Cellular Iron Regulation Research Articles

SS-31 modification alleviates ferroptosis induced by superparamagnetic iron oxide nanoparticles in hypoxia/reoxygenation cardiomyocytes

Superparamagnetic iron oxide nanoparticles (SPION) are widely used in cardiovascular applications. However, their potential to induce ferroptosis in myocardial cells post-ischemia-reperfusion hinders clinical adoption. We investigated the mechanisms behind SPION-induced cytotoxicity in myocardial cells and explored whether co-loading SPION with SS-31 (a kind of mitochondrial-targeted antioxidant peptide) could counteract this toxicity.To create SPION@SS-31, SS-31 was physically adsorbed onto SPION. To study the dose- and time-dependent cytotoxic effects and assess the influence of SS-31 on reducing SPION-induced damage, hypoxia/reoxygenation(H/R) H9C2 cells were treated with either SPION or SPION@SS-31. We examined the relationship between SPION and ferroptosis by measuring mitochondrial ROS, mitochondrial membrane potential (MMP), lipid peroxidation products, ATP, GSH, GPX4, mitochondrial structure, nonheme iron content, cellular iron regulation, and typical ferroptosis markers.The findings showed that SPION induced concentration- and time-dependent toxicity, marked by a significant cell viability loss and an increase in LDH levels. In contrast, SPION@SS-31 produced results comparable to the H/R group, implying that SS-31 can notably reduce cell damage induced by SPION. SPION disrupted cellular iron homeostasis, with FtH and FtMt expression increased and reduced levels of FPN1 and ABCB8, which led to the overload of mitochondrial iron. This iron dysregulation damaged mitochondrial function and integrity, causing ATP depletion, MMP loss, and decreased GPX4 and GSH levels, accompanied by a burst of mitochondrial lipid peroxidation, ultimately resulting in ferroptosis in H/R cardiomyocytes. Notably, SS-31 significantly alleviated SPION-induced ferroptosis by decreasing mitochondrial MDA production and maintaining GSH and GPX4 levels, indicating its possibility to reverse SPION-induced cytotoxicity. The viability of H/R cells and cells treated with SPION and Fer-1 did not differ statistically, whereas cells exposed to SPION along with inhibitors like 3-MA, zVAD, or Nec-1 showed a substantial loss in viability, implying that ferroptosis is the primary mechanism behind SPION-induced myocardial toxicity.SPION triggers mitochondrial lipid peroxidation by causing overload of iron, leading to ferroptosis in H/R H9C2 cells. Mitochondria appear to be the primary target of SPION-induced toxic effects. SS-31 demonstrates potential in inhibiting this ferroptosis by acting as a mitochondria-targeted antioxidant, suggesting that the modification of mitochondria-targeted antioxidant peptides represents an innovative and practical approach to attenuate the myocardial toxicity associated with SPION.

A Review of Literature on Transferrin: Deciphering its Complex Mechanism in Cellular Iron Regulation and Clinical Implications

Iron is a crucial constituent in cellular metabolism, playing a pivotal role in numerous enzymatic activities that are required for the maintenance of life. However, the lack of regulation in iron levels can lead to cellular damage through the Fenton reaction, which produces reactive oxygen species. Transferrin which is a glycoprotein functions crucially in contributing significantly to the movement of iron in biological systems. The polypeptide chain of transferrin, which is made of 700 amino acids, has a crucial role in iron binding and delivery. Transferrin has different N and C lobes that contribute to its exceptional attributes. In this paper, we looked at several aspects of transferrin which were explored, especially its diverse forms, characteristic structures, synthesis mechanisms, and metabolic functions. Various proteins, including lactoferrin, melanotransferrin, serum transferrin, and ovotransferrin, take part in regulating the transportation of iron and the prevention of iron homeostasis in vertebrates. We also explored the role of transferrin in various metabolic processes, which includes its activation of macrophages, antimicrobial attributes, and participation in immunological responses. A detailed assessment of the chemical attributes of transferrin provides useful information about its amino acid constituent, arrangement, and bonds with a broad spectrum of metal ions. This paper delves into taking part in scholarly reviews that address the therapeutic relevance of transferrin, stressing its function as a marker for diagnosing iron deficiency as well as its implications in health conditions such as hemochromatosis and atransferrinemia. This detailed assessment of transferrin that this paper presents makes a renowned development in how we understand its complex mechanisms, thus enhancing how we comprehend iron regulations in cells and how it implies both health and disease.

A Review of Literature on Transferrin: Deciphering its Complex Mechanism in Cellular Iron Regulation and Clinical Implications

A Review of Literature on Transferrin: Deciphering its Complex Mechanism in Cellular Iron Regulation and Clinical Implications

Oxygen modulates iron homeostasis by switching iron sensing of NCOA4

To ensure proper utilization of iron and avoid its toxicity, cells are equipped with iron-sensing proteins to maintain cellular iron homeostasis. We showed previously that nuclear receptor coactivator 4 (NCOA4), a ferritin-specific autophagy adapter, intricately regulates the fate of ferritin; upon binding to Fe3+, NCOA4 forms insoluble condensates and regulates ferritin autophagy in iron-replete conditions. Here, we demonstrate an additional iron-sensing mechanism of NCOA4. Our results indicate that the insertion of an iron-sulfur (Fe-S) cluster enables preferential recognition of NCOA4 by the HERC2 (HECT and RLD domain containing E3 ubiquitin protein ligase 2) ubiquitin ligase in iron-replete conditions, resulting in degradation by the proteasome and subsequent inhibition of ferritinophagy. We also found that both condensation and ubiquitin-mediated degradation of NCOA4 can occur in the same cell, and the cellular oxygen tension determines the selection of these pathways. Fe-S cluster-mediated degradation of NCOA4 is enhanced under hypoxia, whereas NCOA4 forms condensates and degrades ferritin at higher oxygen levels. Considering the involvement of iron in oxygen handling, our findings demonstrate that the NCOA4-ferritin axis is another layer of cellular iron regulation in response to oxygen levels.

Hsp90 and J-protein family chaperones are not directly involved in cellular iron-sulfur protein assembly and iron regulation in yeast.

Systematic studies have revealed interactions between components of the Hsp90 chaperone system and Fe/S protein biogenesis or iron regulation. In addition, two chloroplast-localized DnaJ-like proteins, DJA5 and DJA6, function as specific iron donors in plastidial Fe/S protein biogenesis. Here, we used Saccharomyces cerevisiae to study the impact of both the Hsp90 chaperone and the yeast DJA5-DJA6 homologs, the essential cytosolic Ydj1 and the mitochondrial Mdj1, on cellular iron-related processes. Despite severe phenotypes induced upon depletion of these crucial proteins, there was no critical in vivo impact on Fe/S protein biogenesis or iron regulation. Importantly, unlike the plant DJA5-DJA6 iron chaperones, Ydj1 and Mdj1 did not bind iron in vivo, suggesting that these proteins use zinc for function under normal physiological conditions.

Biochemical control of the mitochondrial protein MitoNEET by biological thiols and lipid-derived electrophiles

MitoNEET is a mitochondrial [2Fe-2S]-containing protein known for its involvement in cellular metabolism, iron regulation, and oxidative stress. The protein has been associated with diseases ranging from diabetes to Parkinson's disease, prompting the development of compounds designed to target mitoNEET selectively. Unfortunately, drug development is limited due to a lack of mechanistic understanding of how mitoNEET integrates into pathophysiological processes, and biological compounds that govern mitoNEET function are still ill-defined. We demonstrate an oxygen-dependent reaction with biological thiols catalyzed by mitoNEET. Specifically, we observed that mitoNEET converts L-cysteine to cystine. Finally, we showed that reduced glutathione (L-GSH) regulates the reactivity of two lipid-derived biomarkers of oxidative stress, 4-HNE and 4-ONE, towards mitoNEET. We found that exposure to L-GSH before treatment with either of the electrophilic aldehydes prevents the formation of a covalently linked mitoNEET dimer. Meanwhile, adding L-GSH after electrophile treatment recovers mitoNEET from the 4-HNE induced modification but not from the modification induced by 4-ONE. These results indicate a possible role for mitoNEET in thiol-mediated oxidative stress and may facilitate the development of drugs designed to modulate mitoNEET activity to improve pathophysiological states.

Label‐free subcellular mapping of iron‐bound proteins in breast cancer cells

Dysregulation of iron transport, storage and utilization is at the basis of a variety of human diseases, since free iron can participate in redox reactions that are deleterious to lipid membranes and signaling pathways. As intracellular iron transport occurs in a pH-dependent manner, changes in endosomal pH, often associated with cancer, could lead to alterations in iron homeostasis. Essential to iron transport is transferrin (Tf), which binds iron at a neutral pH for safe transport throughout the body and releases it upon uptake and delivery to a mildly acidified endosome. Excess iron can be stored in cytoplasmic protein ferritin (Ft) or delivered into mitochondria to be incorporated into proteins with heme or iron-sulfur structures. Recently, we have developed a novel label-free Raman hyperspectral microscopy methodology to identify the subcellular distributions and relative quantities of iron-bound Tf, Ft and heme proteins within heterogenous cell populations. Previously, we have shown that Raman hyperspectral imaging could identify endocytic distributions of iron-bound Tf using a specific Raman peak at 1300 cm-1 in intact breast cancer cells. Moreover, we have shown that alterations in the subcellular localization of iron-bound Tf populations correlated with altered endocytic regulation of iron-bound Tf in two distinct breast cancer cell lines. Based on these results, we propose that breast cancer cells display a disrupted iron homeostasis due to iron release delays caused by alterations in the pH or ionic milieu of the early endosomes. Currently, we are testing a putative correlation between pH gradient alterations and iron release delays in different breast cancer cells. To further investigate cellular iron regulation, Ft cell-specific Raman signature was determined via evaluation of cells incubated in the presence or absence of iron-loaded Ft. Since cells may internalize Ft through an endocytic pathway, this approach allowed for identification of Ft Raman signature. However, the subcellular distribution of internalized iron-loaded Ft may not be reflective of normal cytoplasmic Ft in cells. Internalization of iron-loaded Ft results in multiple significantly different Raman spectra peaks at several wavelengths. Importantly, we have used multivariate analysis to show a significant separation of cell Raman spectra from that of cells incubated with Ft. Further we can map a previously known heme protein peak at 1372 cm-1, which is specifically associated with the ferric iron heme. In the future, following further validation of Ft peaks the mapping of Tf, Ft and heme can be evaluated across multiple cell lines and tissue samples. The application of this research has far reaching potential in understanding how dysregulation of endocytic pH may lead to alterations in intracellular iron transport and storage in breast cancer cells.

On Iron Metabolism and Its Regulation.

Iron is a critical metal for several vital biological processes. Most of the body’s iron is bound to hemoglobin in erythrocytes. Iron from senescent red blood cells is recycled by macrophages in the spleen, liver and bone marrow. Dietary iron is taken up by the divalent metal transporter 1 (DMT1) in enterocytes and transported to portal blood via ferroportin (FPN), where it is bound to transferrin and taken up by hepatocytes, macrophages and bone marrow cells via transferrin receptor 1 (TfR1). While most of the physiologically active iron is bound hemoglobin, the major storage of most iron occurs in the liver in a ferritin-bound fashion. In response to an increased iron load, hepatocytes secrete the peptide hormone hepcidin, which binds to and induces internalization and degradation of the iron transporter FPN, thus controlling the amount of iron released from the cells into the blood. This review summarizes the key mechanisms and players involved in cellular and systemic iron regulation.

Sexually dimorphic impact of the iron-regulating gene, HFE, on survival in glioblastoma.

BackgroundThe median survival for patients with glioblastoma (GBM), the most common primary malignant brain tumor in adults, has remained approximately 1 year for more than 2 decades. Recent advances in the field have identified GBM as a sexually dimorphic disease. It is less prevalent in females and they have better survival compared to males. The molecular mechanism of this difference has not yet been established. Iron is essential for many biological processes supporting tumor growth and its regulation is impacted by sex. Therefore, we interrogated the expression of a key component of cellular iron regulation, the HFE (homeostatic iron regulatory) gene, on sexually dimorphic survival in GBM.MethodsWe analyzed TCGA microarray gene expression and clinical data of all primary GBM patients (IDH-wild type) to compare tumor mRNA expression of HFE with overall survival, stratified by sex.ResultsIn low HFE expressing tumors (below median expression, n = 220), survival is modulated by both sex and MGMT status, with the combination of female sex and MGMT methylation resulting in over a 10-month survival advantage (P < .0001) over the other groups. Alternatively, expression of HFE above the median (high HFE, n = 240) is associated with significantly worse overall survival in GBM, regardless of MGMT methylation status or patient sex. Gene expression analysis uncovered a correlation between high HFE expression and expression of genes associated with immune function.ConclusionsThe level of HFE expression in GBM has a sexually dimorphic impact on survival. Whereas HFE expression below the median imparts a survival benefit to females, high HFE expression is associated with significantly worse overall survival regardless of established prognostic factors such as sex or MGMT methylation.

The multifaceted role of iron in renal health and disease.

Iron is an essential element that is indispensable for life. The delicate physiological body iron balance is maintained by both systemic and cellular regulatory mechanisms. The iron-regulatory hormone hepcidin assures maintenance of adequate systemic iron levels and is regulated by circulating and stored iron levels, inflammation and erythropoiesis. The kidney has an important role in preventing iron loss from the body by means of reabsorption. Cellular iron levels are dependent on iron import, storage, utilization and export, which are mainly regulated by the iron response element-iron regulatory protein (IRE-IRP) system. In the kidney, iron transport mechanisms independent of the IRE-IRP system have been identified, suggesting additional mechanisms for iron handling in this organ. Yet, knowledge gaps on renal iron handling remain in terms of redundancy in transport mechanisms, the roles of the different tubular segments and related regulatory processes. Disturbances in cellular and systemic iron balance are recognized as causes and consequences of kidney injury. Consequently, iron metabolism has become a focus for novel therapeutic interventions for acute kidney injury and chronic kidney disease, which has fuelled interest in the molecular mechanisms of renal iron handling and renal injury, as well as the complex dynamics between systemic and local cellular iron regulation.

Backbone resonance assignments and secondary structure of the apo-Drosophila melanogaster frataxin homolog (Dfh).

Friedreich's ataxia, the most prevalent hereditary ataxia, is caused by a patient's inability to produce a viable form of the protein frataxin. Frataxin plays an essential role in cellular iron regulation and has been shown to participate in the assembly of iron-sulfur (Fe-S) clusters under a variety of roles, including modulating persulfide production and directing Fe(II) delivery to the assembly scaffold protein. While the activity and structure of multiple eukaryotic frataxin orthologs have been characterized, the fly ortholog has numerous advantages over other orthologs with regards to protein stability, its activity towards Fe-S cluster assembly and its stability for forming stable proteins partner assemblies. Given the obvious advantages for studying the Drosophila melanogaster frataxin homolog (Dfh) over its orthologs, we have undertaken a structural characterization of apo-Dfh as the first step towards solving the solution structure of the protein alone and in complex with protein partners within the Fe-S cluster assembly pathway.

Cellular Iron Metabolism and Regulation.

Iron is an essential trace element in the human body, but excess iron is toxic as it contributes to oxidative damage. To keep iron concentration within the optimal physiologic range, iron metabolism at the cellular level and the whole systemic level are tightly regulated. Balance of iron homeostasis depends on the expression levels and activities of iron carriers, iron transporters, and iron regulatory and storage proteins. Divalent metal transporter 1 (DMT1) at the apical membrane of intestinal enterocyte brings in non-heme iron from the diet, whereas ferroportin 1 (FPN1) at the basal membrane exports iron into the circulation. Plasma transferrin (Tf) then carries iron to various tissues and cells. After binding to transferrin receptor 1 (TfR1), the complex is endocytosed into the cell, where iron enters the cytoplasm via DMT1 on the endosomal membrane. Free iron is either utilized in metabolic processes, such as synthesis of hemoglobin and Fe-S cluster, or sequestered in the cytosolic ferritin, serving as a cellular iron store. Excess iron can be exported from the cell via FPN1. The liver-derived peptide hepcidin plays a major regulatory role in controlling FPN1 level in the enterocyte, and thus controls the whole-body iron absorption. Inside the cells, iron regulatory proteins (IRPs) modulate the expressions of DMT1, TfR1, ferritin, and FPN1 via binding to the iron-responsive element (IRE) in their mRNAs. Both the release of hepcidin and the IRP-IRE interaction are coordinated with the fluctuation of the cellular iron level. Therefore, an adequate and steady iron supplement is warranted for the utilization of cells around the body. Investigations on the molecular mechanisms of cellular iron metabolism and regulation could advance the fields of iron physiology and pathophysiology.

Abstract 73: Role of macrophages in the antitumor activity of an anti-transferrin receptor 1 antibody ch128.1 in a xenograft model of multiple myeloma

Abstract The transferrin receptor 1 (TfR1), also known as CD71, is a membrane glycoprotein involved in cellular iron uptake and regulation of cell growth. The high level of TfR1 expression on malignant cells and its key role in cancer cell pathology make this receptor an attractive target for antibody cancer therapy. We previously developed a mouse/human chimeric IgG3 specific for human TfR1 (ch128.1). This antibody exhibits direct cytotoxicity against certain human malignant B cells in vitro through TfR1 degradation and iron deprivation. Importantly, ch128.1 shows remarkable anti-tumor activity in xenograft models of disseminated multiple myeloma (MM) in immunosuppressed mice (SCID-Beige). Interestingly, this anti-tumor protection was observed even against MM cells (KMS-11 cells) that show no sensitivity to this antibody in vitro, suggesting the in vivo contributions of antibody effector functions. This possibility was supported by the lack of anti-tumor protection observed using a ch128.1 Fc mutant with impaired binding to FcγRs and to the complement component C1q. To examine host effector functions involved in ch128.1-mediated protection in our mouse model bearing KMS-11 tumors, depletion studies of complement and macrophages were performed. Complement depletion using cobra venom factor (CVF) did not affect protection, suggesting that complement-mediated cytotoxicity (CDC) is not a relevant mechanism of action. Notably, we now report that macrophage depletion using clodronate liposomes (clodrolip) significantly reduced protection, suggesting that these effector cells play a relevant role in the anti-tumor activity. Consistent with this result, we also report that ch128.1 is capable of eliciting antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) against KMS-11 cells in the presence of the murine macrophage cell line J774.2 or murine bone marrow-derived macrophages. To examine the role of iron deprivation in ch128.1-mediated tumor death in vivo, animals treated with ch128.1 were systemically supplemented with iron in a preliminary study. However, no difference in survival was observed, suggesting that iron deprivation is not a contributor to the effects of ch128.1 in our in vivo model or the iron dose tested was not optimal. Our results suggest that macrophages play a key role in ch128.1-mediated anti-tumor protection in our model and that ch128.1 can be an effective therapy of incurable human B-cell malignancies such as MM. Citation Format: Lai Sum Leoh, Yoon Kyung Kim, Pierre V. Candelaria, Otoniel Martínez-Maza, Tracy R. Daniels-Wells, Manuel L. Penichet. Role of macrophages in the antitumor activity of an anti-transferrin receptor 1 antibody ch128.1 in a xenograft model of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 73. doi:10.1158/1538-7445.AM2017-73

Ferritin, cellular iron storage and regulation.

Ferritin is considered the major iron storage protein which maintains a large iron core in its cavity and has ferroxidase activity. There are many types of ferritin particularly in prokaryotes that include the canonical 24-mer FTN molecules, the heme-containing BFR, the smaller 12-mer DPS and the newly recognized EncFtn of encapsulin that forms a very large iron storage compartment. Recent studies show that ferritin function is more dynamic than previous depicted and new mechanisms of ferritin iron recycling are emerging. They participate to the regulation of cellular iron homeostasis as those of ferritin biosynthesis, cooperating also with the iron-dependent mechanism of cellular iron secretion. Some of these basic processes are in common between unicellular and animal cells, and this review aims at discussing the findings on the connections between iron storage, cellular iron regulation and ferritin iron recycling that have been explored in unicellular organisms and in animals. © 2017 IUBMB Life, 69(6):414-422, 2017.

Abstract 12528: ALR Protein, a Critical Protein in Cardiac Development, Regulates Cellular Iron Homeostasis by Modulating Mitochondrial Transport of ATP-Binding Cassette (ABC)-B8

Introduction: Iron is an essential molecule for normal cellular physiology, and altered cellular iron homeostasis is commonly observed in diseases with disruption of iron/sulfur (Fe/S) cluster maturation, such as cardiomyopathy associated with Friedreich’s ataxia. Inhibition of Augmenter of Liver Regeneration (ALR), a mitochondrial inter-membrane-space protein involved in mitochondrial protein import, results in cardiac developmental defect in zebrafish, and its deletion is associated with increased oxidative stress and cytosolic Fe/S cluster maturation defects. ABCB8 is one of only two mitochondrial membrane proteins known to regulate cytosolic Fe/S cluster maturation. We hypothesized that ALR is critical for cytosolic Fe/S cluster maturation and iron homeostasis by regulating mitochondrial import of ABCB8. Results: Downregulation of ALR in vitro resulted in reduced cytosolic Fe/S cluster-containing enzyme activities and increased cellular iron uptake. Using a knockdown-rescue approach, we further demonstrated that only the mitochondrial, but not the cytosolic, ALR isoform is involved in the maturation of cytosolic Fe/S clusters. Because Fe/S clusters are synthesized in the mitochondria, we then assessed whether ALR can alter the levels or activity of ABCB7 and ABCB8, the two mitochondrial proteins known to regulate the maturation of cytosolic Fe/S clusters. Downregulation of ALR reduced the mitochondrial levels of ABCB8, while ABCB7 levels were not affected. We also identified defects in mitochondrial transport of ABCB8 as the mechanism for reduced mitochondrial ABCB8 levels with ALR knockdown. Finally, we demonstrated that ABCB8 physically interacts with the protein import system consisting of ALR and Mia40, thus providing a mechanism for reduced ABCB8 mitochondrial transport with ALR downregulation. Conclusion: Our results indicate that ALR and its interaction partner Mia40 are involved in the transport of ABCB8 into the mitochondria, which in turn regulates cytoplasmic Fe/S cluster maturation. These findings provide insights into cellular iron regulation, with implications in cardiovascular disease and cardiac development.

Abstract 582: Novel insights into the antitumor activity of an antibody specific for transferrin receptor 1 (ch128.1) in an in vivo model of human multiple myeloma

Abstract The transferrin receptor 1 (TfR1), also known as CD71, is a type II transmembrane homodimeric protein involved in cellular iron uptake and regulation of cell growth. The high levels of TfR1 expression on cancer cells, its extracellular accessibility, and its central role in cancer pathology make it an attractive target for antibody-mediated cancer therapy. We have previously developed a mouse/human chimeric IgG3 specific for human TfR1 (ch128.1), which exhibits direct cytotoxic activity against certain human malignant B cells in vitro through TfR1 degradation and iron deprivation. In addition, we showed that ch128.1 is capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC) against malignant B cells in vitro. Importantly, ch128.1 shows exceptional antitumor activity in xenograft models of disseminated multiple myeloma (MM) in immunosuppressed mice (SCID-Beige) in an early disease setting. Intriguingly, this activity is observed even in malignant cells that show no sensitivity to the direct cytotoxic activity of ch128.1 in vitro. In order to study the mechanism of the in vivo antitumor activity, we generated a ch128.1 mutant with abolished binding to FcγR and the complement component C1q (L234A/L235A/P331S). Interestingly, this antibody mutant exhibited a total lack of in vivo protection against MM. This lack of antitumor activity is not due to increased clearance as determined by bioavailability studies. If fact, the two antibodies showed comparable affinity for the mouse FcRn (the neonatal Fc receptor, also known as the Brambell receptor) as determined via surface plasmon resonance analysis. This result suggests a critical role for the antibody Fc fragment in ch128.1-mediated antitumor activity. However, preliminary in vivo studies showed that depletion of complement using cobra venom factor (CVF) does not decrease the antitumor activity of ch128.1, suggesting that CDC is not a relevant mechanism of action, at least in the model used. Furthermore, we recently found that treatment with ch128.1 also significantly increased survival in late MM disease. Further studies aiming to provide a better understanding of the in vivo activity conferred by this antibody are in progress. Our results suggest that ch128.1 can be effective in the therapy of incurable human B-cell malignancies such as MM. Citation Format: Lai Sum Leoh, Yoon Kyung Kim, Otoniel Martinez-Maza, Tracy R. Daniels-Wells, Manuel L. Penichet. Novel insights into the antitumor activity of an antibody specific for transferrin receptor 1 (ch128.1) in an in vivo model of human multiple myeloma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 582.

Iron management and production of electricity by microorganisms.

The increasing dependency on fossil fuels has driven researchers to seek for alternative energy sources. Renewable energy sources such as sunlight, wind, or water are the most common. However, since the 1990s, other sources for energy production have been studied. The use of microorganisms such as bacteria or archaea to produce energy is currently in great progress. These present several advantages even when compared with other renewable energy sources. Besides the energy production, they are also involved in bioremediation such as the removal of heavy metal contaminants from soils or wastewaters. Several research groups have demonstrated that these organisms are able to interact with electrodes via heme and non-heme iron proteins. Therefore, the role of iron as well as iron metabolism in these species must be of enormous relevance. Recently, the influence of cellular iron regulation by Fur in the Geobacter sulfurreducens growth and ability to produce energy was demonstrated. In this review, we aim to briefly describe the most relevant proteins involved in the iron metabolism of bacteria and archaea and relate them and their biological function with the ability of selected organisms to produce energy.

Insights into the effector functions of human IgG3 in the context of an antibody targeting transferrin receptor 1.

The transferrin receptor 1 (TfR1) is involved in cellular iron uptake and regulation of cell proliferation. The increased expression of TfR1 observed in malignant cells, compared to normal cells, together with its extracellular accessibility, make this receptor an attractive target for antibody-mediated cancer therapy. We have developed a mouse/human chimeric IgG3 specific for human TfR1 (ch128.1), which shows anti-tumor activity against certain malignant B cells in vitro through TfR1 degradation and iron deprivation, and in vivo through a mechanism yet to be defined. To further explore potential mechanisms of action of ch128.1, we examined its ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC). We now report that ch128.1 is capable of mediating ADCC and CDC against malignant B cells, which is consistent with its ability to bind FcγRI, FcγRIIIa, and the complement component C1q. To delineate the residues involved in these effector functions, we developed a panel of three constructs with mutations in the lower hinge region and CH2 domain: 1) L234A/L235A, 2) P331S, and 3) L234A/L235A/P331S. The triple mutant consistently displayed a significant reduction in ADCC, while the L234A/L235A mutant exhibited less reduction in ADCC, and the P331S mutant did not show reduced ADCC. However, all three mutants exhibited impaired binding to FcγRI and FcγRIIIa. These results suggest that all three residues contribute to ADCC, although to different degrees. The P331S mutant showed drastically decreased C1q binding and abolished CDC, confirming the critical role of this residue in complement activation, while the other residues play a less important role in CDC. Our study provides insights into the effector functions of human IgG3 in the context of an antibody targeting TfR1.

Characterisation of iron regulatory protein 1A and 1B in the blood-feeding copepod Lepeophtheirus salmonis

During its parasitic life stages, the marine ectoparasitic copepod Lepeophtheirus salmonis ingests large amounts of host blood, which contains high amounts of iron. Iron is an essential micronutrient, but also toxic in high dosages, and blood-feeding parasites like the salmon louse must thus possess an efficient system to handle the excess iron. Iron regulatory protein 1 and 2 (IRP1 and IRP2) are known to play crucial roles in this process, by regulating several proteins involved in iron transport and storage, depending on the cellular iron concentration. To gain knowledge about the regulation of the iron metabolism in salmon lice, two IRP homologues (LsIRP1A and LsIRP1B) were identified by sequence and predicted structure similarity to known IRPs in other species. In situ hybridisation revealed that LsIRP1A and LsIRP1B mRNAs were expressed in the ovaries, oviducts and vitellogenic oocytes of adult females. Transcription levels of LsIRP1A and LsIRP1B mRNAs did not differ significantly between the different developmental stages of the salmon louse. Adults in the absence of blood as a feed source had decreased levels of LsIRP1A, but not LsIRP1B mRNA. RNA binding experiments indicated the presence of functioning IRP in salmon lice. In order to explore the biological functions of LsIRP1A and LsIRP1B, the mRNAs of both proteins were knocked down by RNA interference (RNAi) in preadult females. The knockdown was confirmed by qRT-PCR. LsIRP1B knockdown lice produced less offspring than control lice due to slightly shorter egg strings and had decreased levels of transcripts involved in egg development. Knockdown of both LsIRP1A and LsIRP1B caused increased expression of a salmon louse Ferritin (LsFer). These results confirm that salmon lice have two IRP1 homologues, LsIRP1A and LsIRP1B, and might suggest a function in cellular iron regulation in the reproductive organs and eggs.

Iron for proliferation of cell lines and hematopoietic progenitors: Nailing down the intracellular functional iron concentration

Iron is an essential nutrient which must be provided in sufficient amounts to support growth of eukaryotic cells. All organisms devote specialized pathways to ensure proper delivery. Yet, a quantitative assessment of the intra-cellular iron concentration needed to allow the cell cycle to proceed in mammalian cells is missing. Starting from iron-depleted cell lines or primary hematopoietic progenitors prepared with clinically implemented iron chelators, replenishment via transferrin and other iron sources has been quantitatively monitored through the main endogenous markers of the cellular iron status, namely proteins involved in the uptake (transferrin receptor), the storage (ferritin), and the sensing (Iron Regulatory Proteins) of iron. When correlated with measurements of iron concentrations and indicators of growth, this minimally intrusive approach provided an unprecedented estimate of the intracellular iron concentration acting upon iron-centered regulatory pathways. The data were analyzed with the help of a previously developed theoretical treatment of cellular iron regulation. The minimal cellular iron concentration required for cell division was named functional iron concentration (FIC) to distinguish it from previous estimates of the cellular labile iron. The FIC falls in the low nanomolar range for all studied cells, including hematopoietic progenitors. These data shed new light on basic aspects of cellular iron homeostasis by demonstrating that sensing and regulation of iron occur well below the concentrations requiring storage or becoming noxious in pathological conditions. The quantitative assessment provided here is relevant for monitoring treatments of conditions in which iron provision must be controlled to avoid unwanted cellular proliferation.