tet1/Tet2 knockout and dimethyloxalylglycine-induced Tet inhibition diminished the 5 hmC increase. These results suggest an iron-regulated Tet-dependent DNA demethylation mechanism mediated by redox-active biomolecules.

Increased expression of ferritin in cerebral cortex after human traumatic brain injury.

Despite numerous researches and improvements in the past few years, the precise mechanisms underlying secondary brain injury after trauma remain obscure. Iron is essential for almost all types of cells, including nerve cells. However, excess of iron has been proved to contribute to the brain injury following trauma in animal models. As a key iron-handling protein in the brain, ferritin might be involved in iron-induced pathophysiological process of various brain disorders. Therefore, the current study was aimed to investigate the expression of ferritin in the human contused brain. Nineteen contused brain samples were obtained from 19 patients undergoing surgery for brain contusions 3 h-17 d after trauma, and three normal temporal pole samples from 3 patients with petroclival meningioma were collected as controls. Expression of ferritin-H-chain was measured by quantitative real-time polymerase chain reaction (PCR), western blot and immunohistochemistry, respectively. Perl's reaction was taken for iron staining. The results showed that human traumatic brain injury (TBI) could up-regulate ferritin-H-chain in pericontusional cortex. A marked increase of ferritin was detected in the early group (≤12 h), and remained elevated for a long time till after 48 h post-injury. The location of ferritin-H-chain was found mainly at the neuron-like cells and seldom at glia-like cells. Perl's reaction showed that most of the iron-positive cells were glia-like cells. These findings suggested that iron and ferritin might be involved in the secondary brain injury and could be therapeutic targets for patients with TBI.

Role of ferritin in the cytodifferentiation of periodontal ligament cells.

This study investigated the expression and functions of ferritin, which is involved in osteoblastogenesis, in the periodontal ligament (PDL). The PDL is one of the most important tissues for maintaining the homeostasis of teeth and tooth-supporting tissues. Real-time PCR analyses of the human PDL revealed abundant expression of ferritin light polypeptide (FTL) and ferritin heavy polypeptide (FTH), which encode the highly-conserved iron storage protein, ferritin. Immunohistochemical staining demonstrated predominant expression of FTL and FTH in mouse PDL tissues in vivo. In in vitro-maintained mouse PDL cells, FTL and FTH expressions were upregulated at both the mRNA and protein levels during the course of cytodifferentiation and mineralization. Interestingly, stimulation of PDL cells with exogenous apoferritin (iron-free ferritin) increased calcified nodule formation and alkaline phosphatase activity as well as the mRNA expressions of mineralization-related genes during the course of cytodifferentiation. On the other hand, RNA interference of FTH inhibited the mineralized nodule formation of PDL cells. This is the first report to demonstrate that ferritin is predominantly expressed in PDL tissues and positively regulates the cytodifferentiation and mineralization of PDL cells.

Imaging and quantification of metastatic melanoma cells in lymph nodes with a ferritin MR reporter in living mice.

Cellular MRI with a reporter gene offers the opportunity to track small numbers of tumor cells and to study metastatic processes in their earliest developmental stages in the target organs of interest. This study demonstrates the feasibility of using the MR reporter ferritin for the noninvasive imaging and quantification of metastatic melanoma cells in the lymph nodes (LNs) of living mice. A B16F10 murine melanoma cell line expressing human ferritin heavy chain (hFTH) and green fluorescent protein (GFP) was constructed to allow the detection of cells by MRI and fluorescence imaging. Stable overexpression of hFTH and GFP in B16F10 murine melanoma cells was feasible and showed no cellular toxicity. In addition, hFTH cells were detectable by 9.4-T MRI in vitro and in vivo, yielding significant changes in T(2)* relative to control cells. In BALB/c nude mice, the presence of hFTH- and GFP-expressing metastatic melanoma cells in deep-seated axillary LNs was demonstrated as areas of low T(2)* on MRI, but the same LNs were not visible by fluorescence imaging because the light was unable to penetrate the tissue. Furthermore, the metastatic volume of each LN, which was assessed by cumulative histogram analysis of the T(2)* MRI data, correlated well with tumor burden, which was determined by histology (r = -0.8773, p = 0.0001). This study is the first to use MRI and an MR reporter gene for both the visualization and quantification of metastatic cancer cells in LNs.

Efficiency of ferritin as an MRI reporter gene in NPC cells is enhanced by iron supplementation.

BACKGROUND: An emerging MRI reporter, ferritin heavy chain (FTH1), is recently applied to enhance the contrast and increase the sensitivity of MRI in the monitoring of solid tumors. However, FTH1-overexpression-related cytotoxicity is required to be explored. METHODS: By using the Tet-Off system, FTH1 overexpression was semi-quantitativiely and dynamicly regulated by doxycycline in a NPC cell line. Effects of FTH1 overexpression on the proliferation, cytotoxicity, apoptosis and migration of NPC cells were investigated in vitro, and MR relaxation rate was measured in vitro and in vivo. RESULTS: In vitro and in vivo overexpression of FTH1 significantly increased the transverse relaxivity (R(2)), which could be enhanced by iron supplementation. In vitro, overexpression of FTH1 reduced cell growth and migration, which were not reduced by iron supplementation. Furthermore, cells were subcutaneously inoculated into the nude mice. Results showed FTH1 overexpression decreased tumor growth in the absence of iron supplementation but not in the presence of iron supplementation. CONCLUSION: To maximize R(2) and minimize the potential adverse effects, supplementation of iron at appropriate dose is recommended during the application of FTH1 as a reporter gene in the monitoring of NPC by MRI.

On the quantification of intracellular proteins in multifluorescence-labeled rat brain slices using slide-based cytometry.

In several brain regions, a subpopulation of neurons exists being characterized by the expression of a peculiar form of extracellular matrix, a so-called perineuronal net (PNN). We have previously shown that the PNN can bind large amounts of iron due to its polyanionic charge. Because free iron can generate reactive oxygen species thus being potentially toxic, the PNN may have a protective function by "scavenging" this free iron. Because of this ability, we have hypothesized that PNN-related neurons have an altered iron-specific metabolism. Thus, to compare the intracellular concentrations of iron containing proteins, specifically, the iron storage protein ferritin H between neurons with and without a PNN, we have used slide-based cytometry with image-based threshold-boundary cell detection on brain sections. In tissue sections, the integrity of the extracellular matrix, especially the characteristic PNNs, is preserved, which is necessary for the identification of the two neuronal subpopulations. A multilabeling approach was chosen to select neurons (neuronal marker NeuN), to classify the neurons according to their subtype (matrix marker Wisteria floribunda agglutinin), and to quantify the protein concentration (protein marker). Using this novel method, we were able to detect a relative difference in protein concentration as low as 12% between the two subpopulations of neurons in the neuronal population of the rat parietal cortex.

Ferritin heavy chain (FTH1) exerts significant antigrowth effects in breast cancer cells by inhibiting the expression of c-MYC.

Overexpression of ferritin heavy chain (FTH1) often associates with good prognosis in breast cancer (BCa), particularly in the triple-negative subtype (triple-negative breast cancer). However, the mechanism by which FTH1 exerts its possible tumor suppressor effects in BCa is not known. Here, we examined the bearing of FTH1 silencing or overexpression on several aspects of BCa cell growth in vitro. FTH1 silencing promoted cell growth and mammosphere formation, increased c-MYC expression, and reduced cell sensitivity to chemotherapy. In contrast, FTH1 overexpression inhibited cell growth, decreased c-MYC expression, and sensitized cancer cells to chemotherapy; silencing of c-MYC recapitulated the effects of FTH1 overexpression. These findings show for the first time that FTH1 suppresses tumor growth by inhibiting the expression of key oncogenes, such as c-MYC.

The Era of Antimicrobial Peptides: Use of Hepcidins to Prevent or Treat Bacterial Infections and Iron Disorders.

The current treatments applied in aquaculture to limit disease dissemination are mostly based on the use of antibiotics, either as prophylactic or therapeutic agents, with vaccines being available for a limited number of fish species and pathogens. Antimicrobial peptides are considered as promising novel substances to be used in aquaculture, due to their antimicrobial and immunomodulatory activities. Hepcidin, the major iron metabolism regulator, is found as a single gene in most mammals, but in certain fish species, including the European sea bass (Dicentrarchus labrax), two different hepcidin types are found, with specialized roles: the single type 1 hepcidin is involved in iron homeostasis trough the regulation of ferroportin, the only known iron exporter; and the various type 2 hepcidins present antimicrobial activity against a number of different pathogens. In this study, we tested the administration of sea bass derived hepcidins in models of infection and iron overload. Administration with hamp2 substantially reduced fish mortalities and bacterial loads, presenting itself as a viable alternative to the use of antibiotics. On the other hand, hamp1 seems to attenuate the effects of iron overload. Further studies are necessary to test the potential protective effects of hamp2 against other pathogens, as well as to understand how hamp2 stimulate the inflammatory responses, leading to an increased fish survival upon infection.

Ferritin overexpression for noninvasive magnetic resonance imaging-based tracking of stem cells transplanted into the heart.

An unmet need in cardiac cell therapy is a noninvasive imaging technique capable of tracking changes in graft size over time and monitoring cell dynamics such as replication and death, factors to which commonly used superparamagnetic nanoparticles are insensitive. Our goal was to explore if overexpression of ferritin, a nontoxic iron-binding protein, can be used for noninvasive magnetic resonance imaging (MRI) of cells transplanted into the infarcted heart. Mouse skeletal myoblasts (C2C12 cells) were engineered to overexpress ferritin. Ferritin overexpression did not interfere with cell viability, proliferation, or differentiation into multinucleated myotubes. Ferritin overexpression caused a 25% decrease in T2 relaxation time in vitro compared to wild-type cells. Transgenic grafts were detected in vivo 3 weeks after transplantation into infarcted hearts of syngeneic mice as areas of hypointensity caused by iron accumulation in overexpressed ferritin complexes. Graft size evaluation by MRI correlated tighly with histologic measurements (R2 = .8). Our studies demonstrated the feasibility of ferritin overexpression in mouse skeletal myoblasts and the successful detection of transgenic cells by MRI in vitro and in vivo after transplantation into the infarcted mouse heart. These experiments lay the groundwork for using the MRI gene reporter ferritin to track stem cells transplanted to the heart.

Toluene diisocyanate (TDI) regulates haem oxygenase-1/ferritin expression: implications for toluene diisocyanate-induced asthma.

Diisocyanate is a leading cause of occupational asthma (OA). Diisocyanate-induced OA is an inflammatory disease of the airways that is associated with airway remodelling. Although the pathogenic mechanisms are unclear, oxidative stress may be related to the pathogenesis of diisocyanate-induced OA. In our previous report, we observed that the expression of ferritin light chain (FTL) was decreased in both of bronchoalveolar lavage fluid and serum of patients with diphenyl-methane diisocyanate (MDI)-induced OA compared to those of asymptomatic exposed controls and unexposed healthy controls. In this study of toluene diisocyanate (TDI)-OA, we found identical findings with increased transferrin and decreased ferritin levels in the serum of patients with TDI-OA. To elucidate whether diisocyanate suppresses FTL synthesis directly, we tested the effect of TDI on the FTL synthesis in A549 cells, a human airway epithelial cell line. We found that haem oxygenase-1 as well as FTL was suppressed by treatment with TDI in dose- and time-dependent manners. We also found that the synthesis of other anti-oxidant proteins such as thioredoxin-1, glutathione peroxidase, peroxiredoxin 1 and catalase were suppressed by TDI. Furthermore, TDI suppressed nuclear translocation of Nrf2 through suppressing the phosphorylation of mitogen-activated protein kinases (MAPKs); extracellular-regulated kinase 1/2 (ERK1/2); p38; and c-Jun N-terminal kinase (JNK). Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonists, 15-deoxy-Delta(12,14)-PGJ2 and rosiglitazone rescued the effect of TDI on HO-1/FTL expression. Collectively, our findings suggest that TDI suppressed HO-1/FTL expression through the MAPK-Nrf2 signalling pathway, which may be involved in the pathogenesis of TDI-induced OA. Therefore, elucidating these observations further should help to develop the therapeutic strategies of diisocyanate-induced OA.

Polarization dictates iron handling by inflammatory and alternatively activated macrophages.

BACKGROUND: Macrophages play a key role in iron homeostasis. In peripheral tissues, they are known to polarize into classically activated (or M1) macrophages and alternatively activated (or M2) macrophages. Little is known on whether the polarization program influences the ability of macrophages to store or recycle iron and the molecular machinery involved in the processes. DESIGN AND METHODS: Inflammatory/M1 and alternatively activated/M2 macrophages were propagated in vitro from mouse bone-marrow precursors and polarized in the presence of recombinant interferon-γ or interleukin-4. We characterized and compared their ability to handle radioactive iron, the characteristics of the intracellular iron pools and the expression of molecules involved in internalization, storage and export of the metal. Moreover we verified the influence of iron on the relative ability of polarized macrophages to activate antigen-specific T cells. RESULTS: M1 macrophages have low iron regulatory protein 1 and 2 binding activity, express high levels of ferritin H, low levels of transferrin receptor 1 and internalize--albeit with low efficiency -iron only when its extracellular concentration is high. In contrast, M2 macrophages have high iron regulatory protein binding activity, express low levels of ferritin H and high levels of transferrin receptor 1. M2 macrophages have a larger intracellular labile iron pool, effectively take up and spontaneously release iron at low concentrations and have limited storage ability. Iron export correlates with the expression of ferroportin, which is higher in M2 macrophages. M1 and M2 cells activate antigen-specific, MHC class II-restricted T cells. In the absence of the metal, only M1 macrophages are effective. CONCLUSIONS: Cytokines that drive macrophage polarization ultimately control iron handling, leading to the differentiation of macrophages into a subset which has a relatively sealed intracellular iron content (M1) or into a subset endowed with the ability to recycle the metal (M2).

Regulation of transferrin receptor 2 in human cancer cell lines.

In a recent study we have explored TfR2 expression in a panel of cancer cell lines and we observed that about 40% of these cell lines clearly express TfR2. Taking advantage of this observation and considering the frequent overexpression of c-Myc in cancer cells we have explored the existence of a possible relationship between c-Myc and TfR2 in these cell lines. Our results provided evidence that TfR2(+) cell lines express low c-Myc levels and low TfR1 levels, while TfR2(-) cell lines express high c-Myc and TfR1 levels. Using the erythroleukemic K562 TfR2(+) cells as a model, we observed that agents that enhance c-Myc expression, such as iron, determine a decrease of TfR2 expression, while molecules that induce a decreased c-Myc expression, such as the iron chelator desferoxamine or the kinase inhibitor ST 1571, induce an enhanced TfR2 expression. On the other hand, we have evaluated a possible effect of hypoxia and nitric oxide on TfR2 expression in erythroleukemia K526 and hepatoma HepG2 cells, providing evidence that: (i) agents inducing cellular hypoxia, such as CoCl(2), elicited a marked upmodulation of TfR1, but a downmodulation of TfR2 expression; (ii) NO(+) donors, such as sodium nitroprusside (SNP), induced a moderate decrease of TfR1, associated with a marked decline of TfR2 expression; (iii) NO donors, such as S-Nitroso-N-Acetylpenicillamine (SNAP), induced a clear increase of TfR1, associated with a moderate upmodulation of TfR2 expression. The ensemble of these observations suggests that in cancer cell lines TfR2 expression can be modulated through stimuli similar to those known to act on TfR1 and these findings may have important implications for our understanding of the role of TfR2 in the regulation of iron homeostasis.

Chlamydia trachomatis alters iron-regulatory protein-1 binding capacity and modulates cellular iron homeostasis in HeLa-229 cells.

Chlamydia trachomatis (CT) is the leading cause of diseases related to reproductive health and iron plays important role in chlamydial pathogenesis. Iron homeostasis in chlamydia-infected cells is not clear thus far. This study shows that expression of the transferrin receptor (TfR) is downregulated, whereas expression of the ferritin heavy chain is upregulated in CT-infected HeLa-229 cells. Expression of iron-regulatory protein (IRP)-1 predominates over IRP-2 in infected cells. In infected cells, attenuated binding activity of IRP-iron responsive elements (IREs) is observed using the electrophoretic mobility-shift assay. These results suggest that iron homeostasis is modulated in CT-infected HeLa cells at the interface of acquisition and commensal use of iron.

[Expression of FTL and FTH genes encoding ferretin subunits in lung and renal carcinomas].

The level of ferritin in serum is known to be increased frequently in most human cancers. Ferritin consists of the heavy and light chains, encoded by FTL and FTH genes. The analysis of the EST database showed that the level of FTL and FTH mRNA is decreased in lung squamous cell carcinomas as compared to the normal tissues, no change in the mRNA level was observed in clear cell renal cell carcinoma. Using real-time PCR we estimated the mRNA level of these genes in primary tumors. It was shown significant and frequent decrease of FTL and FTH mRNA level in lung squamous cell carcinoma: on the average by 11 and 9 times in 83% (33/40) and 73% (11/15) of cases, respectively. In clear cell renal cell carcinoma the changes were not so marked both with respect to the level of decrease (on the average 6 and 3 times) and to its frequency (58 and 27%). In the present work it has been shown for the first time that the FTL mRNA is frequently down-regulated even at the early stages of lung squamous cell carcinoma in all studied samples. This fact permits to consider this gene as potential oncomarker of early diagnosis. The FTL mRNA content may be quantified by non-concurrent hybridization on expression DNA microarrays. The possible causes of a serum ferritin increase in lung cancer and renal cancer are discussed.

Repression of ferritin light chain translation by human eIF3.

A central problem in human biology remains the discovery of causal molecular links between mutations identified in genome-wide association studies (GWAS) and their corresponding disease traits. This challenge is magnified for variants residing in non-coding regions of the genome. Single-nucleotide polymorphisms (SNPs) in the 5' untranslated region (5'-UTR) of the ferritin light chain (FTL) gene that cause hyperferritinemia are reported to disrupt translation repression by altering iron regulatory protein (IRP) interactions with the FTL mRNA 5'-UTR. Here, we show that human eukaryotic translation initiation factor 3 (eIF3) acts as a distinct repressor of FTL mRNA translation, and eIF3-mediated FTL repression is disrupted by a subset of SNPs in FTL that cause hyperferritinemia. These results identify a direct role for eIF3-mediated translational control in a specific human disease.

PM2.5 induces ferroptosis in human endothelial cells through iron overload and redox imbalance.

PM2.5 is becoming a worldwide environmental problem, which profoundly endangers public health, thus progressively capturing public attention this decade. As a fragile target of PM2.5, the underlying mechanisms of endothelial cell damage are still obscure. According to the previous microarray data and signaling pathway analysis, a new form of cell death termed ferroptosis in the current study is proposed following PM2.5 exposure. In order to verify the vital role of ferroptosis in PM2.5-induced endothelial lesion and further understand the potential mechanism involved, intracellular iron content, ROS release and lipid peroxidation, as well as biomarkers of ferroptosis were detected, respectively. As a result, uptake of particles increases cellular iron content and ROS production. Meanwhile, GSH depletion, and the decrease of GSH-Px and NADPH play significant roles in PM2.5-induced endothelial cell ferroptosis. Moreover, significantly changed expression of TFRC, FTL and FTH1 hinted that dysfunction of iron uptake and storage is a major inducer of ferroptosis. Importantly, index monitored above can be partially rescued by lipid peroxidation inhibitor ferrostatin-1 and iron chelator deferoxamine mesylate, which mediated antiferroptosis activity mainly depends on the restoration of antioxidant activity and iron metabolism. In conclusion, our data basically show that PM2.5 enhances ferroptosis sensitivity with increased ferroptotic events in endothelial cells, in which iron overload, lipid peroxidation and redox imbalance act pivotal roles.

Proteomic analysis of recombinant Saccharomyces cerevisiae upon iron deficiency induced via human H-ferritin production.

In our previous study, the expression of active H-ferritins in Saccharomyces cerevisiae was found to reduce cell growth and reactive oxygen species (ROS) generation upon exposure to oxidative stress; such expression enhanced that of high-affinity iron transport genes (FET3 and FTR1). The results suggested that the recombinant cells expressing H-ferritins induced cytosolic iron depletion. The present study analyzes metabolic changes under these circumstances via proteomic methods. The YGH2 yeast strain expressing H-ferritin, the YGH2-KG (E62K and H65G) mutant strain, and the YGT control strain were used. Comparative proteomic analysis showed that the synthesis of 34 proteins was at least stimulated in YGH2, whereas the other 37 proteins were repressed. Among these, the 31 major protein spots were analyzed via nano-LC/MS/MS. The increased proteins included major heat-shock proteins and proteins related to endoplasmic reticulum-associated degradation (ERAD). On the other hand, the proteins involved with folate metabolism, purine and methionine biosynthesis, and translation were reduced. In addition, we analyzed the insoluble protein fractions and identified the fragments of Idh1p and Pgk1p, as well as several ribosomal assembly-related proteins. This suggests that intracellular iron depletion induces imperfect translation of proteins. Although the proteins identified above result from changes in iron metabolism (i.e., iron deficiency), definitive evidence for iron-related proteins remains insufficient. Nevertheless, this study is the first to present a molecular model for iron deficiency, and the results may provide valuable information on the regulatory network of iron metabolism.