Iron binding and release properties of transferrin-1 from Drosophila melanogaster and Manduca sexta: Implications for insect iron homeostasis.

Transferrins belong to an ancient family of extracellular proteins. The best-characterized transferrins are mammalian proteins that function in iron sequestration or iron transport; they accomplish these functions by having a high-affinity iron-binding site in each of their two homologous lobes. Insect hemolymph transferrins (Tsf1s) also function in iron sequestration and transport; however, sequence-based predictions of their iron-binding residues have suggested that most Tsf1s have a single, lower-affinity iron-binding site. To reconcile the apparent contradiction between the known physiological functions and predicted biochemical properties of Tsf1s, we purified and characterized the iron-binding properties of Drosophila melanogaster Tsf1 (DmTsf1), Manduca sexta Tsf1 (MsTsf1), and the amino-lobe of DmTsf1 (DmTsf1N). Using UV-Vis spectroscopy, we found that these proteins bind iron, but they exhibit shifts in their spectra compared to mammalian transferrins. Through equilibrium dialysis experiments, we determined that DmTsf1 and MsTsf1 bind only one ferric ion; their affinity for iron is high (log K' = 18), but less than that of the well-characterized mammalian transferrins (log K' ~ 20); and they release iron under moderately acidic conditions (pH50 = 5.5). Iron release analysis of DmTsf1N suggested that iron binding in the amino-lobe is stabilized by the carboxyl-lobe. These findings will be critical for elucidating the mechanisms of Tsf1 function in iron sequestration and transport in insects.

SPION decorated exosome delivery of TNF-α to cancer cell membranes through magnetism.

Tumor necrosis factor (TNF-α) is capable of inducing apoptosis and is a promising candidate for genetic engineering drugs in cancer therapy; however, the serious side-effects of TNF-α hinder their clinical application. In the present study, a method for preparing fusion proteins of cell-penetrating peptides (CPP) and TNF-α (CTNF-α)-anchored exosomes coupled with superparamagnetic iron oxide nanoparticles (CTNF-α-exosome-SPIONs) with membrane targeting anticancer activity has been demonstrated. To acquire exosomes with TNF-α anchored in its membrane, a CTNF-α expression vector was constructed and a stable mesenchymal stem cell cell line that expressed CTNF-α was established. Conjugating transferrin-modified SPIONs (Tf-SPIONs) onto CTNF-α-exosomes through transferrin-transferrin receptor (Tf-TfR) interaction yields CTNF-α-exosome-SPIONs with good water dispersibility. The incorporation of TNF-α into exosomes and the conjugation of SPIONs significantly enhanced the binding capacity of TNF-α to its membrane-bound receptor TNFR I, thus increasing the therapeutic effects. CTNF-α-exosome-SPIONs significantly enhanced tumor cell growth inhibition via induction of the TNFR I-mediated apoptotic pathway. In vivo studies using murine melanoma subcutaneous cancer models showed that TNF-α-loaded exosome-based vehicle delivery enhanced cancer targeting under an external magnetic field and suppressed tumor growth with mitigating toxicity. Taken together, our results suggest that CTNF-α-exosome-SPIONs showed great potential in membrane targeting therapy.

Biodegradable Nanoprodrugs: "Delivering" ROS to Cancer Cells for Molecular Dynamic Therapy.

Biodegradable nanoprodrugs, inheriting the antitumor effects of chemotherapy drugs and overcoming the inevitable drawback of side effects on normal tissues, hold promise as next-generation cancer therapy candidates. Biodegradable nanoprodrugs of transferrin-modified MgO2 nanosheets are developed to selectively deliver reactive oxygen species to cancer cells for molecular dynamic therapy strategy. The nanosheets favor the acidic and low catalase activity tumor microenvironment to react with proton and release nontoxic Mg2+ . This reaction simultaneously produces abundant H2 O2 to induce cell death and damage the structure of transferrin to release Fe3+ , which will react with H2 O2 to produce highly toxic ·OH to kill tumor cells.

Fe-transferrins or their homologues in ex-vivo mushrooms as identified by ESR spectroscopy and quantum chemical calculations: A full spin-Hamiltonian approach for the ferric sextet state with intermediate zero-field splitting parameters.

Fe-transferrins/their homologues in ex-vivo mushrooms were identified by ESR spectroscopy at liquid helium temperature, 4 K. The ESR fine-structure signals from Grifola frondosa were analyzed by spectral simulation with a full spin-Hamiltonian approach, determining the spin Hamiltonian parameters of the ferric iron species bound in the biological environment: S = 5/2, g = (2.045, 2.01, 2.235), |D| = 0.28 cm-1, |E/D| = 0.05. The zero-field splitting (ZFS) parameters, D- and E-values, are very close to the reported values, |D| = 0.25 cm-1 and |E/D| = 0.06, for an Fe-transferrin with oxalate anion, and to |D| = 0.25 cm-1 and |E/D| = 0.04 for one with malonate anion in human sera, suggesting that the Fe3+ species are from Fe-transferrins or their homologues. Quantum chemical calculations of the ZFS tensors for Fe-transferrins were carried out. Fe-transferrins/homologues have been identified for all the mushrooms under study, suggesting that such Fe3+ enzymes are widely distributed in mushrooms.

Anticancer luminescent gold quantum clusters for in situ cancer-selective marking-imaging-targeting.

Ultrafine Au quantum clusters (QCs) were synthesized by etching host Au nanoparticles in the presence of ethylenediamine (en) and exhibited both strong photoluminescence (PL) and specific anticancer activity. The cutting-edge feature of this QC compound comprises subnanometer-size rhombohedral Au8, which consists of 8 units of the anticancer motif, namely, an Au+(en) complex (Au(en)QCs), which contributes to photo- and physicochemical stability as well as subcellular theranostic activity in intracellular PL imaging and in situ targeting. Moreover, the Au(en)QCs can be surface-encapsulated by transferrins (Tf) to create TfAu(en)QCs as a multipurpose drug carrier owing to numerous merits, which include cancer-selective biolabeling, high loading/release efficiency, high activity against drug-resistant tumor cells, low toxicity to normal cells, and physiological stability against biothiols, e.g., glutathiones. These versatile features, which are due to intrinsic optical and anticancer properties, provide potential as a single-drug delivery PL probe for preclinical applications, which has yet to be achieved using conventional nanoclusters.

Flexibility of the coordination geometry at the N-site of Cu(II)2 human serum-transferrin induced by the different orientations of Arg124.

The ESR spectra of dicupric human serum-transferrin (serum-Tf) were measured from -20 to 37°C in the liquid state (56% glycerol at pH 7.6). Two coordination geometries (types B-1 and B-2) with different ESR parameters were present at the N-site. The contents of the coordination geometry of type B-1 at the N-site increased as the temperature increased. The equilibrium constant between the coordination geometries of types B-1 and B-2 was determined by ESR spectra. The enthalpy value from type B-2 to B-1 was +5.3 kcal/mol, as obtained from a van't Hoff plot. The two conformational energies of the cluster models of the copper-binding site at the N-site of dicupric human serum-Tf, where the Arg124 residue was oriented in two different directions (conformations I and II), were calculated by Density Functional Theory, and the enthalpy value from conformation II to I was +2.1 kcal/mol. The enthalpy value was similar to that (+5.3 kcal/mol) obtained by the coordination geometrical change from type B-2 to B-1 in Cu(II)2 serum-Tf. In conformations I and II, the residue of Arg124 at the N-site is located either far from or near the copper-binding site, respectively, and in both cases the coordination geometry of the cupric ions at the N-site has changed from a flattened tetrahedron to a trigonal bipyramid. This result implies that the ESR spectral change from type B-2 to B-1 is caused by the presence of two different orientations of Arg124 in the change from conformation II to I.

Desalting protein ions in native mass spectrometry using supercharging reagents.

Effects of the supercharging reagents m-NBA and sulfolane on sodium ion adduction to protein ions formed using native mass spectrometry were investigated. There is extensive sodium adduction on protein ions formed by electrospray ionization from aqueous solutions containing millimolar concentrations of NaCl, which can lower sensitivity by distributing the signal of a given charge state over multiple adducted ions and can reduce mass measuring accuracy for large proteins and non-covalent complexes for which individual adducts cannot be resolved. The average number of sodium ions adducted to the most abundant ion formed from ten small (8.6-29 kDa) proteins for which adducts can be resolved is reduced by 58% or 80% on average, respectively, when 1.5% m-NBA or 2.5% sulfolane are added to aqueous solutions containing sodium compared to without the supercharging reagent. Sulfolane is more effective than m-NBA at reducing sodium ion adduction and at preserving non-covalent protein-ligand and protein-protein interactions. Desalting with 2.5% sulfolane enables detection of several glycosylated forms of 79.7 kDa holo-transferrin and NADH bound to the 146 kDa homotetramer LDH, which are otherwise unresolved due to peak broadening from extensive sodium adduction. Although sulfolane is more effective than m-NBA at protein ion desalting, m-NBA reduces salt clusters at high m/z and can increase the signal-to-noise ratios of protein ions by reducing chemical noise. Desalting is likely a result of these supercharging reagents binding sodium ions in solution, thereby reducing the sodium available to adduct to protein ions.

Top-down analysis of 30-80 kDa proteins by electron transfer dissociation time-of-flight mass spectrometry.

Electron transfer dissociation (ETD)-based top-down mass spectrometry (MS) is the method of choice for in-depth structure characterization of large peptides, small- and medium-sized proteins, and non-covalent protein complexes. Here, we describe the performance of this approach for structural analysis of intact proteins as large as the 80 kDa serotransferrin. Current time-of-flight (TOF) MS technologies ensure adequate resolution and mass accuracy to simultaneously analyze intact 30-80 kDa protein ions and the complex mixture of their ETD product ions. Here, we show that ETD TOF MS is efficient and may provide extensive sequence information for unfolded and highly charged (around 1 charge/kDa) proteins of ~30 kDa and structural motifs embedded in larger proteins. Sequence regions protected by disulfide bonds within intact non-reduced proteins oftentimes remain uncharacterized due to the low efficiency of their fragmentation by ETD. For serotransferrin, reduction of S-S bonds leads to significantly varied ETD fragmentation pattern with higher sequence coverage of N- and C-terminal regions, providing a complementary structural information to top-down analysis of its oxidized form.

Targeting polymeric fluorescent nanodiamond-gold/silver multi-functional nanoparticles as a light-transforming hyperthermia reagent for cancer cells.

This work demonstrates a simple route for synthesizing multi-functional fluorescent nanodiamond-gold/silver nanoparticles. The fluorescent nanodiamond is formed by the surface passivation of poly(ethylene glycol) bis(3-aminopropyl) terminated. Urchin-like gold/silver nanoparticles can be obtained via one-pot synthesis, and combined with each other via further thiolation of nanodiamond. The morphology of the nanodiamond-gold/silver nanoparticles thus formed was identified herein by high-resolution transmission electron microscopy, and clarified using diffraction patterns. Fourier transform infrared spectroscopy clearly revealed the surface functionalization of the nanoparticles. The fluorescence of the materials with high photo stability was examined by high power laser irradiation and long-term storage at room temperature. To develop the bio-recognition of fluorescent nanodiamond-gold/silver nanoparticles, pre-modified transferrin was conjugated with the gold/silver nanoparticles, and the specificity and activity were confirmed in vitro using human hepatoma cell line (J5). The cellular uptake analysis that was conducted using flow cytometry and inductively coupled plasma mass spectrometry exhibited that twice as many transferrin-modified nanoparticles as bare nanoparticles were engulfed, revealing the targeting and ease of internalization of the human hepatoma cell. Additionally, the in situ monitoring of photothermal therapeutic behavior reveals that the nanodiamond-gold/silver nanoparticles conjugated with transferrin was more therapeutic than the bare nanodiamond-gold/silver materials, even when exposed to a less energetic laser source. Ultimately, this multi-functional material has great potential for application in simple synthesis. It is non-cytotoxic, supports long-term tracing and can be used in highly efficient photothermal therapy against cancer cells.

Reactive polymer as a versatile toolbox for construction of multifunctional superparamagnetic nanocomposites.

The development of magnetic nanoparticles with multiple functions has been an ever-growing field because of their diverse applications in drug delivery, biosensing, cell labeling, and so on. In this study, a facile method was developed to construct multifunctional magnetic nanocomposites. The approach is based on the use of poly(glycidyl methacrylate), PGMA, with numerous epoxy groups as reactive polymer to combine with fluorescent dye, the surface of magnetic nanoparticles, and targeting ligands directly without expatiatory functionality design. The resultant nanocomposites with good superparamagnetic and fluorescent properties could be exploited for bioimaging. Moreover, after conjugation with a model protein, namely, transferrin, which specifically targets cells overexpressing transferrin receptors, the nanocomposites could be used selectively to recognize Hela cells in comparison with nonconjugated ones. These results indicate that the newly designed magnetic nanocomposites with PGMA as functional polymer could serve as a novel versatile platform to conjugate with various molecules for construction of diverse multifunctional magnetic nanocomposites to meet different requirements and potential uses in nanomedicine and biological chemistry.

Beyond bilobal: transferrin homologs having unusual domain architectures.

BACKGROUND: 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. SCOPE OF REVIEW: 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. MAJOR CONCLUSIONS: 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. GENERAL SIGNIFICANCE: 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.

FbpA--a bacterial transferrin with more to offer.

BACKGROUND: 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. SCOPE OF REVIEW: The relevant literature is reviewed which establishes the molecular mechanism of FbpA mediated iron transport across the periplasm to the inner membrane. MAJOR CONCLUSIONS: 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. GENERAL SIGNIFICANCE: 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.

The thermodynamic and binding properties of the transferrins as studied by isothermal titration calorimetry.

BACKGROUND: 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 Fe(3+) binding to a number of transferrins. However, little attention has been given to the thermodynamic characterization of the interaction of transferrin with its receptor. SCOPE OF REVIEW: 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. GENERAL SIGNIFICANCE: 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. MAJOR CONCLUSIONS: The thermodynamic data revealed stoichiometric binding of all tested metal ions to transferrin with very high affinities ranging between 10(17) and 10(22)M(-1). Iron-loaded transferrin (monoferric or diferric) is shown to bind avidly (K~10(7)-10(8)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.

Molecular evolution of the transferrin family and associated receptors.

BACKGROUND: 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). SCOPE OF REVIEW: This article provides an overview of the known transferrin family members and their associated receptors and interacting partners. MAJOR CONCLUSIONS: 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. GENERAL SIGNIFICANCE: 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.

Anion binding properties of the transferrins. Implications for function.

BACKGROUND: Since the transferrins have been defined by the highly cooperative binding of Fe(3+) and a carbonate anion to form an Fe-CO(3)-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. SCOPE OF REVIEW: 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. GENERAL SIGNIFICANCE: 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. MAJOR CONCLUSIONS: 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.

The long history of iron in the Universe and in health and disease.

BACKGROUND: 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. SCOPE OF REVIEW: 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. GENERAL SIGNIFICANCE: 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. MAJOR CONCLUSIONS: 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.