BBA - General Subjects (v.1820, #3)

Dedication by Saten Kumar (159-160).

The long history of iron in the Universe and in health and disease by Alex D. Sheftel; Anne B. Mason; Prem Ponka (161-187).
Not long after the Big Bang, iron began to play a central role in the Universe and soon became mired in the tangle of biochemistry that is the prima essentia of life. Since life's addiction to iron transcends the oxygenation of the Earth's atmosphere, living things must be protected from the potentially dangerous mix of iron and oxygen. The human being possesses grams of this potentially toxic transition metal, which is shuttling through his oxygen-rich humor. Since long before the birth of modern medicine, the blood—vibrant red from a massive abundance of hemoglobin iron—has been a focus for health experts.We describe the current understanding of iron metabolism, highlight the many important discoveries that accreted this knowledge, and describe the perils of dysfunctional iron handling.Isaac Newton famously penned, “If I have seen further than others, it is by standing upon the shoulders of giants”. We hope that this review will inspire future scientists to develop intellectual pursuits by understanding the research and ideas from many remarkable thinkers of the past.The history of iron research is a long, rich story with early beginnings, and is far from being finished. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.► Iron has been a requirement for nearly all life from the very first organisms. ► We document the history of iron metabolism in health and disease. ► Unique and specific systems for handling iron evolved to control its catalytic nature. ► The plasma protein transferrin plays a key role in iron metabolism. ► Disturbed iron homeostasis can result in catastrophic consequences for humans.
Keywords: Transferrin; Heme; Iron–sulfur cluster; Mitochondrion; Anemia; Hemochromatosis;

Regulation of iron transport and the role of transferrin by Konstantinos Gkouvatsos; George Papanikolaou; Kostas Pantopoulos (188-202).
Iron is utilized by several proteins as cofactor for major biological processes. However, iron may also harm cells by catalyzing the generation of free radicals and promoting oxidative stress. Acquisition, transport, utilization and storage of iron are tightly controlled to meet physiological needs and prevent excessive accumulation of the metal within cells. Plasma transferrin has been known for years as a central player in iron metabolism, assigned to circulate iron in a soluble, non-toxic form and deliver it to the erythron and other tissues. Recent data uncovered an additional role of transferrin as an upstream regulator of hepcidin, a liver-derived peptide hormone that controls systemic iron traffic.Here, we review basic features of iron metabolism, highlighting the function of transferrin in iron transport and cellular iron uptake. We further discuss the role of hepcidin as an orchestrator of systemic iron homeostasis, and the mechanisms underlying hepcidin regulation in response to various physiological cues. Emphasis is given on the role of transferrin on iron-dependent hepcidin regulation.Transferrin exerts a crucial function in the maintenance of systemic iron homeostasis as component of a plasma iron sensing system that modulates hepcidin expression.Proper expression of transferrin and hepcidin are essential for health, and disruption of their regulatory circuits is associated with iron-related disorders. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.► Basic concepts of cellular and systemic iron metabolism. ► The role of transferrin in iron transport and cellular iron uptake. ► Hormonal regulation of systemic iron traffic by hepcidin. ► Mechanisms underlying hepcidin regulation in response to various physiological cues. ► The role of transferrin in iron-dependent regulation of hepcidin.
Keywords: Transferrin; Hepcidin; Iron metabolism;

X-ray structures of transferrins and related proteins by Kimihiko Mizutani; Mayuko Toyoda; Bunzo Mikami (203-211).
Transferrins are a group of iron-binding proteins including serum transferrin, lactoferrin and ovotransferrin.The structures of transferrins are discussed.The typical transferrin molecules are folded into two homologous lobes. X-ray crystallography revealed that each lobe is further divided into two similarly sized domains, and that an iron-binding site is contained within the inter-domain cleft. The six iron coordination sites are occupied by four residues and a bidentate carbonate anion.The structures of the apo- and holo-forms revealed that the transferrins undergo a large-scale conformational change upon the uptake and release of irons: domains rotate as rigid bodies around a screw axis passing through inter-domain contacts. The iron-release mechanism of transferrin N-lobe is also revealed by X-ray crystallography; two basic residues in two domains form an unusual hydrogen bond in neutral pH, and the bond should be broken and facilitate iron release at a low pH of the endosome. For ovotransferrin, the iron release kinetics of two lobes correspond well with the numbers of anion binding sites found in crystal structures. The structures of transferrins bound to other metals revealed that the flexibility of the transferrin structure allows the ability to bind to other metals. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.► X-ray structures of serum transferrin, lactoferrin and ovotransferrin. ► Structural mechanisms for binding and release if iron were introduced. ► Domain movements of transferrins upon metal binding were introduced.
Keywords: Transferrin; Structure; Ovotransferrin;

Beyond bilobal: Transferrin homologs having unusual domain architectures by Jean P. Gaffney; Ann M. Valentine (212-217).
Most transferrin family proteins have a familiar bilobal structure, the result of an ancient gene duplication, with an iron binding site in each of two homologous lobes. Scattered throughout the evolutionary tree from algae to mammals, though, are transferrin homologs having other kinds of domain architectures.This review covers a variety of unusual transferrin forms, including monolobals, bilobals with one or both iron-binding sites abrogated, bilobals accessorized with long insertions or with membrane anchors, and even trilobals. The monolobal transferrin homologs from marine invertebrate ascidians are especially highlighted here.Unusual transferrin homologs appear scattered through much of the evolutionary tree. For some of these proteins, iron binding and/or iron transport appear to be the primary roles; for others they clearly are not. Many are incompletely or not at all studied.Taken together, these proteins begin to offer a glimpse into how the transferrin architecture has been repurposed for a diversity of applications. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.► Most transferrin proteins have a common two-lobed structure. ► Some have between one and three homologous lobes, sometimes with other augmentation. ► These unusual transferrins occur sporadically from algae to mammals. ► They sometimes have functions other than iron binding.
Keywords: Transferrin; Monolobal; Bilobal; Trilobal; Gene duplication;

Physiological roles of ovotransferrin by Francesco Giansanti; Loris Leboffe; Giuseppina Pitari; Rodolfo Ippoliti; Giovanni Antonini (218-225).
Ovotransferrin is an iron-binding glycoprotein, found in avian egg white and in avian serum, belonging to the family of transferrin iron-binding glycoproteins. All transferrins show high sequence homology. In mammals are presents two different soluble glycoproteins with different functions: i) serum transferrin that is present in plasma and committed to iron transport and iron delivery to cells and ii) lactoferrin that is present in extracellular fluids and in specific granules of polymorphonuclear lymphocytes and committed to the so-called natural immunity. To the contrary, in birds, ovotransferrin remained the only soluble glycoprotein of the transferrin family present both in plasma and egg white.Substantial experimental evidences are summarized, illustrating the multiple physiological roles of ovotransferrin in an attempt to overcome the common belief that ovotransferrin is a protein dedicated only to iron transport and to iron withholding antibacterial activity.Similarly to the better known family member protein lactoferrin, ovotransferrin appears to be a multi-functional protein with a major role in avian natural immunity.Biotechnological applications of ovotransferrin and ovotransferrin-related peptides could be considered in the near future, stimulating further research on this remarkable protein. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.► Multiple physiological roles of ovotransferrin are reviewed. ► Ovotransferrin plays a major role in avian natural immunity. ► Experimental evidences support that the ovotransferrin is a multi-functional protein. ► New biotechnolgical applications of ovotransferrin could be envisaged.
Keywords: Ovotransferrin; Serum transferrin; Lactoferrin; Natural immunity; Iron transport; Protein evolution;

Lactoferrin a multiple bioactive protein: An overview by Isui Abril García-Montoya; Tania Siqueiros Cendón; Sigifredo Arévalo-Gallegos; Quintín Rascón-Cruz (226-236).
Lactoferrin (Lf) is an 80 kDa iron-binding glycoprotein of the transferrin family. It is abundant in milk and in most biological fluids and is a cell-secreted molecule that bridges innate and adaptive immune function in mammals. Its protective effects range from anticancer, anti-inflammatory and immune modulator activities to antimicrobial activities against a large number of microorganisms. This wide range of activities is made possible by mechanisms of action involving not only the capacity of Lf to bind iron but also interactions of Lf with molecular and cellular components of both hosts and pathogens.This review summarizes the activities of Lf, its regulation and potential applications.The extensive uses of Lf in the treatment of various infectious diseases in animals and humans has been the driving force in Lf research however, a lot of work is required to obtain a better understanding of its activity.The large potential applications of Lf have led scientists to develop this nutraceutical protein for use in feed, food and pharmaceutical applications. This article is part of a Special Issue entitled Molecular Mechanisms of Iron Transport and Disorders.► It is well known that the mechanisms of action of antimicrobial activity are not fully understood. ► Important advances were made in heterologous Lf expression in prokaryotic systems. ► In the near future we will see the exploitation of the full capacity of lactoferrin expression in plants.
Keywords: Lactoferrin; Iron-binding protein; Transferrin; Functional protein;

Melanotransferrin: Search for a function by Yohan Suryo Rahmanto; Sumeet Bal; Kim H. Loh; Yu Yu; Des R. Richardson (237-243).
Melanotransferrin was discovered in the 1980s as one of the first melanoma tumour antigens. The molecule is a transferrin homologue that is found predominantly bound to the cell membrane by a glycosyl-phosphatidylinositol anchor. MTf was described as an oncofoetal antigen expressed in only small quantities in normal tissues, but in much larger amounts in neoplastic cells. Several diseases are associated with expression of melanotransferrin, including melanoma and Alzheimer's disease, although the significance of the protein to the pathogenesis of these conditions remains unclear.In this review, we discuss the roles of melanotransferrin in physiological and pathological processes and its potential use as an immunotherapy.Although the exact biological functions of melanotransferrin remain elusive, a growing number of roles have been attributed to the protein, including iron transport/metabolism, angiogenesis, proliferation, cellular migration and tumourigenesis.The high expression of melanotransferrin in several disease states, particularly malignant melanoma, remains intriguing and may have clinical significance. Further studies on the biology of this protein may provide new insights as well as potential therapeutic avenues for cancer treatment. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.► Role of melanotransferrin in iron transport and metabolism. ► Melanotransferrin and its role in angiogenesis and plasminogen activation. ► Role of melanotransferrin in cell proliferation, migration and tumourigenesis. ► Role of melanotransferrin in epithelial septal junction assembly. ► The potential of melanotransferrin as therapeutic target.
Keywords: Iron; Melanotransferrin; Cell proliferation;

In vertebrates, serum transferrins are essential iron transporters that have bind and release Fe(III) in response to receptor binding and changes in pH. Some family members such as lactoferrin and melanotransferrin can also bind iron while others have lost this ability and have gained other functions, e.g., inhibitor of carbonic anhydrase (mammals), saxiphilin (frogs) and otolith matrix protein 1 (fish).This article provides an overview of the known transferrin family members and their associated receptors and interacting partners.The number of transferrin genes has proliferated as a result of multiple duplication events, and the resulting paralogs have developed a wide array of new functions. Some homologs in the most primitive metazoan groups resemble both serum and melanotransferrins, but the major yolk proteins show considerable divergence from the rest of the family. Among the transferrin receptors, the lack of TFR2 in birds and reptiles, and the lack of any TFR homologs among the insects draw attention to the differences in iron transport and regulation in those groups.The transferrin family members are important because of their clinical significance, interesting biochemical properties, and evolutionary history. More work is needed to better understand the functions and evolution of the non-vertebrate family members. This article is part of a Special Issue entitled Molecular Mechanisms of Iron Transport and Disorders.► Transferrins (TF) are an ancient family found in all metazoans. ► Multiple intragenic and gene duplications have created numerous variations. ► At least seven orthologs with different functions have been found in vertebrates. ► The number and types of TF receptors differ by ortholog and by family. ► Evolutionary comparisons provide clues to the function of individual residues.
Keywords: Transferrin; Transferrin receptor; Major yolk protein; Iron transport; Molecular evolution; Review;

Hereditary hemochromatosis and transferrin receptor 2 by Juxing Chen; Caroline A. Enns (256-263).
Multicellular organisms regulate the uptake of calories, trace elements, and other nutrients by complex feedback mechanisms. In the case of iron, the body senses internal iron stores, iron requirements for hematopoiesis, and inflammatory status, and regulates iron uptake by modulating the uptake of dietary iron from the intestine. Both the liver and the intestine participate in the coordination of iron uptake and distribution in the body. The liver senses inflammatory signals and iron status of the organism and secretes a peptide hormone, hepcidin. Under high iron or inflammatory conditions hepcidin levels increase. Hepcidin binds to the iron transport protein, ferroportin (FPN), promoting FPN internalization and degradation. Decreased FPN levels reduce iron efflux out of intestinal epithelial cells and macrophages into the circulation. Derangements in iron metabolism result in either the abnormal accumulation of iron in the body, or in anemias. The identification of the mutations that cause the iron overload disease, hereditary hemochromatosis (HH), or iron-refractory iron-deficiency anemia has revealed many of the proteins used to regulate iron uptake.In this review we discuss recent data concerning the regulation of iron homeostasis in the body by the liver and how transferrin receptor 2 (TfR2) affects this process.TfR2 plays a key role in regulating iron homeostasis in the body.The regulation of iron homeostasis is important. One third of the people in the world are anemic. HH is the most common inherited disease in people of Northern European origin and can lead to severe health complications if left untreated. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.► Recent data concerning the regulation of iron homeostasis in the body by the liver is discussed. ► Transferrin receptor 2 plays a distinct role in the regulation of hepcidin. ► Transferrin receptor 2 is controlled both transcriptionally and posttranscriptionally at the level of protein stability. ► Transferrin receptor 2 also plays a role in erythropoiesis. ► The regulation of hepcidin transcription is controlled by both iron through Tf saturation and BMP6 and by inflammation.
Keywords: TfR2; HFE; Hepcidin; Hemojuvelin; BMP; Ferroportin;

The intracellular trafficking pathway of transferrin by Kristine M. Mayle; Alexander M. Le; Daniel T. Kamei (264-281).
Transferrin (Tf) is an iron-binding protein that facilitates iron-uptake in cells. Iron-loaded Tf first binds to the Tf receptor (TfR) and enters the cell through clathrin-mediated endocytosis. Inside the cell, Tf is trafficked to early endosomes, delivers iron, and then is subsequently directed to recycling endosomes to be taken back to the cell surface.We aim to review the various methods and techniques that researchers have employed for elucidating the Tf trafficking pathway and the cell-machinery components involved. These experimental methods can be categorized as microscopy, radioactivity, and surface plasmon resonance (SPR).Qualitative experiments, such as total internal reflectance fluorescence (TIRF), electron, laser-scanning confocal, and spinning-disk confocal microscopy, have been utilized to determine the roles of key components in the Tf trafficking pathway. These techniques allow temporal resolution and are useful for imaging Tf endocytosis and recycling, which occur on the order of seconds to minutes. Additionally, radiolabeling and SPR methods, when combined with mathematical modeling, have enabled researchers to estimate quantitative kinetic parameters and equilibrium constants associated with Tf binding and trafficking.Both qualitative and quantitative data can be used to analyze the Tf trafficking pathway. The valuable information that is obtained about the Tf trafficking pathway can then be combined with mathematical models to identify design criteria to improve the ability of Tf to deliver anticancer drugs. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.► Microscopy can be used to identify cellular components involved in Tf trafficking. ► Radioactivity and SPR can be used to characterize Tf binding and trafficking. ► Developing mathematical models enable evaluation of Tf kinetic parameters. ► Identifying design parameters can improve Tf’s ability to deliver chemotherapeutics.
Keywords: Transferrin; Trafficking; Microscopy; Radioactivity; SPR; Modeling;

Iron mobilization from transferrin by therapeutic iron chelating agents by Robert W. Evans; Xiaole Kong; Robert C. Hider (282-290).
The bacteriostatic activity of the transferrin family has been known since the early 1960’s. The possession of high affinity iron(III)-binding sites and the existence of a specific membrane-bound receptor, have led to the present understanding of serum transferrin acting as the major iron transporter between cells in vertebrate systems. Iron chelators can interact with transferrin, either by directly donating iron or by removing iron from the protein; both interactions have relevance for haematology.Urea polyacrylamide gels and HPLC methods have been developed for the resolution and quantification of the four major forms of transferrin, diferric-transferrin, C-mono Fe-transferrin, N-mono Fe-transferrin and apo transferrin.Negatively charged ligands with pFe values > 20 remove iron from transferrin, preferably from the N-lobe iron-binding site. Some siderophores are capable of removing iron from transferrin. 3-Hydroxypyridin-4-ones, lacking a negative charge are able to remove iron from transferrin with a strong preference for the C- lobe iron-binding site. The donation of iron to apo transferrin by hydroxypyridinone iron(III) complexes has relevance to the treatment of clinical anaemias, because the hydroxypyridinones can also mobilize iron from the reticuloendothelial system and so facilitate the redistribution of iron from macrophages to reticulocytes.Hydroxypyridinones have excellent potential for facilitating the redistribution of iron and this has relevance to the treatment of many disease types, including neurodegeneration and clinical anaemias. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.Display Omitted► Human transferrin binds two iron(III) ions with slightly different affinities. ► Negatively charged chelators (pFe ≥ 20) can remove iron from transferrin. ► Chelators have different abilities to scavenge iron from the 2 binding sites. ► Some siderophores can remove iron from the transferrin. ► Chelators (pFe ≈ 20) can both remove and donate iron from and to transferrin.
Keywords: Transferrin; Chelator; Iron overload;

The transferrin receptor and the targeted delivery of therapeutic agents against cancer by Tracy R. Daniels; Ezequiel Bernabeu; José A. Rodríguez; Shabnum Patel; Maggie Kozman; Diego A. Chiappetta; Eggehard Holler; Julia Y. Ljubimova; Gustavo Helguera; Manuel L. Penichet (291-317).
Traditional cancer therapy can be successful in destroying tumors, but can also cause dangerous side effects. Therefore, many targeted therapies are in development. The transferrin receptor (TfR) functions in cellular iron uptake through its interaction with transferrin. This receptor is an attractive molecule for the targeted therapy of cancer since it is upregulated on the surface of many cancer types and is efficiently internalized. This receptor can be targeted in two ways: 1) for the delivery of therapeutic molecules into malignant cells or 2) to block the natural function of the receptor leading directly to cancer cell death.In the present article we discuss the strategies used to target the TfR for the delivery of therapeutic agents into cancer cells. We provide a summary of the vast types of anti-cancer drugs that have been delivered into cancer cells employing a variety of receptor binding molecules including Tf, anti-TfR antibodies, or TfR-binding peptides alone or in combination with carrier molecules including nanoparticles and viruses.Targeting the TfR has been shown to be effective in delivering many different therapeutic agents and causing cytotoxic effects in cancer cells in vitro and in vivo.The extensive use of TfR for targeted therapy attests to the versatility of targeting this receptor for therapeutic purposes against malignant cells. More advances in this area are expected to further improve the therapeutic potential of targeting the TfR for cancer therapy leading to an increase in the number of clinical trials of molecules targeting this receptor. This article is part of a Special Issue entitled Transferrins: molecular mechanisms of iron transport and disorders.► Summary of delivery strategies targeting the transferrin receptor. ► Summary of complexes with targeting moiety directly conjugated to the therapeutic. ► Summary of approaches where targeted carriers are loaded with the therapeutic.
Keywords: Transferrin receptor; Cancer; Nanoparticle; Immunotoxin; Delivery; Conjugate;

In mammals, serum-transferrins transport iron from the neutral environment of the blood to the cytoplasm by receptor-mediated endocytosis. Extensive in-vitro studies have focused on the thermodynamics and kinetics of Fe3+ binding to a number of transferrins. However, little attention has been given to the thermodynamic characterization of the interaction of transferrin with its receptor.Iron-loaded transferrin (Tf) binds with high affinity to the specific transferrin receptor (TfR) on the cell surface. The Tf–TfR complex is then internalized via receptor mediated endocytosis into an endosome where iron is released. Here, we provide an overview of recent studies that have used ITC to quantify the interaction of various metal ions with transferrin and highlight our current understanding of the thermodynamics of the transferrin–transferrin receptor system at physiological pH.The interaction of the iron-loaded transferrin with the transferrin receptor is a key cellular process that occurs during the normal course of iron metabolism. Understanding the thermodynamics of this interaction is important for iron homeostasis since the physiological requirement of iron must be appropriately maintained to avoid iron-related diseases.The thermodynamic data revealed stoichiometric binding of all tested metal ions to transferrin with very high affinities ranging between 1017 and 1022  M− 1. Iron-loaded transferrin (monoferric or diferric) is shown to bind avidly (K ~ 107–108  M− 1) to the receptor at neutral pH with a stoichiometry of one Tf molecule per TfR monomer. Significantly, both the N- and the C-lobe contribute to the binding interaction which is shown to be both enthalpically and entropically driven. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.► ITC is a valuable tool to characterize the thermodynamics of binding interactions. ► A large number of metal ions exhibit very high binding affinity to transferrin. ► Holo-hTf displays high affinity to TfR and favorable enthalpy and entropy changes. ► Both lobes of hTf appear to contribute to the thermodynamics of hTf-TfR interaction.

Kinetics of iron release from transferrin bound to the transferrin receptor at endosomal pH by Ashley N. Steere; Shaina L. Byrne; N. Dennis Chasteen; Anne B. Mason (326-333).
Human serum transferrin (hTF) is a bilobal glycoprotein that reversibly binds Fe3+ and delivers it to cells by the process of receptor-mediated endocytosis. Despite decades of research, the precise events resulting in iron release from each lobe of hTF within the endosome have not been fully delineated.We provide an overview of the kinetics of iron release from hTF ± the transferrin receptor (TFR) at endosomal pH (5.6). A critical evaluation of the array of biophysical techniques used to determine accurate rate constants is provided.Delivery of Fe3+to actively dividing cells by hTF is essential; too much or too little Fe3+ directly impacts the well-being of an individual. Because the interaction of hTF with the TFR controls iron distribution in the body, an understanding of this process at the molecular level is essential.Not only does TFR direct the delivery of iron to the cell through the binding of hTF, kinetic data demonstrate that it also modulates iron release from the N- and C-lobes of hTF. Specifically, the TFR balances the rate of iron release from each lobe, resulting in efficient Fe3+ release within a physiologically relevant time frame. This article is part of a Special Issue entitled Molecular Mechanisms of Iron Transport and Disorders.► Because the interaction of transferrin with the receptor controls iron distribution, an understanding of this process essential. ► The transferrin receptor modulates iron release from the N- and C-lobes of human serum transferrin. ► The transferrin receptor balances the rate of iron release from each lobe, resulting in efficient Fe3+ release from transferrin.
Keywords: Transferrin; Transferrin receptor; Kinetics; Fluorescence;

Uptake and release of metal ions by transferrin and interaction with receptor 1 by Jean-Michel El Hage Chahine; Miryana Hémadi; Nguyêt-Thanh Ha-Duong (334-347).
For a metal to follow the iron acquisition pathway, four conditions are required: 1—complex formation with transferrin; 2—interaction with receptor 1; 3—metal release in the endosome; and 4—metal transport to cytosol.This review deals with the mechanisms of aluminum(III), cobalt(III), uranium(VI), gallium(III) and bismuth(III) uptake by transferrin and interaction with receptor 1.The interaction of the metal-loaded transferrin with receptor 1 takes place in one or two steps: a very fast first step (μs to ms) between the C-lobe and the helical domain of the receptor, and a second slow step (2–6 h) between the N-lobe and the protease-like domain. In transferrin loaded with metals other than iron, the dissociation constants for the interaction of the C-lobe with TFR are in a comparable range of magnitudes 10 to 0.5 μM, whereas those of the interaction of the N-lobe are several orders of magnitudes lower or not detected. Endocytosis occurs in minutes, which implies a possible internalization of the metal-loaded transferrin with only the C-lobe interacting with the receptor.A competition with iron is possible and implies that metal internalization is more related to kinetics than thermodynamics. As for metal release in the endosome, it is faster than the recycling time of transferrin, which implies its possible liberation in the cell. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.► Interaction of metal-loaded transferrins with Receptor-1. ► Metal release from the transferrin/receptor-1 adduct in the cell. ► Ultra fast interaction of the C-lobe with the helical domain of receptor 1. ► Metal endocytosis occurs mainly with the C-lobe of transferrin.
Keywords: Iron acquisition pathway; Protein/protein interaction; Cobalt; Uranium; Aluminum;

Since the transferrins have been defined by the highly cooperative binding of Fe3+ and a carbonate anion to form an Fe–CO3–Tf ternary complex, the focus has been on synergistic anion binding. However, there are other types of anion binding with both apotransferrin and diferric transferrin that affect metal binding and release.This review covers the binding of anions to the apoprotein, as well as the formation and structure of Fe–anion–transferrin ternary complexes. It also covers interactions between ferric transferrin and non-synergistic anions that appear to be important in vivo.The interaction of anions with apotransferrin can alter the effective metal binding constants, which can affect the transport of metal ions in serum. These interactions also play a role in iron release under physiological conditions.Apotransferrin binds a variety of anions with no special selectivity for carbonate. The selectivity for carbonate as a synergistic anion is associated with the iron binding reaction. Conformational changes in the binding of the synergistic carbonate and competition from non-synergistic anions both play a role in intracellular iron release. Anion competition also occurs in serum and reduces the effective metal binding affinity of Tf. Lastly, anions bind to allosteric sites (KISAB sites) on diferric transferrin and alter the rates of iron release. The KISAB sites have not been well-characterized, but kinetic studies on iron release from mutant transferrins indicate that there are likely to be multiple KISAB sites for each lobe of transferrin. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.Display Omitted► A wide variety of anions bind to apoTf. ► A few of these anions are synergistic and support the binding of iron(III). ► Conformational changes in the synergistic anion are critical for iron release. ► Competition from non-synergistic anions in vivo affects transferrin function. ► There are multiple allosteric anion binding sites that affect iron release.
Keywords: Transferrin; Anion binding; Synergistic anion; Iron release kinetics;

The binding and transport of alternative metals by transferrin by John B. Vincent; Sharifa Love (362-378).
The iron transport protein of the blood plasma, transferrin, is maintained only with about 30% of its capacity to bind Fe3+ ions; this leaves the protein the potential ability to transport other metal ions from the bloodstream to the tissues.This review examines the potential role of transferrin to bind and transport alternative metal ions with possible beneficial and deleterious effects.Transferrin has been postulated to play a significant role in transporting Ti4+, VO2+ (V4+), Cr3+, Ru3+, and Bi3+, all metal ions of potential therapeutic significance. Transferrin may possess a physiological role in the transport of manganese, as the trivalent ion. However, the protein may also play a role in carrying potentially toxic Al3+ and actinide ions, including Pu4+, to the tissues. Attempts to use transferrin in the selective removal of low concentrations of specific metal ions from aqueous mixed ions waste streams using a procedure called metalloprotein affinity metal chromatography are discussed.The binding of alternative metals to transferrins may have therapeutic and toxicological significance. This article is part of a Special Issue entitled Transferrins: Molecular Mechanisms of Iron Transport and Disorders.► Transferrin binds potentially therapeutic Ti4+, VO2+ (V4+), and Cr3+ ions. ► The binding of Mn3+ by transferrin is physiologically significant. ► Transferrin maintains actinide ions in the bloodstream.
Keywords: Transferrin; Vanadium; Chromium; Manganese; Plutonium; Metalloprotein affinity metal chromatography;

FbpA — A bacterial transferrin with more to offer by Claire J. Parker Siburt; Timothy A. Mietzner; Alvin L. Crumbliss (379-392).
Gram negative bacteria require iron for growth and virulence. It has been shown that certain pathogenic bacteria such as Neisseria gonorrhoeae possess a periplasmic protein called ferric binding protein (FbpA), which is a node in the transport of iron from the cell exterior to the cytosol.The relevant literature is reviewed which establishes the molecular mechanism of FbpA mediated iron transport across the periplasm to the inner membrane.Here we establish that FbpA may be considered a bacterial transferrin on structural and functional grounds. Data are presented which suggest a continuum whereby FbpA may be considered as a naked iron carrier, as well as a Fe–chelate carrier, and finally a member of the larger family of periplasmic binding proteins.An investigation of the molecular mechanisms of action of FbpA as a member of the transferrin super family enhances our understanding of bacterial mechanisms for acquisition of the essential nutrient iron, as well as the modes of action of human transferrin, and may provide approaches to the control of pathogenic diseases. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.► FbpA may be considered a structural and functional bacterial transferrin. ► The mechanism of FbpA mediated iron transport across the periplasm is described. ► FbpA is a naked iron carrier, a Fe-chel carrier, and a periplasmic binding protein. ► Studies reviewed here enhance understanding of human transferrin mechanisms. ► Studies reviewed here may provide approaches to the control of pathogenic diseases.
Keywords: Ferric binding protein; Bacterial transferrin; Periplasmic binding protein; Iron transport; Neisseria gonorrhoeae;

Functional roles of transferrin in the brain by Dominique F. Leitner; James R. Connor (393-402).
Transferrin is synthesized in the brain by choroid plexus and oligodendrocytes, but only that in the choroid plexus is secreted. Transferrin is a major iron delivery protein to the brain, but the amount transcytosed across the brain microvasculature is minimal. Transferrin is the major source of iron delivery to neurons. It may deliver iron to immature oligodendrocytes but this trophic effect declines over time while iron requirements for maintaining myelination continue. Finally, transferrin may play an important role in neurodegenerative diseases through its ability to mobilize iron.The role of transferrin in maintaining brain iron homeostasis and the mechanism by which it enters the brain and delivers iron will be discussed. Its relevance to neurological disorders will also be addressed.Transferrin is the major iron delivery protein for neurons and the microvasculature, but has a limited role for glial cells. The main source of transferrin in the brain is likely from the choroid plexus although the concentration of transferrin at any given time in the brain includes that synthesized in oligodendrocytes. Little is known about brain iron egress or the role of transferrin in this process.Neuron survival requires iron, which is predominantly delivered by transferrin. The concentration of transferrin in the cerebrospinal fluid is reflective of brain iron availability and can function as a biomarker in disease. Accumulation of iron in the brain contributes to neurodegenerative processes, thus an understanding of the role that transferrin plays in regulating brain iron homeostasis is essential. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.► Oligodendrocytes and choroid plexus express Tf mRNA in the brain. ► Choroid plexus secretes Tf, while Tf in oligodendrocytes is reportedly not secreted. ► Tf delivers iron to neurons but glial cells do not have mechanisms for Tf uptake. ► The amount of Tf that enters the brain through the BBB may be minimal. ► Tf in the CSF may serve as a biomarker for some neurological diseases.
Keywords: Transferrin; Neuron; Oligodendrocyte; Blood–brain-barrier; Iron; Choroid plexus;

Non-transferrin bound iron: A key role in iron overload and iron toxicity by Pierre Brissot; Martine Ropert; Caroline Le Lan; Olivier Loréal (403-410).
Besides transferrin iron, which represents the normal form of circulating iron, non-transferrin bound iron (NTBI) has been identified in the plasma of patients with various pathological conditions in which transferrin saturation is significantly elevated.To show that: i) NTBI is present not only during chronic iron overload disorders (hemochromatosis, transfusional iron overload) but also in miscellaneous diseases which are not primarily iron overloaded conditions; ii) this iron species represents a potentially toxic iron form due to its high propensity to induce reactive oxygen species and is responsible for cellular damage not only at the plasma membrane level but also towards different intracellular organelles; iii) the NTBI concept may be expanded to include intracytosolic iron forms which are not linked to ferritin, the major storage protein which exerts, at the cellular level, the same type of protective effect towards the intracellular environment as transferrin in the plasma.Plasma NTBI and especially labile plasma iron determinations represent a new important biological tool since elimination of this toxic iron species is a major therapeutic goal.The NTBI approach represents an important mechanistic concept for explaining cellular iron excess and toxicity and provides new important biochemical diagnostic tools. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.► NTBI and its toxic form LPI appear in plasma when transferrin saturation increases. ► NTBI is avidly taken up by parenchymal cells (especially hepatocytes). ► Labile Iron Pool can be considered as an intracytosolic equivalent of plasma NTBI. ► NTBI can be present in diseases not primarily related to iron overload. ► Therapeutic efforts (phlebotomies, iron chelation) must focus on NTBI elimination.
Keywords: Non-transferrin bound iron (NTBI); Hepcidin; Ferroportin; Iron overload; Hemochromatosis; Thalassemia;

The transfer of iron between ceruloplasmin and transferrins by Kenneth N. White; Celia Conesa; Lourdes Sánchez; Maryam Amini; Sebastien Farnaud; Chanakan Lorvoralak; Robert W. Evans (411-416).
It is over 60 years since the discovery and isolation of the serum ferroxidase ceruloplasmin. In that time much basic information about the protein has been elucidated including its catalytic and kinetic properties as an enzyme, expression, sequence and structure. The importance of its biological role is indicated in genetic diseases such as aceruloplasminemia where its function is lost through mutation. Despite this wealth of data, fundamental questions about its action remain unanswered and in this article we address the question of how ferric iron produced by the ferroxidase activity of ceruloplasmin could be taken up by transferrins or lactoferrins.Overlapping peptide libraries for human ceruloplasmin have been probed with a number of different lactoferrins to identify putative lactoferrin-binding regions on human ceruloplasmin. Docking software, 3D-Garden, has been used to model the binding of human lactoferrin to human ceruloplasmin.Upon probing the human ceruloplasmin library with human lactoferrin, three predominantly acidic lactoferrin-binding peptides, located in domains 2, 5 and 6 of human ceruloplasmin, were identified. The docking software identified a complex such that the N-lobe of human apo-lactoferrin interacts with the catalytic ferroxidase centre on human ceruloplasmin. In vitro binding studies and molecular modelling indicate that lactoferrin can bind to ceruloplasmin such that a direct transfer of ferric iron between the two proteins is possible. A direct transfer of ferric iron from ceruloplasmin to lactoferrin would prevent both the formation of potentially toxic hydroxyl radicals and the utilization of iron by pathogenic bacteria.► Human Lf binds three peptides from a human Cp overlapping peptide library. ► Human Lf-binding peptides are located in domains 2, 5 and 6 of human Cp. ► Lf-Cp interactions were modelled using docking software. ► An open N-lobe conformation of human Lf binds via domains 5 and 6 of human Cp. ► A model of ferric iron transfer between human Cp and human Lf is proposed.
Keywords: Ceruloplasmin; Ferroxidase; Transferrin; Lactoferrin;

Transferrin as a model system for method development to study structure, dynamics and interactions of metalloproteins using mass spectrometry by Igor A. Kaltashov; Cedric E. Bobst; Mingxuan Zhang; Rachael Leverence; Dmitry R. Gumerov (417-426).
Transferrin (Tf) is a paradigmatic metalloprotein, which has been extensively studied in the past and still is a focal point of numerous investigation efforts owing to its unique role in iron homeostasis and enormous promise as a component of a wide range of therapies.Electrospray ionization mass spectrometry (ESI MS) is a potent analytical tool that has been used successfully to study various properties of Tf and Tf-based products, ranging from covalent structure and metal binding to conformation and interaction with their physiological partners.Various ESI MS-based techniques produce unique information on Tf properties and behavior that is highly complementary to information provided by other experimental techniques.The experimental ESI MS-based techniques developed for Tf studies are not only useful for understanding of fundamental aspects of the iron-binding properties of this protein and optimizing Tf-based therapeutic products, but can also be applied to study a range of other metalloproteins. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.Display Omitted► Progress in biological mass spectrometry related to transferrin research. ► Unique information highly complementary to other experimental techniques. ► Can also be applied to study a range of other metalloproteins.
Keywords: Transferrin; Siderophore; Metalloprotein; Hydrogen exchange; Native mass spectrometry; Protein therapeutics;

Fibrillation of transferrin by Claire Booyjz̈sen; Charlotte A. Scarff; Ben Moreton; Ian Portman; James H. Scrivens; Giovanni Costantini; Peter J. Sadler (427-436).
The nature of fibrillar deposits from aqueous solutions of human serum and recombinant human transferrin on mica and carbon-coated formvar surfaces has been investigated.Atomic force microscopy showed that the deposition of recombinant transferrin onto the hydrophilic surface of mica resulted in the formation of a monolayer-thick film composed of conformationally-strained flattened protein molecules. Elongated fibres developed on top of this layer and appeared to be composed of single proteins or small clusters thereof. Monomeric and dimeric transferrins were separated by gel permeation chromatography and their states of aggregation confirmed by mass spectrometry and dynamic light scattering. Transmission electron-microscopy showed that dimeric transferrin, but not monomeric transferrin, deposited on carbon-coated formvar grids forms rounded (circular) structures ca. 250 nm in diameter. Small transferrin fibrils ca. 250 nm long appeared to be composed of smaller rounded sub-units. Synchrotron radiation-circular dichroism and, Congo red and thioflavin-T dye-binding experiments suggested that transferrin aggregation in solution does not involve major structural changes to the protein or formation of classical β-sheet amyloid structures. Collisional cross sections determined via ion mobility–mass spectrometry showed little difference between the overall protein shapes of apo- and holo-transferrin in the gas phase.The possibility that transferrin deformation and aggregation are involved in neurological disorders such as Parkinson's and Alzheimer's disease is discussed. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.► Association of transferrin dimers detected by GPC, MS and aggregates by DLS. ► Collisional cross sections of apo- and holo-transferrin determined by ion mobility-MS. ► AFM detects flattened transferrin on mica, aggregation into fibrils in second layer. ► Fibres formed by dimers on carbon surfaces are characterised by TEM. ► Possible relationship between transferrin aggregation, iron deposition, and diseases.
Keywords: Transferrin; Fibrils; Atomic force microscopy; Transmission electron microscopy; Ion mobility–mass spectrometry; Neurological disease;

Insect transferrins: Multifunctional proteins by Dawn L. Geiser; Joy J. Winzerling (437-451).
Many studies have been done evaluating transferrin in insects. Genomic analyses indicate that insects could have more than one transferrin. However, the most commonly studied insect transferrin, Tsf1, shows greatest homology to mammalian blood transferrin.Aspects of insect transferrin structure compared to mammalian transferrin and the roles transferrin serves in insects are discussed in this review.Insect transferrin can have one or two lobes, and can bind iron in one or both. The iron binding ligands identified for the lobes of mammalian blood transferrin are generally conserved in the lobes of insect transferrins that have an iron binding site. Available information supports that the form of dietary iron consumed influences the regulation of insect transferrin. Although message is expressed in several tissues in many insects, fat body is the likely source of hemolymph transferrin. Insect transferrin is a vitellogenic protein that is down-regulated by Juvenile Hormone. It serves a role in transporting iron to eggs in some insects, and transferrin found in eggs appears to be endowed from the female. In addition to the roles of transferrin in iron delivery, this protein also functions to reduce oxidative stress and to enhance survival of infection.Future studies in Tsf1 as well as the other insect transferrins that bind iron are warranted because of the roles of transferrin in preventing oxidative stress, enhancing survival to infections and delivering iron to eggs for development. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.► Insect transferrins have domains similar to mammalian Tsf that bind iron. ► The fat body is the likely source of insect hemolymph transferrin. ► Female insects endow Tsf1 to developing eggs as a vitellogenic protein. ► Insect transferrins transport dietary iron to tissues and developing embryos. ► Insect transferrins are increased during infection and limit free iron in hemolymph.
Keywords: Development; Ferritin; Immunity; Insect; Oxidative stress; Transferrin;