In Situ Insights into the Nucleation and Growth Mechanisms of Gold Nanoparticles on Tobacco Mosaic Virus.

Biotemplated syntheses have emerged as an efficient strategy to control the assembly of metal nanoparticles (NPs) and generate promising plasmonic properties for sensing or biomedical applications. However, understanding the nucleation and growth mechanisms of metallic nanostructures on biotemplate is an essential prerequisite to developing well-controlled nanotechnologies. Here, we used liquid cell Transmission Electron Microscopy (TEM) to reveal how the formation kinetics of gold NPs affects their size and density on Tobacco Mosaic Virus (TMV). These in situ insights are used as a guideline to optimize bench-scale synthesis with the possibility to homogenize the coverage and tune the density of gold NPs on TMV. In line with in situ TEM observations, fluorescence spectroscopy confirms that the nucleation of NPs occurs on the virus capsid rather than in solution. The proximity of gold NPs on TMV allows shifting the plasmonic resonance of the assembly in the biological window.

Interaction between carbon dots from folic acid and their cellular receptor: a qualitative physicochemical approach.

According to the World Health Organization, the number of cancers (all cancers, both sexes, all ages and worldwide) in 2020 reached a total of 19 292 789 new cases leading to 9 958 133 deaths during the same period. Many cancers could be cured if detected early. Preventing cancer and detecting it early are two essential strategies for controlling this pathology. For this purpose, several strategies have been described for imaging cancer cells. One of them is based on the use of carbon nanoparticles called carbon dots, tools of physical chemistry. The literature describes that cancer cells can be imaged using carbon dots obtained from folic acid and that the in cellulo observed photoluminescence probably results from the interaction of these nanoparticles with the folic acid-receptor, a cell surface protein overexpressed in many malignant cells. However, this interaction has never been directly demonstrated yet. We investigated it, for the first time, using (i) freshly synthesized and fully characterized carbon dots, (ii) folate binding protein, a folic acid-receptor model protein and (iii) fluorescence spectroscopy and isothermal titration calorimetry, two powerful methods for detecting molecular interactions. Our results even highlight a selective interaction between these carbon made nano-objects and their biological target.

Modified Fluoroquinolones as Antimicrobial Compounds Targeting Chlamydia trachomatis.

Chlamydia trachomatis causes the most common sexually transmitted bacterial infection and trachoma, an eye infection. Untreated infections can lead to sequelae, such as infertility and ectopic pregnancy in women and blindness. We previously enhanced the antichlamydial activity of the fluoroquinolone ciprofloxacin by grafting a metal chelating moiety onto it. In the present study, we pursued this pharmacomodulation and obtained nanomolar active molecules (EC50) against this pathogen. This gain in activity prompted us to evaluate the antibacterial activity of this family of molecules against other pathogenic bacteria, such as Neisseria gonorrhoeae and bacteria from the ESKAPE group. The results show that the novel molecules have selectively improved activity against C. trachomatis and demonstrate how the antichlamydial effect of fluoroquinolones can be enhanced.

Project ACTIVE: a Randomized Controlled Trial of Personalized and Patient-Centered Preventive Care in an Urban Safety-Net Setting.

BACKGROUND: Evidence-based preventive care in the USA is underutilized, diminishing population health and worsening health disparities. We developed Project ACTIVE, a program to improve adherence with preventive care goals through personalized and patient-centered care. OBJECTIVE: To determine whether Project ACTIVE improved utilization of preventive care and/or estimated life expectancy compared to usual care. DESIGN: Single-site randomized controlled trial. PARTICIPANTS: Cluster-randomized 140 English or Spanish speaking adult patients in primary care with at least one of twelve unfulfilled preventive care goals based on USPSTF grade A and B recommendations. INTERVENTION: Project ACTIVE employs a validated mathematical model to predict and rank individualized estimates of health benefit that would arise from improved adherence to different preventive care guidelines. Clinical staff engaged the participant in a shared medical decision-making (SMD) process to identify highest priority unfulfilled clinical goals, and health coaching staff engaged the participant to develop and monitor action steps to reach those goals. MAIN MEASURES: Change in number of unfulfilled preventive care goals from USPSTF grade A and B recommendations and change in overall gain in estimated life expectancy. KEY RESULTS: In an intent-to-treat analysis, Project ACTIVE increased the average number of fulfilled preventive care goals out of 12 by 0.68 in the intervention arm compared with 0.15 in the control arm (mean difference [95% CI] 0.53 [0.19-0.86]), yielding a gain in estimated life expectancy of 8.8 months (3.8, 14.2). In a per-protocol analysis, Project ACTIVE increased fulfilled preventive care goals by 0.80 in the intervention arm compared with 0.16 in the control arm (mean difference [95% CI], 0.65 [0.25-1.04]), yielding a gain in estimated life expectancy of 13.7 months (6.2, 21.2). Among the 12 preventive care goals, more improvement occurred for alcohol use, hypertension, hyperlipidemia, depression, and smoking. CONCLUSIONS: Project ACTIVE improved unfulfilled preventive care goals and improved estimated life expectancy. CLINICAL TRIAL REGISTRATION NUMBER: NCT04211883.

In vitro activities of a new fluoroquinolone derivative highly active against Chlamydia trachomatis.

Chlamydia trachomatis is a bacterial human pathogen responsible for the development of trachoma, an infection leading to blindness, and is also the cause of the main bacterial sexually transmitted infection worldwide. We designed a new inhibitor of this bacterium with, however, some prerequisites using (i) the iron dependency of the bacterium, (ii) a commercially available broad-spectrum antibiotic and (iii) a short synthetic pathway. The corresponding 8-hydroxyquinoline-ciprofloxacin conjugate was evaluated against a panel of pathogenic bacteria, including C. trachomatis but also the ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species). Its anti-Chlamydia activity is higher than that of ciprofloxacin and seems to be related to the fluoroquinolone moiety of the molecule, which is also responsible for the complexation of iron(III), as demonstrated by spectrophotometric titration.

Iron uptake and transfer from ceruloplasmin to transferrin.

BACKGROUND: Dietary and recycled iron are in the Fe(2+) oxidation state. However, the metal is transported in serum by transferrin as Fe(3+). The multi-copper ferroxidase ceruloplasmin is suspected to be the missing link between acquired Fe(2+) and transported Fe(3+). METHODS: This study uses the techniques of chemical relaxation and spectrophotometric detection. RESULTS: Under anaerobic conditions, ceruloplasmin captures and oxidizes two Fe(2+). The first uptake occurs in domain 6 (<1ms) at the divalent iron-binding site. It is accompanied by Fe(2+) oxidation by Cu(2+)D6. Fe(3+) is then transferred from the binding site to the holding site. Cu(+)D6 is then re-oxidized by a Cu(2+) of the trinuclear cluster in about 200ms. The second Fe(2+) uptake and oxidation involve domain 4 and are under the kinetic control of a 200s change in the protein conformation. With transferrin and in the formed ceruloplasmin-transferrin adduct, two Fe(3+) are transferred from their holding sites to two C-lobes of two transferrins. The first transfer (~100s) is followed by conformation changes (500s) leading to the release of monoferric transferrin. The second transfer occurs in two steps in the 1000-10,000second range. CONCLUSION: Fe(3+) is transferred after Fe(2+) uptake and oxidation by ceruloplasmin to the C-lobe of transferrin in a protein-protein adduct. This adduct is in a permanent state of equilibrium with all the metal-free or bounded ceruloplasmin and transferrin species present in the medium. GENERAL SIGNIFICANCE: Ceruloplasmin is a go-between dietary or recycled Fe(2+) and transferrin transported Fe(3+).

Transferrin receptor-1 iron-acquisition pathway - synthesis, kinetics, thermodynamics and rapid cellular internalization of a holotransferrin-maghemite nanoparticle construct.

BACKGROUND: Targeting nanoobjects via the iron-acquisition pathway is always reported slower than the transferrin/receptor endocytosis. Is there a remedy? METHODS: Maghemite superparamagnetic and theragnostic nanoparticles (diameter 8.6nm) were synthesized, coated with 3-aminopropyltriethoxysilane (NP) and coupled to four holotransferrin (TFe2) by amide bonds (TFe2-NP). The constructs were characterized by X-ray diffraction, transmission electron microscopy, FTIR, X-ray Electron Spectroscopy, Inductively Coupled Plasma with Atomic Emission Spectrometry. The in-vitro protein/protein interaction of TFe2-NP with transferrin receptor-1 (R1) and endocytosis in HeLa cells were investigated spectrophotometrically, by fast T-jump kinetics and confocal microscopy. RESULTS: In-vitro, R1 interacts with TFe2-NP with an overall dissociation constant KD=11nM. This interaction occurs in two steps: in the first, the C-lobe of the TFe2-NP interacts with R1 in 50µs: second-order rate constant, k1=6×10(10)M(-1)s(-1); first-order rate constant, k-1=9×10(4)s(-1); dissociation constant, K1d=1.5µM. In the second step, the protein/protein adduct undergoes a slow (10,000s) change in conformation to reach equilibrium. This mechanism is identical to that occurring with the free TFe2. In HeLa cells, TFe2-NP is internalized in the cytosol in less than 15min. CONCLUSION: This is the first time that a nanoparticle-transferrin construct is shown to interact with R1 and is internalized in time scales similar to those of the free holotransferrin. GENERAL SIGNIFICANCE: TFe2-NP behaves as free TFe2 and constitutes a model for rapidly targeting theragnostic devices via the main iron-acquisition pathway.

Uptake and release of metal ions by transferrin and interaction with receptor 1.

BACKGROUND: 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. SCOPE OF THE REVIEW: 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. MAJOR CONCLUSIONS: 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 (2-6h) 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 0.5µM, 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. GENERAL SIGNIFICANCE: 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.

Gold and Iron Oxide Nanoparticle Assemblies on Turnip Yellow Mosaic Virus for In-Solution Photothermal Experiments.

The ability to construct three-dimensional architectures via nanoscale engineering is important for emerging applications in sensors, catalysis, controlled drug delivery, microelectronics, and medical diagnostics nanotechnologies. Because of their well-defined and highly organized symmetric structures, viral plant capsids provide a 3D scaffold for the precise placement of functional inorganic particles yielding advanced hierarchical hybrid nanomaterials. In this study, we used turnip yellow mosaic virus (TYMV), grafting gold nanoparticles (AuNP) or iron oxide nanoparticles (IONP) onto its outer surface. It is the first time that such an assembly was obtained with IONP. After purification, the resulting nano-biohybrids were characterized by different technics (dynamic light scattering, transmission electron microcopy, X-ray photoelectron spectroscopy…), showing the robustness of the architectures and their colloidal stability in water. In-solution photothermal experiments were then successfully conducted on TYMV-AuNP and TYMV-IONP, the related nano-biohybrids, evidencing a net enhancement of the heating capability of these systems compared to their free NP counterparts. These results suggest that these virus-based materials could be used as photothermal therapeutic agents.

In vitro interaction between ceruloplasmin and human serum transferrin.

The thermodynamics of the interactions of serum apotransferrin (T) and holotransferrin (TFe(2)) with ceruloplasmin (Cp), as well as those of human lactoferrin (Lf), were assessed by fluorescence emission spectroscopy. Cp interacts with two Lf molecules. The first interaction depends on pH and µ, whereas the second does not. Dissociation constants were as follows: K(11Lf) = 1.5 ± 0.2 µM, and K(12Lf) = 11 ± 2 µM. Two slightly different interactions of T or TFe(2) with Cp are detected for the first time. They are both independent of pH and µ and occur with 1:1 stoichiometry: K(1T) = 19 ± 7 µM, and K(1TFe2) = 12 ± 4 µM. These results can improve our understanding of the probable process of the transfer of iron from Cp to T in iron and copper transport and homeostasis.

Can uranium follow the iron-acquisition pathway? Interaction of uranyl-loaded transferrin with receptor 1.

Transferrin receptor 1 (R(D)) binds iron-loaded transferrin and allows its internalization in the cytoplasm. Human serum transferrin also forms complexes with metals other than iron, including uranium in the uranyl form (UO(2)(2+)). Can the uranyl-saturated transferrin (TUr(2)) follow the receptor-mediated iron-acquisition pathway? In cell-free assays, TUr(2) interacts with R(D) in two different steps. The first is fast, direct rate constant, k(1) = (5.2 +/- 0.8) x 10(6) M(-1) s(-1); reverse rate constant, k(-1) = 95 +/- 5 s(-1); and dissociation constant K(1) = 18 +/- 6 microM. The second occurs in the 100-s range and leads to an increase in the stability of the protein-protein adduct, with an average overall dissociation constant K(d) = 6 +/- 2 microM. This kinetic analysis implies in the proposed in vitro model possible but weak competition between TUr(2) and the C-lobe of iron-loaded transferrin toward the interaction with R(D).

Thermodynamics and kinetics of the complexation reaction of lead by calix-DANS4.

The thermodynamics and kinetics of the complexation reaction between lead ions and the fluorescent sensor Calix-DANS4 are determined to optimize the geometry of the microreactor used for the flow-injection analysis of lead and to tune the working conditions of this microdevice. Under our experimental conditions (pH 3.2, low concentration of Calix-DANS4) the 1:1 Pb(2+)-Calix-DANS4 complex is predominantly formed with a high stability constant (log K(1:1)=6.82) and a slow second-order rate constant (k=9.4×10(4) L mol(-1) s(-1)). Due to this sluggish complexation reaction, the microchannel length must be longer than 130 mm and the flow rate lower than 0.25 mL h(-1) to have an almost complete reaction at the output of the microchannel and a high sensitivity for the heavy metal detection. After determination of the values of the reaction times in our different microdevices, it is possible to simulate the calibration curves for the fluorimetric detection of lead under different conditions. An original method is also presented to determine mixing times in microreactors.

In Vitro interaction between yeast frataxin and superoxide dismutases: Influence of mitochondrial metals.

BACKGROUND: Friedreich's ataxia results from a decreased expression of the nuclear gene encoding the mitochondrial protein, frataxin. Frataxin participates in the biosynthesis of iron-sulfur clusters and heme cofactors, as well as in iron storage and protection against oxidative stress. How frataxin interacts with the antioxidant defence components is poorly understood. METHODS: Therefore, we have investigated by kinetic, thermodynamic and modelling approaches the molecular interactions between yeast frataxin (Yfh1) and superoxide dismutases, Sod1 and Sod2, and the influence of Yfh1 on their enzymatic activities. RESULTS: Yfh1 interacts with cytosolic Sod1 with a dissociation constant, Kd = 1.3 ±â€¯0.3 µM, in two kinetic steps. The first step occurs in the 200 ms range and corresponds to the Yfh1-Sod1 interaction, whereas the second is slow and is assumed to be a change in the conformation of the protein-protein adduct. Furthermore, computational investigations confirm the stability of the Yfh1-Sod1 complex. Yfh1 forms two protein complexes with mitochondrial Sod2 with 1:1 and 2:1 Yfh1/Sod2 stoichiometry (Kd1 = 1.05 ±â€¯0.05 and Kd2 = 6.6 ±â€¯0.1 µM). Furthermore, Yfh1 increases the enzymatic activity of Sod1 while slightly affecting that of Sod2. Finally, the stabilities of the protein-protein adducts and the effect of Yfh1 on superoxide dismutase activities depend on the nature of the mitochondrial metal. CONCLUSIONS: This work confirms the participation of Yfh1 in cellular defence against oxidative stress.

Assembly of gold nanoparticles using turnip yellow mosaic virus as an in-solution SERS sensor.

A common challenge in nanotechnology is the conception of materials with well-defined nanoscale structure. In recent years, virus capsids have been used as templates to create a network to organize 3D nano-objects, building thus new functional nanomaterials and then devices. In this work, we synthetized 3D gold nanoclusters and we used them as Surface Enhanced Raman Scattering (SERS) sensor substrates in solution. In practice, gold nanoparticles (AuNPs) were grafted on turnip yellow mosaic virus (TYMV) capsid, an icosahedral plant virus. Two strategies were considered to covalently bind AuNPs of different sizes (5, 10 and 20 nm) to TYMV. After purification by agarose electrophoresis and digestion by agarase, the resulting nano-bio-hybrid AuNP-TYVM was characterized by different tools. Typically, dynamic light scattering (DLS) confirmed the grafting through the hydrodynamic size increase by comparing AuNPs alone to AuNP-TYMV (up to 33, 50 and 68 nm for 5, 10 and 20 nm sized AuNPs, respectively) or capsids alone (28 nm). Transmission electronic microscopy (TEM) observations revealed that AuNPs were arranged with 5-fold symmetry, in agreement with their grafting around icosahedral capsids. Moreover, UV-vis absorption spectroscopy showed a red-shift of the plasmon absorption band on the grafted AuNP spectrum (530 nm) compared to that of the non-grafted one (520 nm). Finally, by recording in solution the Raman spectra of a dissolved probe molecule, namely 1,2-bis(4-pyridyl)ethane (BPE), in the presence of AuNP-TYVM and bare AuNPs or capsids, a net enhancement of the Raman signal was observed when BPE is adsorbed on AuNP-TYVM. The analytical enhancement factor (AEF) value of AuNP-TYMV is 5 times higher than that of AuNPs. These results revealed that AuNPs organized around virus capsid are able to serve as in-solution SERS-substrates, which is very interesting for the conception of ultrasensitive sensors in biological media.

Kinetics and thermodynamics of metal-loaded transferrins: transferrin receptor 1 interactions.

Transferrin receptor 1 (R) and human serum transferrin (T) are the two main actors in iron acquisition by the cell. R binds TFe(2) (iron-loaded transferrin), which allows its internalization in the cytoplasm by endocytosis. T also forms complexes with metals other than iron. In order to follow the iron-acquisition pathway, these metals should obey at least two essential rules: (i) formation of a strong complex with T; and (ii) interaction of this complex with R. In the present paper, we propose a general mechanism for the interaction of five metal-loaded Ts [Fe(III), Al(III), Bi(III), Ga(III) and Co(III)] with R and we discuss their potential incorporation by the iron-acquisition pathway. With iron- and cobalt-loaded Ts, the interaction of R takes place in two steps: the first is detected by the T-jump technique and occurs in the 100 micros range, whereas the second is slow and occurs in the hour range. Bi(III)- and Ga(III)-loaded Ts interact with R in a single fast kinetic step, which occurs in the 100-500 micros range. No interaction is detected between R and aluminium-saturated T. The fast steps are ascribed to the interaction of the C-lobe of metal-loaded T with the helical domain of R: dissociation constant, K'(1), of 0.50+/-0.07, 0.82+/-0.25, 4+/-0.4 and 1.10+/-0.12 microM for Fe(III), Co(III), Bi(III) and Ga(III) respectively. The second slow steps are ascribed to changes in the conformation of the protein-protein adducts which increase the stability to achieve, at thermodynamic equilibrium, an overall dissociation constant, K(1), of 2.3 and 25 nM for Fe(III) and Co(III) respectively. This last step occurs over several hours, whereas endocytosis takes place in several minutes. This implies that metal-loaded Ts are internalized with only the C-lobe interacting with R. This suggests that, despite a lower affinity for R when compared with TFe(2), some metal-loaded Ts can compete kinetically with TFe(2) for the interaction with R and thus follow the iron-acquisition pathway.

The mechanism of iron release from the transferrin-receptor 1 adduct.

We report the determination in cell-free assays of the mechanism of iron release from the N-lobe and C-lobe of human serum transferrin in interaction with intact transferrin receptor 1 at 4.3< or =pH< or =6.5. Iron is first released from the N-lobe in the tens of milliseconds range and then from the C-lobe in the hundreds of seconds range. In both cases, iron loss is rate-controlled by slow proton transfers, rate constant for the N-lobe k(1)=1.20(+/-0.05)x10(6)M(-1)s(-1) and for the C-lobe k(2)=1.6(+/-0.1)x10(3)M(-1)s(-1). This iron loss is subsequent to a fast proton-driven decarbonation and is followed by two proton gains, (pK(1a))/2=5.28 per proton for the N-lobe and (pK(2a))/2=5.10 per proton for the C-lobe. Under similar experimental conditions, iron loss is about 17-fold faster from the N-lobe and is at least 200-fold faster from the C-lobe when compared to holotransferrin in the absence of receptor 1. After iron release, the apotransferrin-receptor adduct undergoes a slow partial dissociation controlled by a change in the conformation of the receptor; rate constant k(3)=1.7(+/-0.1)x10(-3)s(-1). At endosomic pH, the final equilibrated state is attained in about 1000 s, after which the free apotransferrin, two prototropic species of the acidic form of the receptor and apotransferrin interacting with the receptor coexist simultaneously. However, since recycling of the vesicle containing the receptor to the cell surface takes a few minutes, the major part of transferrin will still be forwarded to the biological fluid in the form of the apotransferrin-receptor protein-protein adduct.

Cd2+ and Pb2+ complexation by glutathione and the phytochelatins.

Phytochelatins or PCn, (γGlu-Cys)n-Gly, and their glutathione (GSH) precursor are thiol-rich peptides that play an important role in heavy metal detoxification in plants and microorganisms. Complex formation between Cd2+ and Pb2+ and GSH or PCn (n = 2, 4 and 6) are investigated by microcalorimetry, absorption spectrophotometry and T-jump kinetics. Complex formation with Pb2+ or Cd2+ is exothermic, and induces ligand metal charge transfer bands in UV absorption spectral range, which implies the formation of a coordination bond between the metal and the thiol groups of the phytochelatins. Absorption spectra and microcalorimetry experiments allow the determination of the affinity constants and the stoichiometry of the complexes. We show that the three PCn interact with Pb2+ to form the 1:1 and 2:1 M:L complexes, with similar affinity constants (log K11Pb∼4.6, log K21Pb∼11.4). These affinities are independent of the number of thiols and are, moreover, lower than those determined for complex formation with Cd2+. On the other hand, with Cd2+, PC2-Cd, PC2-Cd2, (PC2)3-Cd2, PC4-Cd, PC4-Cd2, PC6-Cd, (PC6)2-Cd3 and PC6-Cd3 complexes are detected. Furthermore, for PC4-Cd, the 1:1 complex is the most stable: affinity constant (log K11Cd∼7.5). Kinetic studies indicate that complex formation between Cd2+ and GSH occurs in the ms range; direct rate constant kobs = (6.8 ± 0.3) 106 M-1 s-1 and reverse rate constant k-obs = 340 ± 210 s-1. Thus, when encapsulated in a silica matrix, PCn can be good candidates for heavy metal detection.

Direct thermodynamic and kinetic measurements of Fe²âº and Zn²âº binding to human serum transferrin.

Human serum transferrin (hTf) is a single-chain bilobal glycoprotein that efficiently delivers iron to mammalian cells by endocytosis via the transferrin/transferrin receptor system. While extensive studies have been directed towards the study of ferric ion binding to hTf, ferrous ion interactions with the protein have never been firmly investigated owing to the rapid oxidation of Fe(II) to Fe(III) and the difficulty in maintaining a fully anaerobic environment. Here, the binding of Fe(2+) and Zn(2+) ions to hTf has been studied under anaerobic and aerobic conditions, respectively, in the presence and absence of bicarbonate by means of isothermal titration calorimetry (ITC) and fluorescence spectroscopy. The ITC data indicate the presence of one class of strong binding sites with dissociation constants of 25.2 nM for Fe(2+) and 6.7 nM for Zn(2+) and maximum binding stoichiometries of 1 Zn(2+) (or 1 Fe(2+)) per hTf molecule. With either metal, the binding interaction was achieved by both favorable enthalpy and entropy changes (ΔH(0)~-12 kJ/mol and ΔS(0)~106 J/mol·K for Fe(2+) and ΔH(0)~-18 kJ/mol and ΔS(0)~97 J/mol·K for Zn(2+)). The large and positive entropy values are most likely due to the change in the hydration of the protein and the metal ions upon interaction. Rapid kinetics stopped-flow fluorescence spectroscopy revealed two different complexation mechanisms with different degrees of conformational changes upon metal ion binding. Our results are discussed in terms of a plausible scenario for iron dissociation from transferrin by which the highly stable Fe(3+)-hTf complex might be reduced to the more labile Fe(2+) ion before iron is released to the cytosol.

Can uranium be transported by the iron-acquisition pathway? Ur uptake by transferrin.

Transferrin (T) is one of the major protein targets of uranyl (Ur) and the Ur-loaded protein (TUr(2)) interacts with receptor 1. In vitro, Ur is transferred from one of the major plasma complexes, tricarbonated Ur (Ur(CO(3))(3)(4-)) to T in four kinetically differentiated steps. The first is very fast and accompanied by HCO(3)(-) loss. It yields a first intermediate ternary complex between dicarbonated Ur and the phenolate of one of the two tyrosine ligands in the C-lobe; direct rate constant, k(1) = (7.0 ± 0.4) × 10(5) M(-1) s(-1); reverse rate constant, k(-1) = (4.7 ± 0.2) × 10(3) M(-1) s(-1); dissociation constant, K(1) = (6.7 ± 0.6) × 10(-3) and an affinity of the T for the dicarbonated Ur (Ur(CO(3))(2)(2-)) close to that of the latter to CO(3)(2-), K'(3) ~ 1 × 10(4). This first kinetic product undertakes a fast rate-limiting conformation change leading to the loss of a second HCO(3)(-): direct rate constant, k(2) = 33 ± 14 s(-1). This second ternary complex undergoes two very slow conformation changes (1 and 5 h), at the end of which both C- and N-lobes become loaded with Ur. When unexposed to uranium, the Ur concentrations in the bloodstream are much too low to favor receptor-mediated transport. However, in the case of exposure, these concentrations can grow considerably. This, added to the fast Ur complex formation with the C-lobe and the fast interaction of the Ur-loaded T with the receptor, can allow a possible internalization by the iron-acquisition pathway.

Selenodiglutathione uptake by the Saccharomyces cerevisiae vacuolar ATP-binding cassette transporter Ycf1p.

The Saccharomyces cerevisiae vacuolar ATP-binding cassette transporter Ycf1p is involved in heavy metal detoxification by mediating the ATP-dependent transport of glutathione-metal conjugates to the vacuole. In the case of selenite toxicity, deletion of YCF1 was shown to confer increased resistance, rather than sensitivity, to selenite exposure [Pinson B, Sagot I & Daignan-Fornier B (2000) Mol Microbiol36, 679-687]. Here, we show that when Ycf1p is expressed from a multicopy plasmid, the toxicity of selenite is exacerbated. Using secretory vesicles isolated from a sec6-4 mutant transformed either with the plasmid harbouring YCF1 or the control plasmid, we establish that the glutathione-conjugate selenodigluthatione is a high-affinity substrate of this ATP-binding cassette transporter and that oxidized glutathione is also efficiently transported. Finally, we show that the presence of Ycf1p impairs the glutathione/oxidized glutathione ratio of cells subjected to a selenite stress. Possible mechanisms by which Ycf1p-mediated vacuolar uptake of selenodiglutathione and oxidized glutathione enhances selenite toxicity are discussed.